Soil Microbiology and Sustainable Crop Production

Soil Microbiology and Sustainable
Crop Production
Geoffrey R. Dixon • Emma L. Tilston
Editors
Soil Microbiology and

Sustainable Crop Production
Editors
Geoffrey R. Dixon Emma L. Tilston
Centre for Horticulture & Landscape Scottish Universities Environmental
School of Biological Sciences Research Centre
University of Reading, Whiteknights Scottish Enterprise Technology Park,
Reading, Berkshire, RG6 6AS, UK East Kilbride, G75 0QF, UK
[email protected] [email protected]
ISBN 978-90-481-9478-0 e-ISBN 978-90-481-9479-7

DOI 10.1007/978-90-481-9479-7
Springer Dordrecht Heidelberg London New York
Library of Congress Control Number: 2010934733
© Springer Science+Business Media B.V. 2010

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Cover illustration:
Background: Sustainable crop production as represented by bed production of salad leaves with adjacent
wildlife refuge and trees (copyright: Geoff Dixon)
Small left photo: Three roots of swede showing galling caused by Plasmodiophora brassicae (Clubroot);
(copyright: Geoff Dixon)
Small middle photo: Roots of pea plant showing nodules caused by nitrifying bacteria (copyright: Oksana
Shtark)
Small right photo: Three roots of winter wheat showing moderate to severe damage caused by
Gaeumannomyces graminis var. tritici (Take-All) (copyright: Emma Tilston).
Printed on acid-free paper
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Preface
Nothing is so fatal to the progress of the human mind as to suppose ........ that there are no
new worlds to conquer. (Humphrey Davy, English engineer and physicist, public lecture
1810)
Soil and in particular its microbial diversity remains largely an unexplored world.
A few researchers have provided insights into the outer edges of this world but it
remains mostly unknown and inhabited by a huge diversity of organisms whose
biology is open to speculation. Yet it is this world and its inhabitants which argu-
ably hold many of the properties which will enable mankind to surmount the huge
problems resulting from a burgeoning population and diminishing land supply.
Increasing food production in parallel with conserving and protecting our environ-
ment while allowing producers adequate financial returns are the primary challenges
facing agricultural science research in the twenty-first century. These factors of
food production, environmental protection and producers’ profit form a triangle
which defines agrarian sustainability. Sustainably raising crop production will only
be achieved by gaining far greater understanding of the physics and chemistry of
the soil environment in which roots grow and the impact of benign and pathogenic
microbes on them. It is at least as important that we understand the world beneath
our feet as we do the Earth’s atmosphere and oceans and those of neighbouring
planets. Increasing the benefits obtained from soil microbes must be linked with
cautious care for the world which they inhabit. Ill-judged and ignorant exploitation
has lead to, and continues leading to, devastated land where the soil is degraded into
lifeless dust-bowls where structure, texture and biological activity are lost and
salinity rises. The very nature of soils has slowed their study until recently. Now the
tools of molecular biology are offering powerful new ways of unravelling complex
relationships and simplifying interactions.
This Book provides an insight into the developing knowledge of soil microbes
and points to ways by which they can be utilised in support of agronomically and
environmentally sustainable crop production. This context is introduced in the first
chapter which sets out the parameters of sustainable production. It is succeeded by
analyses of microbiology in natural, unfarmed soil defining the baselines from
which agriculture has modified soil resources. Chapters describing nutrient cycling
and the development of soil organic matter clearly demonstrate the impact of man-
kind’s activities and means by which these may be tailored to achieve sustainable
v
vi Preface
objectives. Detailed studies of beneficial and pathogenic soil- and root-borne

microbes follow identifying the continuous interactions between plants and the
organisms with which they co-exist. Outcomes of this co-existence can be either
immensely valuable in terms of raising crop health and productivity or totally disas-
trous leading to disease and death. Husbandry practices affect the balance between
these outcomes. Gradually the significance of the way in which land-use interacts
with crop production and the potential for its manipulation to raise sustainable
yields is being extending into mainstream crop production. This philosophy is not
new but had been ignored and side-lined for at least the latter part of the twentieth
century by crop production methods based almost solely on approaches which have
targeted the plant as opposed to the soil. Crop agronomy using a soil-based
approach demands as a priority the revision of plant breeders’ targets. A result
should be new cultivars tailored to attain maximum yields and quality in harmony
with beneficial soil-borne microbes. This approach will enable crop producers to
make far greater use of integrated systems for the control of pests and pathogens.
Biological control used with cultivars fitted for increased productivity growing in
soil managed by husbandry systems which enhance beneficial microbial popula-
tions could deliver the sustainable yield enhancements needed by population
growth. Greater understanding and manipulation of the soil environment must
include knowledge of the aerial environment and the manner by which it is chang-
ing. Both environments interact and influence each other consequently consider-
ation must be given to the impact of global climate change both directly and
indirectly on soil microbial populations. Changes to air temperature, precipitation
and wind will have direct and substantial effects on soil borne microbes. Evidence
for this is already apparent in the movements of aggressive pathogenic species in to
previously un-colonised regions. Ultimately, increasing crop productivity and car-
ing for the soil environment can only be considered to be fully sustainable if farm-
ers and growers are able to maintain viable and successful businesses. Soil is the
first resource which suffers from lack of care where economic sustainability is
absent. Ensuring adequate incomes for soil users is integral and essential for the
achievement of sustainability in both crop production and environmental care and
conservation. Science and its practitioners can, given the necessary resources, open
up and more fully explore soils for the greater benefit of mankind and the Earth’s
environment and its natural biodiversity. It is the task of politicians to understand
the opportunities which this offers and ensure that the general public, the tax-pay-
ers, recognise why financial resources should be applied to the crucial task of
acquiring knowledge of the microbes in soil. Regrettably, for at least the past gen-
eration there has been a worldwide failure by politicians and their advisors to pro-
vide adequately for studies of the agricultural and soil sciences. Unless this
situation is reversed in the very short-term none of the opportunities outlined in this
Book will be realised. That will exacerbate the famines which afflict mankind and
continue the destruction of the Earth’s soil, its environment and its inhabitants.
This Book was conceived as a contribution towards the international debate on
population growth, food insecurity and the conservation of biodiversity. Gradually
there is a recognition that soils demand as much attention as the atmosphere as an
Preface vii
integral part of the biosphere. It has been written by an international team of

researchers recruited from around the world. Substantial effort, expert knowledge
and energy have brought this project to fruition for which the Editors Geoffrey R.
Dixon and Emma L. Tilston sincerely thank each member of the team of authors.
All have been prepared to devote considerable time to fulfilling this task and have
shared with the Editors the joys and frustrations inherent in writing such a Book.
We thank all of them for the care with which they have submitted excellently
authoritative manuscripts. The Editors have been supported by many family and
friends. Particular thanks go to Mrs Kathy Dixon who with much patience and good
humour aids and accepts her husband’s literary activities. Both Editors have made
much use of the facilities in the Library and our respective Departments
(Horticulture and Landscape and Soil Science) at the University of Reading for
which we offer very grateful thanks and acknowledgement. Authors have offered
acknowledgements at the ends of their Chapters.
Professor Geoffrey R. Dixon and Dr. Emma L. Tilston
March 2010 Sherborne, Dorset and Glasgow
Contents
1 The Nature of Sustainable Agriculture.................................................. 1

Andrew D. Noble and Sawaeng Ruaysoongnern
2 The Microbiology of Natural Soils......................................................... 27

Teri C. Balser, Devin Wixon, Lindsey K. Moritz, and Laura Lipps
3 Soil Microbiology and Nutrient Cycling................................................ 59

David W. Hopkins and Jennifer A.J. Dungait
4 The Role of Microbial Communities in the Formation

and Decomposition of Soil Organic Matter .......................................... 81
Leo Condron, Christine Stark, Maureen O’Callaghan,
Peter Clinton, and Zhiqun Huang
5 Intimate Associations of Beneficial Soil Microbes

with Host Plants....................................................................................... 119
Oksana Y. Shtark, Alexey Y. Borisov, Vladimir A. Zhukov,
Nikolay A. Provorov, and Igor A. Tikhonovich
6 Soil-Borne Pathogens and Their Interactions

with the Soil Environment....................................................................... 197
Geoffrey R. Dixon and Emma L. Tilston
7 The Impact of Land-Use Practices on Soil Microbes........................... 273

Emma L. Tilston, Tom Sizmur, Geoffrey R. Dixon,
Wilfred Otten, and Jim A. Harris
8 The Effects of Plant Breeding on Soil Microbes.................................... 297

Petra Marschner and Zed Rengel
9 Utilizing Soil Microbes for Biocontrol................................................... 315

Alison Stewart, Michael Brownbridge, Robert A. Hill,
and Trevor A. Jackson
ix
x Contents
10 How Will Climate Change Impact Soil

Microbial Communities? . ...................................................................... 373
Teri C. Balser, Jessica L.M. Gutknecht, and Chao Liang
11 Evaluating the Economic and Social Impact

of Soil Microbes........................................................................................ 399
Harpinder S. Sandhu, Vadakattu V.S.R. Gupta,
and Stephen D. Wratten
Index.................................................................................................................. 419
Contributors
Teri C. Balser
Department of Soil Science, University of Wisconsin-Madison,
1525 Observatory Drive, Madison, WI 53706, USA
[email protected]
Alexey Y. Borisov
All Russia Research Institute for Agricultural Microbiology (ARRIAM),
Laboratory of Genetics of Plant-Microbe Interactions, Department of Biotechnology,
Podbelsky chausee 3, St. Petersburg, Pushkin 8, 196608, Russia
[email protected]
Michael Brownbridge
Vineland Research and Innovation Centre,
P.O. Box 8000, Vineland Station, ON LOR 2EO, Canada
[email protected]
Peter Clinton
Scion, P.O. Box 29-237, Fendalton, Christchurch 8041, New Zealand
[email protected]
Leo Condron
Agriculture and Life Sciences, Lincoln University,
P.O. Box 84, Lincoln 7647, Christchurch, New Zealand
[email protected]
Geoffrey R. Dixon
Centre for Horticulture & Landscape, School of Biological Sciences,
Whiteknights, University of Reading, Berkshire, RG6 6AS, UK
[email protected]
Jennifer A.J. Dungait
Rothamsted Research – North Wyke, Okehampton, Devon, EX20 2SB, UK
[email protected]
Vadakattu V.S.R. Gupta
CSIRO Entomology, PMB No. 2, Glen Osmond South Australia 5064, Australia
[email protected]
xi
xii Contributors
Jessica L.M. Gutknecht

Department of Soil Ecology, Helmholtz-Centre for Environmental Research-UFZ,
Theodor-Lieser-Str, 4, D06120 Halle (Saale), Germany
[email protected]
Jim A. Harris
Natural Resources Department, School of Applied Sciences, Cranfield University,
Cranfield, Bedfordshire, MK43 0AL, UK
[email protected]
Robert A. Hill
Bio-protection Research Centre, Lincoln University, PO Box 84,
Lincoln University, Lincoln 7647, Christchurch, New Zealand
[email protected]
David W. Hopkins
Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, UK
[email protected]
Zhiqun Huang
Scion, PO Box 29-237, Fendalton, Christchurch 8041, New Zealand
[email protected]
Trevor A. Jackson
AgResearch, Private Bag 4749, Christchurch 8140, New Zealand
[email protected]
Chao Liang
1525 Observatory Drive, Madison, WI 53706, USA
[email protected]
Laura Lipps
Gaylord Nelson Institute for Environmental Studies,
University of Wisconsin-Madison, Madison, WI 53706, USA
[email protected]
Petra Marschner
Soils, School of Agriculture, Food & Wine, The University of Adelaide,
Adelaide, South Australia 5005, Australia
[email protected]
Lindsey K. Moritz
1525 Observatory Drive, Madison WI, 53706, USA
[email protected]
Contributors xiii
Andrew D. Noble
International Water Management Institute,
c/o National Agriculture and Forestry Research Institute (NAFRI),
Ban Nongviengkham, Xaythany District, Vientiane, Lao PDR
[email protected]
Maureen O’Callaghan
Maureen.o’[email protected]
Wilfred Otten
SIMBIOS Centre, University of Abertay, Bell Street, Dundee, DD1 1HG, UK
[email protected]
Nikolay A. Provorov
[email protected]
Zed Rengel
School of Earth and Environment, The University of Western Australia,
Crawley, Western Australia 6009, Australia
[email protected]
Sawaeng Ruaysoongnern
Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand
[email protected]
Harpinder S. Sandhu
CSIRO Sustainable Ecosystems, PMB No. 2, Glen Osmond,
South Australia 5064, Australia
[email protected]
Oksana Y. Shtark
Laboratory of Genetics of Plant-Microbe Interactions, Department of
Biotechnology, Podbelsky chausee 3, St. Petersburg, Pushkin 8, 196608, Russia
[email protected]
Tom Sizmur
Department of Soil Science, University of Reading, Whiteknights,
Berkshire, RG6 6DW, UK
[email protected]
Christine Stark
Environmental and Geographical Sciences,
Manchester Metropolitan University, Manchester, UK
[email protected]
xiv Contributors
Alison Stewart
Bio-protection Research Centre, Lincoln University,
PO Box 84, Lincoln University, Lincoln 7647, New Zealand
[email protected]
Igor A. Tikhonovich
Podbelsky chausee 3, St. Petersburg,
Pushkin 8, 196608, Russia
[email protected]
Emma L. Tilston
Scottish Universities Environmental Research Centre,
Scottish Enterprise Technology Park, East Kilbride, G75 0QF, UK
[email protected]
Devin Wixon
Department of Botany, University of Wisconsin-Madison,
430 Lincoln Drive, Madison, WI 53706, USA
[email protected]
Stephen D. Wratten
Bio-Protection Research Centre, Lincoln University,
P.O. Box 84, Lincoln 7647, New Zealand
[email protected]
Vladimir A. Zhukov
Laboratory of Genetics of Plant-Microbe Interactions,
Department of Biotechnology, Podbelsky chausee 3,
St. Petersburg, Pushkin 8, 196608, Russia
[email protected]
Chapter 1
The Nature of Sustainable Agriculture
Andrew D. Noble and Sawaeng Ruaysoongnern
Introduction
By 2050 the world’s population will have increased by a third and demand for
agricultural products will rise by 70% with meat consumption doubling. Lateral
expansion of the agricultural sector through the clearing of land is untenable with-
out significant negative implications on already stressed natural ecosystems and the
range of drivers, including climate variability, that farmers will have to cope with,
will require changes in the way we undertake agriculture. In meeting future food
production demands without consuming more land and water will require techno-
logical innovation and changes in the way agriculture is undertaken. The chapter
discusses the future global demands for food and highlights the importance of
addressing soil chemical and physical constraints in increasing the productivity of
degraded production systems. The role of clays in permanently changing the sur-
face charge characteristics of soils and the potential for selected grass species to
remediate compacted soil layers are presented as possible options in addressing the
sustainability of degraded production systems.
The fragility of our current agriculture systems was brought into sharp focus
during the recent global food crisis of 2007–2008. Not since the 1970s when the
tumultuous famines of south Asia and the Horn of Africa rocked the world, have
we seen such turmoil in global food supply and prices. National governments
imposed restrictions on grain exports and people came out on the streets protesting
dramatic increases in the prices of basic commodities, with the poorest and most
marginalized sectors of the global community being most vulnerable. Governments
A.D. Noble (*)

International Water Management Institute, c/o National Agriculture and Forestry Research
Institute (NAFRI), Ban Nongviengkham, Xaythany District, Vientiane, Lao PDR
e-mail: [email protected]
S. Ruaysoongnern
Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand
G.R. Dixon and E.L. Tilston (eds.), Soil Microbiology and Sustainable Crop Production, 1
DOI 10.1007/978-90-481-9479-7_1, © Springer Science+Business Media B.V. 2010
2 A.D. Noble and S. Ruaysoongnern
around the world were caught off guard and scrambled to address the problem.
How could this happen in the twenty-first century?
It has been argued that the recent food crisis was the confluence of a range of
drivers that included but was not limited to, the effects of competition for cropland
from the growth in biofuels; low global cereal stocks; high oil prices; speculation
in food markets; restrictions on grain exports in key countries; and extreme weather
events (Nellemann et al. 2009). The impacts of reduced food availability, higher
food prices and thus lower access to food by many people have been dramatic. It is
estimated that the crisis resulted in a dramatic increase in several central commod-
ity prices, drove an estimated 110 million people into poverty and added 44 million
more to the already undernourished (World Bank 2008).
The underlying supply and demand tensions are still present today as evidenced
by the elevated food price index and price indices of major commodities when
compared to the recent past (Fig. 1) (FAO Statistics 2009). These recent failures
may reflect our inability to adapt to transformative changes as we push our agro-
ecosystems towards a threshold. Coping with short-term food price volatility is
daunting enough, particularly for low and middle income countries, but the
long-term challenge of avoiding a perpetual food crisis under conditions of climate
change and global warming is far more serious (Battisti and Naylor 2009) and will
possibly challenge our notion of civilization. We are living in times of great uncer-
tainty with respect to food supply and hence the sustainable management of our
agricultural production systems is an imperative and one that will challenge farm-
ers, scientist and policy makers over the coming decades.
260
240 Food Price Index

Meat Price Index
220 Dairy Price Index
Cereal Price Index
200 Sugar Price Index
180
Price Index
160
140
120
100
80
60
1990 1995 2000 2005 2010
Year
Fig. 1 Food price indices for a range of commodities (FAO Statistics 2009)
1 The Nature of Sustainable Agriculture 3
Recent Challenges in the Global Foodscape
Food and feed crop demand will nearly double in the coming 50 years. The two
main factors driving how much food we will need are population growth and
dietary change (Comprehensive Assessment of Water Management and Agriculture
2007). Over a 24 h period the global population grows by 200,000 with a concomi-
tant impact on overall food demand. Projections in global population growth
indicate that by 2050, a further 2.5 billion people will be added to the current
6.7 billion people that inhabit the planet (UN Population Division 2007). These new
additions to the global community will require adequate housing and food placing
greater demands on already stressed and compromised agro-ecosystems and natural
resources. This growth in populations is geographically specific with the largest
projected increases occurring in China, India and Southeast Asia. On a relative
basis, Africa will experience the most rapid growth, over 70% faster than that in
Asia (annual growth of 2.4% versus 1.4% in Asia) and in sub-Saharan Africa alone,
the population is projected to increase from about 770 million to nearly 1.7 billion
by 2050 (UN Population Division 2007).
The implications of global population growth on poverty are highlighted in a
report released in 2008 by the World Bank (2008). It concluded that the number of
people living in extreme poverty may be higher than previously thought. With a
threshold of extreme poverty set at US$1.25 a day (2005 prices), the number of
people living in extreme poverty in 2005 was estimated 1.4 billion. Reflecting on
the consequences of extreme poverty, it is estimated that approximately ten million
die of hunger and hunger-related diseases annually (World Bank 2008). This dire
state of affairs is compounded by the fact that the number of children that are
underweight exceeds 140 million in developing countries. The legacy of poor
nutrition at critical stages in a child’s development is evident throughout an entire
generation with significant implications on GDP, productivity and health services.
There are large regional disparities in the aforementioned trends. For example,
China has been extremely successful in more than halving the proportion of under-
weight children between 1990 and 2006. Contrasting this and despite improve-
ments since 1990, it is estimated that 50% of children are underweight in South
Asia, a region that alone accounts for more than half the worlds malnourished
children (Nellemann et al. 2009).
Concomitant with the rising demand for food associated with population growth
there is an observable and significant shift in the dietary intake of communities.
With rising incomes and continuing urbanization, food habits change toward more
nutritious and more varied diets – not only toward increasing consumption of staple
cereals but also to a shift in consumption patterns among cereal crops and away
from cereals towards livestock and fish products and high-value crops (Comprehensive
Assessment of Water Management and Agriculture 2007).
The global production of cereals is the cornerstone of world food supply
accounting for approximately 50% of the calorie intake of humans (FAO 2003).
Any changes in the production of or in the use of cereals for non-human consumption
has a direct effect on the calorie intake of a large fraction of the world’s population.
As nearly half of the world’s cereal production is used to produce animal feed, the
dietary proportion of meat has a major influence on global food demand (Keyzer
et al. 2005). With meat consumption projected to increase from 37.4 kg person−1
year−1 in 2000 to over 52 kg person−1 year−1 by 2050 (FAO 2006), cereal require-
ments for more intensive meat production may increase substantially to more than
50% of total cereal production (Keyzer et al. 2005). They conclude that compared
to other factors that are generally expected to affect the future world food situation,
the quantitative impact of the increased cereal feed demand will greatly exceed that
of genetically modified organisms (GMOs) and climate change in the coming 3
decades (Keyzer et al. 2005).
With urbanization, industrialization, energy demand and population growth there
has been a growing trend in converting cropland to other uses. This is best exempli-
fied in China where 14.5 million hectares of arable land has gone out of production
between 1979 and 1995 (ICIMOD 2008). It is estimated that 120 million hectares
will be needed to support the traditional growth in food production by 2030, mainly
in developing countries (FAO 2003). These estimates of cropland and associated
yield increases to meet future demand for food will inevitably contribute to environ-
mental degradation and losses in the provision of ecosystem services.
The production of biofuels has grown exponentially over the past decade fueled
by higher global oil prices and an initial perception of their role in reducing CO2
emissions (FAO 2008). It is estimated that biofuels, including biodiesel from palm
oil and ethanol from sugarcane, corn and soybean, accounted for about 1% of the
total road transport in 2005, and could potentially reach 25% by 2050, with the EU
having set targets as high as 10% by 2020 (World Bank 2008; FAO 2008). For
several countries including Indonesia and Malaysia, biofuels are seen as an oppor-
tunity to improve rural livelihoods and contribute to economic growth through
exports (Fitzherbert et al. 2008).
While biofuels are a potential low-carbon energy source, the conversion of rain-
forests, peatlands, savannas, or grasslands to produce biofuels in the US, Brazil and
Southeast Asia may create a “biofuel carbon debt” by releasing 17 to 420 times
more carbon dioxide than the annual greenhouse gas reductions that these biofuels
would provide by displacing fossil fuels (Fargione et al. 2008; Searchinger et al.
2008). In contrast, biofuels made from waste biomass or from biomass grown on
degraded and abandoned agricultural lands planted with perennials incur little or no
carbon debt and can offer immediate and sustained green house gas advantages
(Fargione et al. 2008).
There are a range of drivers that will influence global food supply in this millen-
nium. However, the greatest challenge for the global community will undoubtedly
be adapting to climate change. The impacts of a changing climate are already being
experienced with increased frequencies in the occurrence of droughts, floods, and
heat waves bringing greater risks to our agricultural production systems. Before
discussing the notion of sustainable agriculture it is pertinent to briefly reflect upon
the dominant drivers that will influence the nature of agricultural systems and the
challenges that the sector faces in the coming decades.
Land Degradation and It Impact on Sustainability
Central to the discussion of the notion of sustainable agriculture is the impact of a

range of drivers on soil degradation and quality. Changes in land use associated
deforestation and inappropriate land management has had a negative impact on
approximately 2 billion hectares of agricultural (Pinstrup-Andersen and Pandya-
Lorch 1998). However, this estimate hides large differences between zones and the
particular vulnerabilities of soils in the tropics (Stocking 2003). It is estimated that
unsustainable land use practices result in net losses of cropland productivity that
averages approximately 0.2%year−1. The combined effects of competition for land
from growing populations, reduced opportunity for migration and rotation along
with higher livestock densities, result in frequent overgrazing and, hence, loss of
long-term productivity (Nellemann et al. 2009). Satellite measurements show that
between 1981 and 2003, there was an absolute decline in the productive land area
(as Net Primary Productivity) across 12% of the global land area. The areas affected
are home to about 1–1.5 billion people, some 15–20% of the global population (Bai
et al. 2008).
Land degradation effectively results in a decline in the productive capacity of
soils that can be attributed to changes in physical, chemical, and biological attri-
butes from some ideal state brought about by natural or anthropogenic influences
(Latham 1994; Lal 1990). These all contribute to what in general terms are referred
to as soil quality. Land degradation is often assessed as the amount of soil material
that is removed from a landscape by water and wind erosion, since such physical
changes are obvious and quantifiable. Effects of degradation on chemical proper-
ties, soil nutrient cycling, and biological activity of soils may be less visible, but are
functionally as important as the former, particularly in developing countries
(Drechsel et al. 2004; Eswaran et al. 2000).
At its most fundamental level, soil degradation is caused when natural factors
such as wind, rain, evaporation and gravity combine with human management
practices to remove, or add, soil or its nutrients at a given site leading to reduced
productivity of land, water and/or other ecosystem services. Whilst soil degradation
could be considered a ‘natural’ process associated with pedological development
within a geological timeframe, it is human interventions which often results in
accelerated rates of these processes that are of social concern at the human time
scale. Of the range of human actions associated with soil degradation, deforestation
and the removal of natural vegetation, overgrazing, and poor agricultural practices
have been cited as the most common causes (Olderman et al. 1991). As Lal
(Lal et al. 2004) shows, ‘ploughing’ is a particularly destructive human intervention
in tropical agricultural systems. Inappropriate water and waste management and
use of heavy equipment can also lead to degradation in the form of salinization,
pollution and compaction. The human role in soil degradation is now relatively well
understood. Globally, degradation of land resources, and as a consequence water
resources, threaten food security, particularly in the hundreds of millions of house-
holds on marginal lands, and they reduce the level of environmental services that
societies derive from the natural resources, including the provision of clean water
(Penning de Vries et al. 2002).
The resilience of an ecosystem to buffer change, including resistance to a shock,
is a fundamental property of ecosystems and provides a measure of vulnerability to
degradation (Stocking and Murnaghan 2001). The concept suggests that a system,
such as an agro-ecosystem, responds more or less gradually to changes in its man-
agement or environment (climate) but only up to a certain threshold. Once this
threshold is surpassed, adjustments cannot take place rapidly enough and the sys-
tem collapses. Realization of the existence of a ‘resilience threshold’ for natural
resources has important consequences for their management (Sayer and Campbell
2003). Resilient soils (e.g. Vertisols) may be degraded to a significant degree and
still be restored to some equilibrium state (e.g. in traditional slash and burn sys-
tems). Other soil types (e.g. Ultisols) are much less resilient. Once a threshold is
reached, these systems collapse and cannot easily be brought back to their former
state. This has been referred to as retrogressive succession as species diversity and
functionality changes due to pedogenesis and landscape age (Walker et al. 2000;
Jenny 1941). Soils that degrade beyond some threshold level are often damaged
beyond repair at time scales of human relevance and may never recover.
Fluxes between soil organic carbon and the atmosphere are of importance when
considering overall changes in global carbon budgets. Changes in land use and
associated conversion to agriculture have over the last 50 years contributed an esti-
mated 50,000,000 Mg C to the atmosphere which represents approximately one
third of the total loss from soil and vegetation (IPCC 2000). The rate of soil organic
matter (SOM) mineralization depends predominantly on temperature and oxygen
availability, land use, cropping system, soil and crop management (Lal 1990).
Clearly inappropriate management of soil resources that result in a loss in SOM
represent resource degradation and have contributed significantly to overall carbon
emissions. Through the adoption of regenerative sustainable production systems that
focus on the conservation of organic matter and its enhancement in soils, it is esti-
mated that globally 1,302 Mt C year−1 could be achieved by 2010 (IPCC 2000).
Whilst it is customary to focus on the solid soil component when discussing
land and soil degradation such approaches ignore an important dimension in the
land degradation equation, since the loss of functionality of the agro-ecosystem refers
as much to ‘water’ as to ‘soil’. Degradation of soil has direct impacts on soil water
holding capacity, erosion, increased sediment loads and siltation of water storage
facilities and a decline in water quality, and often has significant landscape impacts.
These impacts on water productivity have long been significant, especially in arid
regions, but are increasingly important globally as levels of water stress increase.
To place soil degradation in perspective, selective examples are explored that
focus on soil chemical and physical degradation and its remediation. Whilst bio-
logical components associated with the functionality of soils is equally important
in soil quality, this aspect will be covered in several chapters that follow in this
Book. What is important in discussing the sustainability of our agricultural soils is
that all three soil attributes, namely chemical, physical and biological, need to be
considered holistically and not as distinct components.
Chemical Degradation
In their natural state, fertile soils provide nutrients and water to sustain plant bio-
mass production. Both water and nutrients are held by the inorganic soil substrate
(ISS) and soil organic matter (SOM). The water and nutrient holding capacities of
both of these components can be reduced both naturally and through anthropo-
genic activities. With low holding capacities, soil water drains easily down below
the rooting zone and nutrients are leached. At a pedological level, a reduction in
the water and nutrient holding capacities of a profile constitutes soil degradation.
The nutrient holding capacity of the ISS and SOM is related to the electrical
charge characteristics of these two substrates. As a consequence of chemical degrada-
tion, the surface charge properties of soils are reduced, thereby affecting their ability
to retain base cations. This has significant implications for the retention and supply
of essential plant nutrients, and their subsequent leaching. For quantitative assessment
of this decline in base cation retention, a method termed ‘surface charge fingerprinting’
(SCF) has been developed. A SCF is the graphical response curves of the soil’s cation
and anion exchange capacity (CEC, AEC) to changes in pH. The full concept and
methodology for determining these fundamental properties are comprehensively
described elsewhere (Gillman and Sumpter 1986a, b; Menzies and Gillman 1997).
The CEC can be separated into two components, namely, the basic cation exchange
capacity (CECB) which is the total amount of basic cations (normally measured using
Ca2+ as the exchanging ion) that can be retained in an exchangeable form at any par-
ticular solution pH and ionic strength; and total cation exchange capacity (CECT)
which is the total amount of basic and acidic cations (i.e. Al3+) that can be retained in
an exchangeable form at any particular solution pH and ionic strength (Gillman and
Sumpter 1986a, b). By comparing the CEC of degraded and non-degraded soils, a
quantifiable measure of the degree of chemical degradation a soil has undergone can
be established. Using the concept of SCF, the degree of chemical degradation that
occurs both naturally as well as through human intervention can be demonstrated.
In general, in their natural state soils maintain productive and diverse ecosys-
tems that are dependent on efficient resource utilization. A characteristic of these
systems is their reliance on SOM to cycle nutrients from the soil through the plant
and back to the soil through plant debris. Soil organic matter effectively acts as a
slow release nutrient delivery system that mediates the cycling of nutrients between
soil and vegetation. However, vegetation structure and functionality of natural eco-
systems are a function of pedogenesis and landscape age (Walker et al. 2000). In
northern Queensland Australia, soils developed from recent quaternary basalt flows
that are comparatively young having undergone limited pedogenesis are nutrient-
rich, have high levels of organic carbon in their pristine state, high CECT (Table 1)
and support complex notophyll vine forests (Walker et al. 2000). Contrasting this,
soils that are formed from highly weathered metamorphic schist are inherently
infertile when compared to younger soils, have lower levels of organic carbon,
limited CECT and support simple notopyll vine forests (Table 1). These soils are
naturally low in basic cation content associated with advanced weathering, with the
Table 1 Differences in selected soil chemical properties of contrasting Notophyll Vine Forests on
soils derived from metamorphic schist and basalt in Northern Queensland, Australia (Gillman and
Abel 1986; Noble et al. 2001)
Basalt derived soils Metamorphic schist derived soils
Complex Simple
notophyll notophyll
Soil attribute forest Tea plantation forest Sugarcane
Soil pH 5.2 4.9 4.3 4.4
Organic carbon (g kg−1) 66.0 35.0 19.0 10.0
Ca2+ (cmolc kg−1) 1.30 0.24 0.07 0.07
Mg2+ (cmolc kg−1) 1.02 0.17 0.26 0.05
K+ (cmolc kg−1) 0.23 0.26 0.09 0.07
Na+ (cmolc kg−1) 0.09 0.04 0.06 0.02
Al3+ (cmolc kg−1) 0.25 0.15 1.40 1.00
CECT at pH 5.5 (cmolc kg−1) 4.94 1.85 2.68 1.52
CEC largely being satisfied by exchangeable aluminium (Table 1). The fragile
nature of these relatively infertile schist derived soils can be masked by climax
rainforest vegetation in their undisturbed state, where a delicate balance is main-
tained between nutrients in the biomass, organic matter in surface soil horizons and
the efficient cycling of nutrients between these components and the soil. If a pedo-
logically young system is disturbed, it will tend to return to something similar to its
previous state relatively quickly, a process/concept termed progressive succession
(Walker et al. 2000). However, in an old system (highly weathered and in an
advanced state of soil formation) such returns to a pre-disturbance state or a variant
thereof, is highly unlikely, this being termed retrogressive succession.
The importance of soil organic carbon under these two contrasting vegetation
types is clearly demonstrated when these systems of converted to agriculture
(Table 1). Declines in soil organic carbon, exchangeable cations and CECT are
clearly evident. The decline in CECT for every percent decline in organic matter is
1.28 and 0.99 cmolc kg−1 for the highly weathered metamorphic schist and basalt
derived soils respectively, reflecting differences in the quality of the organic matter
produced under these contrasting forest systems. Hence conversion of soils that
have undergone a greater degree of pedogenesis requires high levels of inputs in
order to maintain productivity, an issue that is discussed below. When climax eco-
systems are disturbed through clearing and continuous cultivation, the productivity
of strongly weathered soils often declines rapidly due to a loss in SOM, accelerated
soil acidification, crusting and erosion (Gillman and Abel 1986; Aweto et al. 1992;
Kang and Juo 1986; Kooistra et al. 1990; Willett 1995; Gillman et al. 1985;
Plamondon et al. 1991; Van der Watt and Valentin 1992). Consequences of such a
catastrophic decline in fertility can best be illustrated by comparing the abundance of
vegetation in undisturbed systems (pristine systems) with the meager and unpalatable
vegetation of those that have undergone some form of changed land use. The
influence of changed land management induced by man on soil chemical degrada-
tion can be found in two representative sites from Northeast Thailand both of which
Table 2 Selected soil chemical properties of the surface 0–10 cm depth interval from paired sites
in Northeast Thailand indicating differences in attributes associated with conversion to agricul-
tural production (Noble et al. 2003)
Attribute Undisturbed Disturbed Undisturbed Disturbed
Location RS4 CS1
Dipterocarp Dipterocarp
Cropping/plant system forest Rice forest Cassava
Parent material Alluvium Alluvium
Soil pH 4.87 5.18 5.18 5.00
Organic carbon (g kg−1) 8.5 2.1 6.7 3.3
Ca2+ (cmolc kg−1) 0.34 0.14 0.74 0.25
Mg2+ (cmolc kg−1) 0.20 0.03 0.40 0.11
K+ (cmolc kg−1) 0.07 0.04 0.09 0.03
Na+ (cmolc kg−1) 0.03 0.01 0.00 0.00
Exch. acidity (cmolc kg−1) 0.57 0.19 0.12 0.35
CECB pH 5.5 (cmolc kg−1) 1.33 0.48 1.57 0.83
Sum bases 0.65 0.22 1.24 0.39
Clay content (%) 4.9 2.9 4.9 4.7
pH buffer capacity (cmol 1.07 0.41 0.92 0.62
H+ kg−1.unit pH−1)
are on light textured sandy soils (Table 2) (Noble et al. 2003). Over the past 3
decades significant land clearing has occurred throughout Northeast Thailand asso-
ciated with the expansion of upland cropping systems. The original climax
Dipterocarp forests have been replaced by intensive cropping systems that are
dominated by rice, cassava and sugarcane. Remnant Dipterocarp forests can be
found in isolated pockets that allow direct comparisons to be made between an
undisturbed state and the current agronomic enterprise that is in close proximity.
Changes in soil chemical properties for two of these sites under climax Dipterocarp
forest and adjacent farmer fields under production for the past 40 years are pre-
sented in Table 2 for the 0–10 cm depth interval. Assuming a soil bulk density of
1,300 kg m−3, the loss in soil organic carbon (OC) is equivalent to 8.3 and 4.4 t C ha−1
in the top 0–10 cm depth interval for sites RS4 and CS1 respectively. Accompanied
with this decline in OC are a loss of exchangeable cations and an increase in
exchangeable acidity in the case of site CS1. By comparing the difference in CEC
between the undisturbed site and the adjacent agronomic production system at
pH 5.5, the degree of permanent change in the charge characteristics associated
with changed land management becomes clear. There has been a 64% and 47%
decline in the CECB with conversion to agriculture in the previous examples. The
loss of plant nutrients in the same layer has been even more dramatic: as much as
70% of the nutrients are removed, due to enhanced leaching and to extraction by
crops without full replenishment by inorganic fertilization or other approaches.
Soil degradation associated with changed land use is not confined to light
textured soils. Highly weathered clay based soils of the humid tropics converted to
intensive agricultural systems exhibit significant degradation of both physical and
chemical attributes. The properties of an Oxisol cleared of climax rain forest 53

years previously and currently under tea production in north Queensland were
compared with an adjacent undisturbed forest (Noble et al. 2001). Soil pH declined
by approximately 0.6 units under the tea production system. The loss of soil organic
carbon from the tea site amounted to an equivalent of 58.1 t ha−1 over the 0–20 cm
depth. This dramatic decline in soil organic carbon has had a significant impact on
the soils ability to retain cations and hence the inherent fertility of the disturbed site
as evident in the charge fingerprint for the 0–10 cm depth interval (Fig. 2).
However, with depth, differences between the disturbed and undisturbed systems
declined so that at 50 cm there were no differences between systems (Fig. 2).
Contrasting the previous examples, which represent strongly weathered soils
(Ultisols and Oxisols) of the tropics whose mineralogy is dominated by either kao-
linite or sesquoxides, an example of a soil whose ISS is dominated by smectite-
vermiculite (Vertisol) is presented from the sub-tropics. The soils are from a
long-term trash management trial established in 1939 at Mt Edgecombe, South
Africa and are part of the oldest sugarcane soil management trials in the world. For
brevity, selected results of SCF from soil samples collected in 1996 are presented.
Figure 3 represents the charge fingerprints of composite samples from the control
treatment (under grass for the duration of the trial) and an extreme treatment that
had been burnt, harvest residues removed, and an annual fertilizer dressing of 140
kg N ha−1, 20 kg P ha−1 and 140 kg K ha−1 applied. Over the duration of the study
there has been a significant decline in soil pH and associated base stripping in the
0–5 cm depth interval (Van Antwerpen and Meyer 1998). It is of note that the inher-
ent charge characteristics of these soils are an order of magnitude larger than the
previously discussed examples but clearly show a decline associated with changed
management which is predominantly driven by a decline in soil organic matter
(Graham et al. 2002). Intuitively these soils have a greater capacity to resist charge
diminution due to contrasting management systems as they are dominated by per-
manently charged smectitic clays. This is clearly demonstrated when one compares
the decline in percentage charge between the grass ley managed system compared
to the burnt cane managed system at a comparable pH of 5.5. The burnt system has
undergone a mere 14% reduction in charge over a 57 year period whilst under con-
tinuous cane production. This demonstrates the resilience of these soils to buffer
changed management and contrasts dramatically with the previous two examples.
The examples presented above demonstrate the contrasting impact of changed
land management on soil chemical properties. The degree of impact is in part con-
tingent upon the clay mineralogy of the soils. If soils are dominated by low activity
kaolinitic and sesquioxidic clays as in the first two examples, the amount of charge
associated with this inorganic phase is small. Consequently these soils have a small
‘permanent’ charge component. In permanent surface-charge minerals, the charge
is due to imperfections in the lattice structure resulting from isomorphous substitution.
As is evidenced by the change in surface charge with an increase in pH, there is a
second form of charge generation namely ‘variable’ charge. In variable charge soil
systems, the surface charge is created by the adsorption of the potential-determining
ions (p.d.i.) or other specifically adsorbed ions onto the surface. The charging process
a
9
8
7
6
5
4
3
2
1
0
b 9
8
Exchange capacity (cmolc /kg)
7
6
5
4
3
2
1
0
c 9
8
7
6
5
4
3
2
1
0
3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0
pH
Fig. 2 Cation exchange characteristics for the 0–10 cm (a), 20–30 cm (b) and 50–70 cm
(c) of soils collected from a tea () and adjacent climax tropical rain forest () collected from
north Queensland. CECT is represented by the dotted lines and CECB by the solid lines (Noble
et al. 2001)
requires the presence of these ions in the soil solution in sufficient quantities
for adsorption to occur. Variable charge or amphoteric constituents in soils may
have organic (i.e. organic matter) and inorganic origin (i.e. sesquioxidic surfaces).
21
20
CECT (cmolc kg−1) 19
18
17
16
15
14
13
4.0 4.5 5.0 5.5 6.0 6.5
pH
Fig. 3 Surface charge fingerprints of soil collected from the control () and trash burnt/fertilized ()
treatments of a long-term trial established at Mt Edgecombe, South Africa on a Vertisol in 1939
With the loss of soil organic matter through mineralization and accelerated acidifica-
tion due to crop removal and the application of acidifying nitrogenous fertilizers, the
variable charge component declines dramatically upon conversion to agriculture. The
first three cases discussed above would typify soils that are dependent on this form
of charge. Contrasting this, soils that are dominated by permanently charged clay
minerals, as demonstrated in the previous example, show significantly higher inher-
ent charge capacity and relatively small declines in this attribute over a significant
period of crop production. The major difference in this case is that these soils are
dominated by high-activity 2:1 layer smectitic clays that have ‘permanent’ charge.
This charge is not affected by changes in pH and is little influenced by soil organic
matter. Clearly, soils that are dominated by these clay minerals are more resilient to
changed management without a significant decline in productive capacity.
Amelioration of Charge Degradation
Declining productivity on degraded soils can in part be ascribed to a reduction in

the water holding capacity of the profile, a decline in the physical characteristics
(i.e. reduced infiltration, greater runoff, or increased drainage) and a reduction in
the fertility status of the soil largely driven by a decline in the cation exchange
capacity. In degraded soils, in order to sustain agronomic production, larger inputs
of water and inorganic fertilizers are required to sustain yield levels, leading to
greater losses associated with leaching and potentially to lower nutrient efficien-
cies. Organic amendments including manures and composted sewage-sludge have
been used to add nutrients in a slow release form as well as increase the available
water holding capacity of soils (Tester 1990; Bauer and Black 1992). However, in
the case of organic amendments the positive effects on soil chemical and physical
attributes are often transient and require ongoing additions to sustain these effects.
Alternative strategies such as the application of crushed basalt rock, zeolites and
other geological materials have been tested with varying degrees of success
(Gillman 1980; Chesworth et al. 1987). Using these techniques the CEC is
increased with a concomitant increase in basic cations. Studies have shown that
remediation of chemical degradation and its associated declining productivity can
be achieved through the addition of bentonite that has been shown to be highly
economic (Noble et al. 2001; Saleth et al. 2009).
Changing the production system to one that incorporates soil organic carbon
conservation can lead to significant improvements in the fertility and nutrient holding
capacity of soils. Changing land use so as to conserve SOM, fundamental surface
charge characteristicscan be manipulated to one’s advantage. Following a change in
harvesting practice in the sugar industry in north Queensland, Australia, from burnt
cane harvesting to green cane trash blanketing, there have been recorded instances of
SOM enhancement (Wood 1991; Prove et al. 1986; Spain and Hodgen 1994). For
instance, on an Inceptisol under a high rainfall regime in tropical Queensland, changed
management practice through green cane trash blanketing caused organic carbon to
increase from 1.33% to 2.26% in the top 0–5 cm interval over a relatively short period
of 7 years (Noble et al. 2003). This in turn favorably altered surface charge character-
istics as evidenced in the charge fingerprints (Fig. 4). The degree of increase in charge
characteristics of the surface soil under the green cane trash blanketing system was
estimated to be 150%. It is important to note that improvements in the overall CEC
were confined to the surface layer and that below 5 cm there was no evidence of
improvements in charge. This generation of increased surface charge can only be
viewed as beneficial. However, it should be realized that the increase in charge char-
acteristics associated with elevated soil organic matter is transient under these tropical
conditions. When the system is perturbed through cultivation a significant proportion
of this increased charge will be lost through organic matter mineralization.
Alternative strategies such as a grass ley in rotation with crops would also
increase the soil organic carbon content and have a direct benefit on the charge
characteristics of the soil (Noble et al. 1998, 2001). However, to resource poor
farmers in developing countries whose primary objective is house-hold food secu-
rity, the implementation of a long term grass ley into their farming systems is
remote. There is mounting empirical evidence to suggest that ‘conservation agricul-
ture’ systems are an effective alternative to conventional farming (tillage based)
systems that effectively exploit the natural resources upon which they are based
without degrading them, and in some cases allowing their restoration (Bot and
Benites 2001). The two essential features of ‘conservation agriculture’ are no-tillage
and the maintenance of a cover on the soil surface. Significant increases in produc-
tivity have been achieved through the adoption of ‘conservation agriculture’
(Bot and Benites 2001). However, these responses are contingent on the inherent
fertility status of the soil. Furthermore on highly degraded production systems,
the positive productivity benefits associated with the adoption of organic matter
Fig. 4 Surface charge fingerprints a

of the 0–2 cm (a), 2–5 cm (b) and 7
5–10 cm (c) Depth intervals under
a long-term Green Cane Trash GCTB CECB
6 GCTB CECT
Blanketing and Burnt Cane System
Burnt CECB
(Noble et al. 2003) 5 Burnt CECT
b
7
Exchange capacity (cmolc /kg)
c
7
1
4.0 4.5 5.0 5.5 6.0 6.5 7.0
pH0.002
conservation systems may have a lag phase associated with a buildup of SOM to
some critical level. This may influence adoption of such practices under particular
socio-economic circumstances.
As alluded to in the previous discussion, one constraint in the adoption of sus-
tainable agronomic practices is the lag time between implementation of a practice(s)
and associated increases in productivity. In some cases there is often a slight reduction
in productivity before significant increases are achieved. In an effort to mitigate this

lag effect, the concept of directly addressing the key chemical constraints of
degraded soils (namely low nutrient and water holding capacity), has been effected
through the incorporation of permanently charged high activity clays to degraded
soils (Noble et al. 2001). Through the addition of cation beneficiated bentonite to
degraded soils, Noble (Noble et al. 2001) have shown significant responses in yield
and permanent changes in the charge characteristics of soils. Surface charge
characteristics from a subsequent study where varying rates of beneficiated benton-
ite (0, 20, 40 and 60 t ha−1 containing a ratio 4:2:1 of Ca/Mg/K) were applied to two
contrasting soil types are presented in Fig. 5. The cation exchange capacity at soil
a 8
Col 45 vs Col 46
7 Col 47 vs Col 48
Col 49 vs Col 50
Charge capacity (cmolc /kg)
6 Col 51 vs Col 52
Col 53 vs Col 54
Col 55 vs Col 56
5
Col 57 vs Col 58
Col 59 vs Col 60
4
0
4.0 4.5 5.0 5.5 6.0 6.5
b 6
5
CECB (cmolc kg−1)
0
4.0 4.5 5.0 5.5 6.0 6.5 7.0
pH
Fig. 5 Surface charge characteristics of (a) an Oxisol and (b) Ultisol after the addition of varying
rates of Bentonite (Noble et al. 2001)
Cumulative yield (g/pot) 4

Ultisol
Oxisol
3
0
0 20 40 60
Treatments
Fig. 6 Total above ground yield response of forage sorghum to varying additions of beneficiated
bentonite (t/ha) on two contrasting soil types (Noble et al. 2001). Vertical bar represents the LSD
at the 5% level
pH was increased from 0.97 to 4.08 cmolc kg−1 on a light textured Ultisol and from
0.58 to 2.95 cmolc kg−1 on an Oxisol through the addition of an equivalent of 60 t
ha−1 of beneficiated bentonites. Since the surface charge on bentonite clay is per-
manently negative, i.e. not affected by changes in soil pH, the additional CEC now
present in these soils will be a permanent feature. Concomitant with the improved
charge characteristics was a significant and sustained increase in forage sorghum
biomass production with increasing additions of these materials on both soil types
(Fig. 6). The cumulative increase in yield between the control and 60 t ha−1 benton-
ite was 5.6 and 9.0 fold for the Ultisol and Oxisol soil types respectively.
In studies undertaken in Northeast Thailand to assess the efficacy of a range of
soil amendment techniques in rejuvenating a degraded light textured sandy soils,
highly significant increases in biomass production were observed in those treat-
ments receiving the traditional termite mound material, bentonite and bentonite +
compost when compared to the control, dredged material and composted leaf
litter (Fig. 7) (Noble et al. 2004). In the case of the 2003 growing season it was
not possible to establish a forage sorghum crop even after repeated plantings on
the control, dredged and compost treatments this being ascribed to the variable
precipitation that was experienced. The responses with respect to the termite
mound materials and bentonite treatments can in part be attributed to the enhanced
nutrient supplying capacity and increased CEC associated with these materials
(Table 3). Total above ground dry matter (DM) production over the 2 years ranged
from 0.22 t DM ha−1 in the control treatments where plant production completely
failed in the second year of cropping to 10.8 and 22.4 t DM ha−1 for the locally
sourced termite mound material (120 t ha−1) and local bentonite (50 t ha−1) + leaf
16000
14000 Yield 2002
Dry matter production (kg ha−1)

Yield 2003
12000
10000
8000
6000
4000
2000
0
p. . . .
tro
l
m ed rm nt. mp
n Co Dr Te Be o
Co +C
nt.
Be
Treatments
Fig. 7 Yield response of forage sorghum to selected treatments applied to a degraded light
textured soil in Northeast Thailand over two consecutive years. Treatments include Control =
Control (current farmer practice); Dred. = Dredged Material at 240 t ha−1; Comp. = Leaf Litter
Compost at 10 t ha−1; Term. = Termite Mound Soil at 120 t ha−1; Bent. = Local Bentonite at 50 t
ha−1; and Bent. + Comp. = Local Bentonite at 50 t ha−1 with Leaf Litter Compost of 10 t ha−1.
Vertical bars are the standard errors of treatment means (Noble et al. 2004)
Table 3 Selected chemical characteristics of Bentonite clay and Termite mound material used in
a field study in Northeast Thailand. Values in brackets are the standard error of the means of four
replicates
Exchangeable cations (cmolc kg−1) Sum bases CEC
Source Ca2+ Mg 2+
K +
Na +
(cmolc kg ) (cmolc kg−1)
−1
Termite 17.04 (±0.48) 5.92 (±0.11) 0.41 (±0.03) 1.23 (±0.17) 24.62 (±0.30) 13.68 (±0.66)
mound
Bentonite 25.59 (±0.36) 12.78 (±0.09) 0.55 (±0.09) 6.44 (±0.10) 45.37 (±0.44) 27.68 (±0.53)
litter compost (10 t ha−1), respectively. The responses to the treatments persisted
into the second season and continued to increase under the bentonite treatments.
It is of note that the CEC of the bentonite was approximately double that of the
termite mound material, and similarly, the sum of exchangeable bases in each
case was approximately double that of the CEC indicating a significant propor-
tion of the bases were not being held on the exchange complex (Table 3). A further
significant benefit associated with the application of bentonite to these light tex-
tured soils is the potential to increase the water holding capacity of these soils
(Suzuki et al. 2007).
Addressing Soil Physical Degradation
In addition to the unfavorable chemical properties of soils, many soils are prone to
sub-surface compaction which is often observed as a high resistance to penetration
that inhibits the development of crop root systems below 20–40 cm (Bruand et al.
2004; Hartmann et al. 2001). This has a significant impact on the crops ability to
take up nutrients and stored subsurface moisture leaving the crop vulnerable to
drought stress. Deep ploughing and sub-soiling have been shown to be ineffective
in overcoming this problem on coarse textured soils due to their low structural
stability (Ball-Coelho et al. 2000) and hence re-establishment of compacted layers
occurs within the following growing season. The vast majority of soil compaction
in agricultural production systems is attributed to traffic load associated with heavy
machinery.
Comparisons of penetrometer resistance between remnant Dipterocarp forest in an
upland landscape position in Northeast Thailand and adjacent crop land show the
effect of changed land use on soil physical properties (Fig. 8). A layer of extreme
resistance is present over the 20–40 cm depth interval that would significantly reduce
a crops ability to penetrate this layer in order to accesses stored water. The depth of
this compacted region in the profile corresponds with the working depth of most till-
age implements and therefore is attributed to both the increased loads associated with
wheel traffic and smearing effect of tillage implements (i.e. disc plough). The conse-
quences of these areas of high soil strength are reduced root proliferation and enhanced
risk of the crop being predisposed to drought stress that is common to the region.
Penetrometer resistance (MPa)

0.0 0.5 1.0 1.5 2.0 2.5 3.0
0
10
20
30
40
Depth (cm)
50
60
70
Agriculture
80 Forest
90
100
Fig. 8 Effect of changed land use on penetrometer resistance for an upland farming systems in
Northeast Thailand. Horizontal bars represented the standard deviation of the mean
The impact of soil structure on root growth and proliferation has received
considerable attention over the past decade (Passioura 1991). It has been shown
that root proliferation in the soil is closely dependent on the presence of macropo-
res (Hatano and Sakuma 1990; Hatano et al. 1988; Stewart et al. 1999).
Mechanical modification of the soil profile, through deep-ploughing or subsoiling
will significantly increase the porosity of soils. However, such profile modifica-
tions require high energy inputs that is often beyond the means of resource-poor
farmers and invariably, the benefits diminish rapidly after the first heavy rains due
to the inherently unstable nature of these soils (Hartmann et al. 2002). Actively
growing plant root systems have the potential to ameliorate subsoils in poor
physical condition by biological drilling (Cresswell and Kirkegaard 1995).
Decaying roots leave a continuous network of vertically-oriented macropores that
subsequent plants can use. In studies conducted with the grass species Andropogen
gayanus cv Kent (Gamba) and the legume Stylosanthes hamata (Stylo) on a light
textured sandy soil with a distinct compacted layer between 20 and 40 cm have
clearly demonstrated the contrasting potential of these species in penetrating
these compacted layers (Fig. 9). Despite the existence of the dense compacted
layer, development and proliferation of Gamba roots to depth were significantly
higher when compared to Stylo with a rooting depth reaching down to 1.0 m.
Taking advantage of the ability of Gamba root to penetrate compacted layer,
Gamba is a highly effective biological soil remediation agent on these compacted
Root frequency impact (%)

0 20 40 60 80 100 120 140 160
0.0
0.1
0.2
0.3
0.4
Depth (m)
0.5
0.6
0.7
0.8
0.9 Gamba
Stylo
1.0
Fig. 9 Mean root frequency impacts for Andropogen gayanus cv Kent (Gamba) and Stylosanthes
hamata (Stylo) treatments. Horizontal bars are the least significant difference between treatment
means (n = 3)
soils and would fit into a grass ley system. Moreover, the establishment of stable
macropores through these compacted layers could potentially provide subsequent
crops with biological channels through which roots could grow and access stored
sub-surface moisture thereby enhancing water productivity under these rainfed
production system.
In a similar series of studies with the same physical impediments using the
aforementioned species, soil organic carbon (OC) levels were increased to depth
with the establishment of pasture species within these profiles (Noble et al. 2008).
Of importance in assessing the amount of carbon stored in soil profiles is the net
carbon contents that fall below the plough layer (0–30 cm) that is assumed to be
fixed with a much greater residence time. Figure 10 represents the net storage of
carbon over the 0–30 cm and 30–110 cm depth intervals in studies undertaken in
Northeast Thailand. The mean increase in soil organic carbon in the surface 0–30
cm was 776 kg ha−1 from that of the 2001 with all treatments having significantly
higher levels of OC than the initial values. What is important is the over sixfold
increase in soil organic carbon stored at >30 cm from that of the 2001 (Fig. 10).
Fisher (Fisher et al. 1994) observed substantial increases in organic carbon under
Gamba pastures in South American savannas.
The role of permanent leys, whether legume or grass, in cropping systems
clearly demonstrates the ability of these species to penetrate compacted layers
2500
0-30 cm
30-110 cm
2000
Organic carbon (kg ha−1)
1500
1000
500
0
Control 2001 Gamba Gamba NH4 Gamba NO3 Stylo
Treatments
Fig. 10 Total organic carbon stored in the 0–30 cm and 30–110 cm depth intervals 3 years after
the implementation of a range of treatments that included a control (bare fallow); Gamba (no
fertilizer); Gamba NH4 (Gamba fertilized 278 kg N as NH4 over 3 years); and Gamba NO3 (Gamba
fertilized 278 kg N as NO3 over 3 years). Vertical bars represent the LSD0.05 between treatment
means over the same depth interval (Noble et al. 2008)
resulting in the development of permanent stable macropores and the storage of

organic carbon to depth. This has positive benefits under rainfed production sys-
tems since stored subsoil moisture can be accessed reducing the risk of drought
stress as well as increasing the productivity of these systems.
Transforming the Agricultural Landscape
In 1974 Henry Kissinger, the then USA secretary of state, announced at a global
food conference that no child would go hungry within 10 years. Just over 35 years
later a United Nations food summit in Rome stated one billion people will go to bed
hungry (The Economist 2009). By 2050 the world’s population will have increased
by a third and demand for agricultural produce will rise by 70% with meat con-
sumption doubling. Lateral expansion of the agricultural sector through the clearing
of land is untenable without significant negative implications on already stressed
natural ecosystems and the range of drivers, including climate variability, that farmers
will have to cope with, will require changes in the way we undertake agriculture.
In meeting future food production demands without consuming more land and
water will require technological innovation. This may include changes in the way
we use water resources through more efficient irrigation; the adoption of no-till
farming systems; better use of fertilizers and pesticides; and raising yield potential
through genetically modified (GM) crops that are adapted to a range of environ-
mental stresses.
From a soils perspective in the introduction of conservation agriculture (CA)
that is founded upon three guiding principles: (1) minimum or no mechanical soil
disturbance; (2) permanent organic soil cover (consisting of a growing crop or a
dead mulch of crop residues); and (3) diversified crop rotations has been success-
fully implemented in North and South America with positive impacts on soil con-
servation (Bolliger et al. 2006; Triplett and Warren 2008). However, recent issues
over claims of increased yields under CA and its role in the context of Sub-Saharan
African (SSA) agriculture highlight the necessity for further review of where this
approach to soil resource management is applicable (Giller et al. 2009). Concerns
raised include decreased yields often observed with CA, increased labour require-
ments when herbicides are not used, an important gender shift of the labour burden
to women and a lack of mulch due to poor productivity and due to the priority given
to feeding of livestock with crop residues (Giller et al. 2009). Despite the publicity
claiming widespread adoption of CA, the available evidence suggests virtually no
uptake of CA in most SSA countries, with only small groups of adopters in South
Africa, Ghana and Zambia. We conclude that there is an urgent need for critical
assessment under which ecological and socio-economic conditions CA is best
suited for smallholder farming in SSA. Critical constraints to adoption appear to be
competing uses for crop residues, increased labour demand for weeding, and lack
of access to, and use of external inputs. These are significant challenges that are
faced by the majority of global farmers, namely smallholder poor farmers.
While there is considerable uncertainty about the impacts of climate change,

there is little doubt that its effects will be felt most strongly by the poor. Climate
change impacts on the habitats that marginal producers depend on will further
exacerbate their livelihood insecurity stemming from historical neglect and dis-
crimination. This underscores the gravity of their vulnerability to the cumulative
impacts of climate and other anthropogenic activities that degrade the natural
resources and result in a decline in ecosystem services (ES).
Agriculture does not of itself compromise ecosystem services. On the
contrary, agriculture is essentially a means of concentrating and enhancing
provisioning services – but this can often come at the cost of other functions,
particularly regulating services which are intimately related to land cover. For
example: deforestation reduces the ability to regulate erosion; loss of wetlands
reduces the ability of streams to regulate floods and water quality; and clear-
ance of mangroves reduces the ability to regulate storm impacts.
Landscapes can be modified to protect or enhance a specific ES; for example:
afforestation in watersheds to prevent erosion and improve water quality; man-made
wetlands for sewage treatment; restoring environmental flows in rivers to preserve
aquatic ecosystems; and construction of man-made reefs and replanting of man-
groves to mitigate storm damage in coastal zones. An important aspect of protecting
ES is identifying critical thresholds, beyond which the resilience of the ecosystem
is compromised, and managing agricultural systems within these limits.
The challenge for the future is to create productive agro-ecosystems that
maintain and enhance a range of ecosystem services that are delivered. In
many cases, this can best be achieved by mimicking aspects of natural
systems (Lefroy et al. 1999); for example:
• Paddy fields mimic the water retention of natural wetlands to provide multiple
benefits including rice and fish production, flood mitigation, ground water
recharge, soil erosion control, water purification.
• Plantations can mimic natural forests, with well developed understorey to pre-
vent soil erosion, provide habitat and promote biodiversity.
It is important that actions to preserve and improve the quality of ecosystem ser-
vices enhance, rather than compete with, livelihoods either directly, through
improvements in local environments and production, or indirectly through financial
compensation or payments for ecosystem services.
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Food-Fuel
Chapter 2
The Microbiology of Natural Soils
Teri C. Balser, Devin Wixon, Lindsey K. Moritz, and Laura Lipps
Introduction
Soil microorganisms, such as bacteria and fungi, control ecosystem functioning

through decomposition and nutrient cycling and may serve as indicators of land-use
change and ecosystem health (Doran and Zeiss 2000; Waldrop et al. 2000; Yao et al.
2000). However, the study of soil microorganisms is difficult and our current under-
standing limited. The sheer number, astonishing diversity and small size of these
communities become more apparent as our technologies to explore them have improved
in recent years (Cardon and Gage 2006). With the rapid rise of molecular techniques,
microbial ecologists are now able to walk through the world with the equivalent of the
naturalist’s “field notebook,” cataloguing and classifying species. However, we often
do not know what they do functionally or ecologically, or why they are found in some
soils and not others (Balser et al. 2006). What we do know is that soil microbial com-
munities are dynamic and diverse (Sylvia et al. 2005) almost beyond measure (Schloss
and Handelsman 2006), and that some patterns seem to hold on a global scale.
In this chapter we survey a variety of ecosystems and summarize what we know
about their underground microscopic inhabitants. Our knowledge is limited by the
T.C. Balser (*) and L.K. Moritz

Department of Soil Science, University of Wisconsin-Madison, 1525 Observatory Drive,
Madison, WI 53706, USA
D. Wixon
Department of Botany, University of Wisconsin-Madison, 430 Lincoln Drive, Madison,
WI 53706, USA
L. Lipps
Gaylord Nelson Institute for Environmental Studies, University of Wisconsin-Madison,
28 T.C. Balser et al.
relative lack of studies and the particular challenge of linking microbial function
with identity. However, trends such as astounding belowground diversity in the
tropics, and the importance of fungi in forest soils, are gaining wider attention.
Methods of Study
Any treatment of microbial communities or ecology must include consideration of how

we study them. More than most areas of endeavour, the study of soil microbiology is
methodologically constrained: the size and fantastic diversity of microorganisms have
long been a challenge to researchers. Historically, most techniques have relied on grow-
ing the organisms in a laboratory (culturing), but we now know that less than 1% of
bacteria and an estimated 17% of fungi are culturable (Torsvik et al. 1990; Amann et al.
1995; Bridge and Spooner 2001; Horner-Devine et al. 2004). Currently we have many
new tools for biochemical and molecular analysis of microorganisms, including
phospholipid fatty acid (PLFA) analysis and genetic sequencing. These tools allow the
targeting of the entire microbial community, rather than just those that grow in culture
(Zelles 1999; Nannipieri et al. 2003; Kirk et al. 2004; Leckie 2005). In PLFA (lipid
biomarker) analysis, soil microbial community members are identified based on the
structure of fatty acids in the microbial cell membrane (Vestal and White 1989). Cell
membranes of microorganisms degrade rapidly upon cell death and thus the fatty acids
extracted from a soil sample provide an estimate of viable microbial community
biomass and composition (Balser 2000; White et al. 1979). Amino sugar analysis
instead assesses compounds from cell walls that persist for a very long time after cell
death in the soil (Liang et al. 2008). In contrast to lipid biomarker analysis, molecular
methods use DNA or RNA sequencing to identify genes unique to microorganism
groups, potentially providing a higher level of resolution of the soil microbial community
(Nannipieri et al. 2003). New molecular methods attempt to consider the entire comm-
unity genome rather than that of particular inhabitants (metagenomics, Handelsman 2005).
Regardless of what biochemical or molecular method is used to examine the struc-
ture of a microbial community, they usually have the same shortcoming: they primarily
provide information about what groups of microorganisms are present, rather than
allowing insight into the functionality, or what the microorganisms are doing in the soil
(Zelles 1999; Nannipieri et al. 2003; Kirk et al. 2004; Leckie 2005). Other methods do
allow determination of functionality, i.e., we know what they consume based on how
rapidly they consume food (substrate-induced respiration, or SIR), community-level
physiological profiling (CLPP), or the enzymes microbes produce (extracellular
enzyme assays). However, while these methods can assess the potential and actual
activities of the soil biota, they don’t tell us exactly who is doing what. In other words –
using a given method we can determine who is there, but not what they are doing, or
we can determine what they are doing, but not who it is. Currently, key studies often
combine identification-based methods with activity-based methods. Future research
will depend on finding better ways to do both simultaneously.
2 The Microbiology of Natural Soils 29
Soil as a Habitat
Soil microbial community structure and activity depend to a large extent on the
status of their soil habitat. Within this habitat, soil organisms are eating, respiring,
competing, cooperating, and responding to changes in their immediate environment.
Indeed, the majority of the microbial community may be dormant at any given time
in most soils, ready to respond as conditions for a particular group become favor-
able (Stenström et al. 2001). The soil habitat is perhaps best envisioned as a
complex matrix with pores and soil aggregates of differing sizes (Sylvia et al.
2005). Certain bacteria and fungi tend to congregate in the soil immediately
adjacent to plant roots (the rhizosphere), where they may feed off the sugars that
plant roots exude or actually physically associate with the plant root system and
exchange sugars and nutrients in a (usually) mutualistic relationship (mycorrhizas).
The soil community and its habitat are influenced by an interconnected web of
variables that differ among ecosystems, making each ecosystem somewhat unique
in its microbial community (Wixon and Balser 2009). Across the globe, as with
vegetation, community structure is perhaps most influenced by soil temperature
and moisture (Sylvia et al. 2005), though it changes with the seasons (e.g. Lipson
2007), and is strongly affected by soil acidity or alkalinity (pH).
Within a given ecosystem, depth in soil is a primary consideration for microbial
habitat, and many key habitat characteristics (e.g. oxygen levels, availability of food
and nutrients) change through the soil profile. Carbon availability (and often quality)
declines, as does overall microbial biomass (Fig. 1). Soil structure, such as particle
size fractions and stable aggregates also change with depth and impact the soil bio-
logical habitat (Van Gestel et al. 1996; Ranjard et al. 2000; Sessitsch et al. 2001; Poll
et al. 2003). Few studies have considered, however, the importance of both soil parent
material and resultant soil texture (Ulrich and Becker 2006; Rasmussen et al. 2007).
Soil organic carbon (SOC) is the largest terrestrial component of the global
carbon budget (Jobbágy and Jackson 2000). Worldwide, the top 1 m of soil contains
two to three times more carbon than the amount stored in all aboveground vegetation
(Brady and Weil 2002). Studies of soil carbon and microbial communities often
concentrate on the upper 20–30 cm of soil, as this is considered to be the most bio-
logically active portion of the soil profile (Fierer et al. 2007; Jobbágy and Jackson
2000; Veldkamp et al. 2003; Baisden and Parfitt 2007; Goberna et al. 2006).
However, the majority of carbon in soil occurs below 20 cm, and thus by discounting
lower depths we are missing up to 50–65% of the carbon (Jobbágy and Jackson
2000). As a result, many current ecosystem models of land-use and climate change
inadequately model carbon turnover and microbial communities because they
disregard the carbon stocks and biological activities of deeper soil horizons (Baisden
and Parfitt 2007). Generally, most microbial community studies down the soil
profile have occurred in grasslands and agricultural lands (Fierer et al. 2007;
Lavahun et al. 1996; Blume et al. 2002; Taylor et al. 2002; Allison et al. 2007),
with fewer studies in boreal and temperate forests (Goberna et al. 2005; 2006;
Fig. 1 Microbial and soil properties vary with depth. Depth profile graphs from Sedimentary and
Ultrabasic sites in Borneo (See Moritz, 2008). (a) Total soil C and carbon:nitrogen (C/N) ratios
with depth. Soil C/N differed significantly (p < 0.05) between sites under the conditions of two-
way ANOVA. (b) The number of identified lipid peaks (detectable PLFAs) at each soil sampling
depth, representing PLFA richness. Error bars are ± 1 SEM and N = 3. Error bars are ± 1 standard
error of the mean (SEM) and N = 3
Ekelund et al. 2001), and even fewer in tropical forests (Veldkamp et al. 2003). In
general, studies demonstrate that fungal-to-bacterial ratios decline with depth, as
does overall microbial biomass. Deeper soils become more dominated by bacteria,
particularly the slower growing Gram-positive types. In exceptionally deep soils
bacterial diversity declines, though organisms are still capable of living at great
depths.
Survey of Ecosystems
Soil formation is generally considered to be driven by five factors: parent material,

climate, biota, topography and time (Brady and Weil 2002). As we saw briefly above,
parent material provides the framework for soil ‘architecture’, which provides habit-
able space for microorganisms. Parent material and climate influence weathering rates,
which influence soil chemical properties. Over time soils evolve and change and
develop in accordance with their environmental and biological factors. The resulting
global mix of soil-ecosystems differs in many ways that impact microbial communi-
ties. Tropical, temperate, agricultural and wetland ecosystems each have distinct soils,
and pose strikingly different challenges for soil microbial life. For example, tropical
systems often have low levels of nutrients in the soil, and generally constant warm,
moist temperatures, while high-latitude systems store massive amounts of carbon and
can experience dramatic temperature shifts (Brady and Weil 2002). Finally, human
land use can also greatly alter ecosystems and thus soil life and its functioning.
Although some generalization by ecosystem is possible, the diversity of factors
impacting microbial community habitats implies some caution is needed. Even in
adjacent areas, the microbial community of soils of differing types can be strikingly
different. For example, two studies in the Pacific Northwest of the United States
have compared microbial communities between adjacent soils of differing types
(serpentine and non-serpentine) and found that microbial communities in the
former were more similar to each other than non-serpentine communities (Oline
2006). The serpentine soils had reduced microbial biomass (DeGrood et al. 2005),
suggesting that these offer an inferior environment for microbial growth.
Among the least well understood areas in terms of soils and their microbial
community structure and function are the tropical and wetland ecosystems. The
vast majority of studies are focused on temperate grasslands and forests, with some
emphasis on taiga or boreal forest communities. However, tropical forest and
wetland soils are some of the most endangered in the world. They are critical in
their potential response to current and future climate change, and as potential sites
for agriculture in a world with ever increasing human population size. Below we
review some of what is known about the microbiology of these soils.
Tropical Forests
Tropical forests are complex natural ecosystems endowed with unrivaled biodiver-
sity. Located between the Tropics of Cancer and Capricorn, and comprising only
7–12% of the earth’s total landmass, tropical forests contain approximately 50% of
all known species (Laurance 1999; FAO 2001). In addition to supporting these high
levels of biodiversity, tropical forests offer a multitude of ecosystem services includ-
ing: human habitat, pharmaceuticals, food, and other natural products; watershed
stability, flood amelioration, and soil conservation; control of regional climate
patterns through evapotranspiration; and regulation of global carbon and nutrient
budgets (Laurance 1999; Reiners et al. 1994; Serrão et al. 1996; Bawa et al. 2004;
Davidson and Artaxo 2004; Fearnside 2005).
The importance of tropical forests has recently taken on greater importance as the
scale of global climate change becomes appreciated. The tropics are at the forefront
of concerns for the risks posed by global change because of the high levels of
de-forestation and rapid rates of land conversion to cropping, with subsequent loss
of biodiversity and capacities for carbon sequestration. From 1981 to 2000, 21% of
the area occupied by tropical forests was de-forested (FAO 2001; Bawa et al. 2004)
and these rates have not slowed (FAO 2005); if anything they have accelerated.
Efforts are being made to catalogue the enormous species diversity in tropical forests
and assess what ecosystem characteristics are altered by land-use changes in order
to assess the implications of their loss. In 2001, tropical biologists convened in
Bangalore, India, to establish research priorities for the tropics and refined these
objectives and directions over subsequent years by additional meetings, workshops,
and retreats (Bawa et al. 2004). A primary broad ecological research goal that
emerged from these efforts aimed to achieve a better understanding of the structure
and function of tropical ecosystems. Emphasis was placed on studies of soil micro-
bial communities and belowground ecosystem structure and function including
specifically: describing genetic and species diversity in poorly known regions,
groups, and habitats of the tropics. This included defining the fungi and other
microorganisms in soil; exploring relationships between components of soil biodi-
versity, nutrient cycling, and productivity; and predicting the functional response of
tropical ecosystems to naturally and anthropogenically induced change.
The tropics are an especially understudied microbial ecosystem. Those studies
which do exist have focused on tropical soil microbial communities in the Neotropics,
primarily in the Amazon, Costa Rica, and Hawaii, with infrequent scattered studies
in other locations (Veldkamp et al. 2003; Borneman and Triplett 1997; Nüsslein and
Tiedje 1999; Burke et al. 2003; Carney and Matson 2006; Gomez-Alvarez et al.
2007; Kim et al. 2007; Cleveland et al. 2003). There has been little research on soil
microorganisms in the Tropics of Africa, Southeast Asia, and Oceania (Waldrop
et al. 2000; Amir and Pineau 1998; Krave et al. 2002; Bossio et al. 2005; Venkatesan
and Senthurpandian 2006), with no studies of microbial communities yet published
for tropical Australia. The majority of these studies have examined shifts in micro-
bial community composition and function when tropical forest is converted to other
land uses, such as pasture, plantations, and agriculture (e.g. Waldrop et al. 2000;
Borneman and Triplett 1997; Burke et al. 2003). Other studies have examined
seasonal influences of tropical wet and dry seasons (Carney and Matson 2006; Krave
et al. 2002), and the effects of plant species and aboveground diversity on soil micro-
bial communities (Carney and Matson 2005; 2006). Below we consider several
studies from around the world that have investigated soil microbial communities in
the tropics using culture-independent methods (Table 1).
Neotropics: Amazonia, Costa Rica, Hawaii
Amazonia. The Amazon Basin is the largest area of contiguous primary rainforest
in the world (Davidson and Artaxo 2004; FAO 2005; Rudel 2005). Amazonia is
four times larger than the world’s second major expanse of rainforest in the African
Congo Basin, and spans nine countries across the northern portion of South
America (FAO 2005). Brazil contains nearly two thirds of Amazonian tropical
forests and is home to an estimated 7,780 native tree species (FAO 2001; 2005;
Rudel 2005). From 2000 to 2005, Brazil had the largest annual net loss of forested
area worldwide with 3.1 million hectares lost per year (FAO 2005). The dominant
land-use change is conversion to pasture, accounting for approximately 50–70% of
the de-forestation, with broadacre cropping and logging being the other major
forms of land-use change (Serrão et al. 1996; Fearnside 2005).
Table 1 Summary of tropical soil microbiology studies included in review
Authors Location Study scheme Sampling depth Microbiological methods
Borneman and Triplett 1997 Eastern Amazon (Para, Brazil) Mature forest; Pasture 0–10 cm SSU rDNA
Kim et al. 2007 Western Amazon (Rondonia, Pristine forest; Terra preta 0–10 cm 16S rRNA
Brazil) soils
Cleveland et al. 2003 Costa Rica Oxisol forest and pasture; 0–10 cm Chloroform fumigation-
Mollisol forest and pasture extraction; phosphatase
enzyme assay;
substrate-induced
growth response
Veldkamp et al. 2003 Costa Rica (La Selva) Alluvial forest and pasture; 0–3 m (alluvial) Basal respiration;
residual forest and pasture 0–4 m (residual) substrate-induced
2 The Microbiology of Natural Soils
respiration
Carney and Matson 2005; Costa Rica (La Selva) Plant diversity gradient of 0–10 cm Phospholipid fatty acid
2006 1, 3, 5, or >25 species analysis (PLFA)
Nüsslein and Tiedje 1998 Hawaii (Big island of Hawaii) 200 year old volcanic ash 0–7.5 cm Guanine-plus-Cytosine
deposit with low plant (G+C)of DNA;
diversity SSU rDNA;
denaturing gradient
gel electrophoresis
(DGGE)
Nüsslein and Tiedje 1999 Hawaii (Big island of Hawaii) Pristine forest; Pasture 0–10 cm G+C of DNA; SSU rDNA
Gomez-Alvarez et al. 2007 Hawaii (Big island of Hawaii) Volcanic deposits of four Litter + 1 cm 16S rRNA
different ages mineral soil
Burke et al. 2003 Hawaii Pasture; sugarcane 0–20 cm PLFA
Brazil Forest; sugarcane 0–20 cm
Ecuador Forest; pasture; SC 0–10 cm
Bossio et al. 2005 Western Kenya Range of soil textures; forest, 0–5 cm Chloroform fumigation-
woodlot, or agriculture extraction; five enzyme
land use; improved fallow, assays; BIOLOG;
traditional ag., or tea PLFA; 16S rRNA
33
DGGE
(continued)
Table 1 (continued)
34
Authors Location Study scheme Sampling depth Microbiological methods

Venkatesan and Western Ghats, India Pristine forest; tea plantations 0–200 cm Five enzyme assays
Senthurpandian 2006
Amir and Pineau 1998 New Caledonia Five ultrabasic soils of varying 0–20 cm Dilute plate count;
plant cover fluorescein diacetate
hydrolysis
Waldrop et al. 2000 Tahiti, French Polynesia Forest; pineapple plantations 0–12.5 cm Chloroform fumigation-
of varying ages extraction; seven
enzyme assays;
BIOLOG; PLFA
Krave et al. 2002 Java, Indonesia Seasonal effects (wet and dry Litter + 0–10 cm 16S rRNA DGGE
season) mineral soil
Moritz 2008 Borneo (Mt. Kinabalu, Sabah, Pristine forest on ultrabasic or 0–120 cm Two lignocellulose
Malaysia) meta-sedimentary bedrock degrading enzyme
assays; PLFA; amino
sugar analysis
T.C. Balser et al.
The traditional first step in conversion of primary forest to some other land-use
is to slash and burn, which may or may not be preceded by selective logging
(Hölscher et al. 1997; Fearnside and Imbrozio Barbosa 1998; Desjardins et al.
2004). The slash and burn process results in a rapid redistribution of nutrients
because of the substantial removal of aboveground biomass. Large amounts of
carbon are removed from the system during burning while other nutrients, including
phosphorus, potassium, calcium, magnesium, and sometimes ammonium-nitrogen,
are concentrated in the ash layer. These nutrients are deposited on top of the soil
and incorporated throughout the surface layer, resulting in a temporary influx of
basic nutrients and an increase in alkalinity (Reiners et al. 1994; de Moraes et al.
1996; Müller et al. 2004). This temporary enrichment of soil nutrients allows alter-
nate land uses on the otherwise nutrient-poor soils of the tropics (Hölscher et al.
1997; Müller et al. 2004). Over time, the conversion from primary tropical forest to
pasture decreases the bulk density and porosity of the soil (Reiners et al. 1994;
Eden et al. 1991), and has varying effects on carbon content (Fearnside and Imbrozio
Barbosa 1998). Some studies in Amazonia have quantified increases in soil carbon
under pasture management (Desjardins et al. 2004; de Moraes et al. 1996; Trumbore
et al. 1995; Neill et al. 1996); others report decreases in soil carbon (Desjardins
et al. 2004; Eden et al. 1991). Some studies document the absence of changes to
soil carbon after conversion (Müller et al. 2004; Buschbacher et al. 1988). These
conflicting results appear to be related to forms of pasture management and age of
pasture, though there may be an as-yet unspecified role for microorganisms in car-
bon cycling in such changes to land-use (Balser and Firestone 2005).
While the effects of conversion of primary tropical forest to pasture on soil
properties have been relatively well documented in Amazonia, comparatively very
little is known about the effects of conversion and anthropogenic influences on soil
microbial communities in the Region. Few enough studies have examined soil
microorganisms in the tropics at all, while even fewer have been conducted using
culture-independent techniques (Borneman and Triplett 1997; Kim et al. 2007). In
the first published study using molecular methods which surveyed Amazonian
soils, Borneman and Triplett (1997) analyzed 100 small-subunit rRNA gene
sequences (SSU rDNA) from northeastern Pará, Brazil, from active pasture and
mature forest (50 clones from each land-use). They found 98 bacterial and two
archaeal sequences that were unique (with no duplicate sequences), and all were
novel sequences that had not been previously documented. Representative major
bacterial phyla included Planctomyces, Clostridium, Fibrobacterium, and
Proteobacteria. Phylogenetic analysis of the sequences indicated that in general,
most clones were distinct from other organisms that had been previously described
in sequence libraries and 18% of the sequences could not be placed into a known
bacterial kingdom. Accordingly, Borneman and Triplett (1997) suggested that these
tropical soils contained high levels of microbial diversity composed of some
unusual microorganisms. The authors then used rRNA intergenic spacer analysis
(RISA) to investigate the effects of vegetative cover and land-use changes on
microbial communities and found different banding patterns for the mature forest
and pasture soils, indicating that distinct microbial communities were present under
each form of land-use. This study made a major impact in soil and tropical
microbiology by providing evidence of novel soil microorganisms and vast
microbial diversity. Further, this study showed that de-forestation in the tropics and
subsequent conversion to pasture resulted in changes in the composition of the
bacterial community, which may also indicate or result in altered nutrient cycling
and ecosystem functioning. Unfortunately, the authors did not assay microbial
activity together with the community composition. The influence of de-forestation
on the role of microbial communities in ecosystem processes (such as nutrient
cycling) under each land-use remains unknown.
The next molecular survey of Amazonian soils was published a decade later by
Kim et al. (2007). In this work, the authors used oligonucleotide fingerprint group-
ings (OFRG) to sort through 1,500 16S rRNA clone libraries and DNA sequencing
to compare bacterial communities of pristine tropical forest soils and ‘terra preta’
forest soils in Rondonia, Brazil, in Western Amazonia. Terra preta soils were
anthropogenically created by disturbance during pre-Colombian times by the indig-
enous practice of “slash and char” agriculture. This alternative to slash and burn
involves converting the biomass to charcoal, or charring, rather than burning, and
results in a lower impact on the environment. Today these soils contain thick epipe-
dons (e.g. the ‘A’ horizon was 1 m thick at the study site of Kim et al.) with high
organic matter contents (Lima et al. 2002; Mann 2002). There is interest in terra preta
soils for their ability to stabilize large amounts of organic matter and maintain high
fertility in spite of the presence of favorable conditions for rapid organic matter
decomposition (Amundson 2001; Lehmann et al. 2003). Kim et al. (2007) found that
terra preta soils had greater bacterial diversity and were significantly different from
pristine forest soils, with approximately 25% greater species richness. This is particu-
larly interesting given that both soils had similar aboveground species composition
and structure, and indicates that the legacy effect from past land-use and the alteration
of soil properties remained apparent in the extant microbial community, regardless
of subsequent vegetational cover. Phylogenetic analysis showed 14 major bacterial
groups were present on terra preta, compared with nine in the pristine forest soil. Phyla
common to both soils included the predominant Acidobacterium, Proteobacteria,
Actinobacteria, Planctomycetes, and Verrucomicrobia. Similarly, Borneman and
Triplett (1997) found Planctomycetes and Proteobacteria dominated in eastern
Amazonian soils. An important difference between this study by Kim et al. (2007)
and Borneman and Triplett (1997) was that the methodology and technology for
clone analysis had greatly improved in a decade, thereby facilitating a more compre-
hensive survey using 1,500 clones in 2007 versus the 100 sequenced by Borneman
and Triplet in 1997. In the same way, the sequence databases were much larger
following 10 years of additional research, and consequently, fewer novel sequences
were found in the study by Kim et al. (2007), although they did identify three poten-
tial new subgroups of Acidobacterium. A similarity between the two studies was that
neither employed a functional measure of the microbial community alongside their
molecular analysis and therefore the role of each microbial community remained
unknown. The taxonomic information generated using molecular methods only
allows us to quantify the diversity of soil microorganisms and facilitates comparison,
but cannot currently give information about ecological functions (Fierer et al. 2007).
Nevertheless, both studies showed that soil microbial communities in Amazonia

changed alongside anthropogenic disturbance, be it a soil legacy effect from hun-
dreds of years ago or recent de-forestation and pasture conversion. Further, both
studies indicated that Amazonian tropical soils harbour distinct microbial communi-
ties belowground, paralleling the distinctness found in aboveground biodiversity.
Costa Rica. Central America contains approximately 4.5% of the world’s tropical
forests and houses an estimated 7% of the world’s biodiversity (FAO 2001). From
1990 to 2005, Central America experienced one of the highest rates of de-forestation in
the world (FAO 2001; 2005). As in Amazonia, cattle ranching and broadacre cropping
have generally been the forces driving land-use change, with urban expansion playing
a lesser but still important role (FAO 2001; Rudel 2005). Costa Rica contains just over
2.5% of Central American tropical forests and has the lowest rate of de-forestation in
the region (FAO 2001). Costa Rica is one of the pre-eminent locations for tropical
forest research in Central America and the world. In the 1960s, pristine tropical forest
was set aside in northeastern Costa Rica for ecological research and named La Selva
Biological Station of the Organization for Tropical Studies (Veldkamp et al. 2003).
Three studies have focused on soil microbial activities and communities at La Selva
(Veldkamp et al. 2003; Carney and Matson 2005; 2006), with one additional study of
soil microbial dynamics located elsewhere in Costa Rica (Cleveland et al. 2003).
At La Selva Biological Station in 1991, an abandoned cacao plantation was
cleared, the merchantable trees extracted, and then the area was slash burned
(Haggar and Ewel 1995). Subsequently, plant communities with gradients of
diversity were established, including plots planted with one, three and five species,
and >25 species (natural regeneration). Carney and Matson (2005; 2006) utilized
these experimental plots to investigate the influence of aboveground diversity on
belowground soil microbial communities using PLFA (Carney and Matson 2005;
2006), catabolic potential and litter decomposition assays (Carney and Matson
2005). They found that microbial communities shifted in composition with each
level of plant diversity and bacterial communities (not fungal) were primarily
responsible for these differences (Carney and Matson 2005). Catabolic and litter
decomposition assays suggested that each microbial community under different
levels of plant diversity harboured distinct functional characteristics. The authors
suggested that these shifts were related to the diversity of secondary compounds
available for microorganisms to use in their metabolism; for example, a monoculture
that produces a carbon source of uniform composition results in a microbial
community with lower enzyme diversity. Thus, a reduction in aboveground
diversity may affect the catabolic capacity of a belowground microbial community,
thereby altering carbon and nutrient cycling and ecosystem functioning. This could
indicate an important linkage between aboveground and belowground biodiversity.
This finding is particularly relevant to de-forestation issues in the tropics, where
primary forest is often cut down and replaced with monocultural plantations (e.g.
Waldrop et al. 2000; Krave et al. 2002; Venkatesan and Senthurpandian 2006).
In 2006, Carney and Matson published a follow-up to their 2005 study, where
they presented comparisons among different monocultures and focal members in the
three species combinations and ranges of sampling dates (representative of wet and
dry seasons). They found that microbial community composition differed significantly
among different monocultures and focal species, further supporting the suggested
influence of individual plant species in shaping microbial communities. Examination
of the microbial communities from different sampling dates showed no significant
effect of season relating to community composition. Taking the results of both
studies together, it may be concluded that land-use change that alters plant species
diversity and composition affects soil microbial communities and their functional
roles in ecosystems. These are notable studies because Carney and Matson (2005;
2006) successfully utilized functional and compositional measures of the microbial
community to provide an insight into altered ecosystem functioning under land-use
change (an important goal of tropical ecology [Bawa et al. 2004]).
At La Selva, in addition to the experimental set-up for studies of aboveground and
belowground diversity used by Carney and Matson (2005; 2006), there is primary
forest that lies adjacent to de-forested areas cleared for pasture during the 1970s
(Veldkamp et al. 2003). Thus, an existing system is in place to study the ecological
effects of pasture conversion on native tropical forests. Veldkamp et al. (2003) used
the pristine forest and adjacent pasture to look at microbial activity throughout the
profile (0–3 m) using basal respiration (BR) and substrate-induced respiration (SIR).
Both BR and SIR measure the carbon dioxide production of a soil sample over a
period of time, but SIR adds a labile substrate (e.g. glucose) to maximize the respira-
tion rate (representative of the “potential” of the soil microbial community), while
BR represents the capacity of the microbial community in situ. Soil profiles in the
tropics are highly weathered and often many metres deep, containing substantial
stores of soil carbon (Veldkamp et al. 2003; Trumbore et al. 1995; Nepstad et al.
1994). Thus, an energy source is available for microorganisms at depth, but very
little is known about their activities in the subsoil. Veldkamp et al. (2003) detected
both BR and SIR down to 3 m depth in both forest and pasture. The pasture had
30–50% higher SIR than the forest above 2 m, below which there was no significant
difference between land-use cover types. By contrast, BR was higher in the forest
than the pasture in surface sampling depths (10–50 cm), with no significant dif
ference below 50 cm. It is interesting that the potential community activity under
pasture was greater than under forest cover, but the actual activity of the community
was greater under forest cover than pasture. However, the focus of the Veldkamp
et al. (2003) study was primarily to assess the existence of substantial carbon pools
and microbial activity at depth in tropical soils and therefore soil microbial
communities and the ecology of microorganisms were not generally addressed.
A final study in Costa Rica was located outside of La Selva on the Osa Peninsula,
and also examined soil microorganisms under forest and pasture (Cleveland et al.
2003). In contrast to the depth study by Veldkamp et al. (2003), the focus of
Cleveland et al. (2003) was to investigate how land conversion to pasture affects soil
microbial communities using two contrasting soil types, Oxisols and Mollisols.
(Oxisols are highly weathered, low-nutrient tropical soils, while Mollisols are gener-
ally higher-nutrient grassland soils) (Brady and Weil 2002). Cleveland et al. (2003)
used chloroform fumigation-extraction to quantify microbial biomass, and phos-
phatase enzyme assays and substrate-induced growth response (SIGR) to measure
microbial activities. They found that both soil carbon and microbial biomass
decreased upon conversion to pasture, with a more pronounced effect on Oxisols.
The authors suggested that low fertility soils (e.g. Oxisols and Ultisols; also the most
common in the tropics) are more susceptible to reductions in microbial biomass than
soils of higher fertility (e.g. Mollisols). Analysis of microbial activities between
pasture and forest showed different physiological capacities, with forest communi-
ties being more responsive to changes in resource availability. Further, forest
communities appeared to be better adapted to decompose recalcitrant carbon com-
pounds (as evidenced by SIGR using salicylate) than pasture soils. This indicates
alterations in microbial community composition after land-use conversion from high
biodiversity forest vegetation to less diverse pasture vegetation, and supports the
conclusions of Carney and Matson (2005) who suggested shifts in microbial
communities under different levels of plant diversity were related to the diversity of
carbon sources. However, Carney and Matson (2005) used both compositional and
functional measures of the microbial community, while Cleveland et al. (2003) did
not use a compositional measure of the microbial community, but rather inferred it
from shifts in the microbial activity with different types of cover.
Hawaii. The Hawaiian Islands are the most secluded archipelago in the world
(Gomez-Alvarez et al. 2007), formed in the Pacific Ocean over millions of years
from basaltic magma that originated from a “hotspot” in the earth’s crust, creating
a chain of volcanic islands that migrate outward from the centre, activity that
continues today (Crews et al. 1995). The geographic isolation and continuing
volcanic activity of the Hawaiian Islands make the archipelago an attractive system
for ecological studies (Gomez-Alvarez et al. 2007; Crews et al. 1995; Nüsslein and
Tiedje 1998). Several studies of soil microbial communities have taken place in the
Hawaiian Islands, including examination of soil bacteria in volcanic deposits
(Gomez-Alvarez et al. 2007; Nüsslein and Tiedje 1998) and the effects of land-use
change on soil microorganisms (Nüsslein and Tiedje 1999; Burke et al. 2003).
Nüsslein and Tiedje (1998) were the first to study soil microbial communities in
Hawaii using molecular methods. For their study site, they selected a 200 year-old
volcanic ash deposit with low plant diversity and used genetic nucletotide guanine-
plus-cytosine (G+C) content to distinguish members of the community. An advan-
tage of G+C content is that it is comprehensive for all DNA and not subject to
methodological bias (Kirk et al. 2004; Nüsslein and Tiedje 1999). Prior to the study,
they hypothesized there would also be a low level of bacterial diversity on the
parent material because of its youth and lack of aboveground diversity. By contrast,
they found such high bacterial diversity that they could not determine the commu-
nity structure using G+C content, as this measure of microbial diversity has
relatively coarse resolution. To attempt to reduce the diversity to more manageable
levels, they fractionated the G+C content into 63% and 35% (indicative of certain
bacterial groups), but still found few duplicates and high diversity. The 63% fraction
represented the dominant bacterial biomass and consisted of fewer bacterial taxa,
including Pseudomonas, Rhizobium-Agrobacterium, and Rhodospirillum, while
the 35% fraction was not dominant but had higher bacterial diversity. The authors
suggested that this reflects the ecological paradigm (Levine 1976) where the most
successful competitors consist of fewer species, while the less competitive minority
consists of more diverse organisms that occupy smaller, more defined niches. This
initial study of microbial communities in Hawaii identified high levels of bacterial
diversity, even on a young substrate.
The next study of bacterial diversity in Hawaii, by Gomez-Alvarez et al. (2007)
was conducted nearly a decade later. The authors used molecular methods with higher
resolution and compared soil bacterial communities on three volcanic deposits
ranging in age and included an established old-growth tropical forest (Gomez-Alvarez
et al. 2007). The study sites ranged in vegetative cover from a complete absence of
vegetation to mature forest. The sequencing and phylogenetic analysis of 16S rRNA
showed 56% of the sequences were unclassified and largely distinct. The three volca-
nic deposits had 60–80% of sequences unclassified, while the mature forest had only
6% unclassified. The oldest volcanic deposit had the greatest bacterial diversity, while
the intermediate-age volcanic deposit with an absence of vegetative cover showed the
least. Only Acidobacteria and Actinobacteria were common to all four study sites and
no phylotype showed >97% homology among sites. The results of Gomez-Alvarez
et al. (2007) indicated a high diversity of soil bacteria within a relatively small area
of the Kilauea volcano caldera and found a large number of previously un-described
clone sequences in Hawaiian Andisols. Similar to the study of Nüsslein and Tiedje
(1998), the work of Gomez-Alvarez et al. (2007) emphasized the diverse and ubiqui-
tous abilities of bacteria to colonize young, even un-vegetated soil, while providing
more detailed genetic sequencing of the microbial community.
Another study in Hawaii by Nüsslein and Tiedje (1999) used G+C content frac-
tionation to compare soil bacterial communities under land conversion from native
tropical forest to pasture. Similar to other studies in other tropical areas, they found
a significant G+C shift (49%) in microbial community structure with the change in
vegetative cover (Veldkamp et al. 2003; Borneman and Triplett 1997; Cleveland
et al. 2003). Fibrobacter, an organism that specializes in cellulose degradation,
dominated the 63% G+C content in the forest, with a shift to Proteobacteria (general
Gram-negative type bacteria) dominance in the pasture. This shift in dominance may
indicate that the physiological capacity of the soil bacterial community also changed
upon land-use conversion, but there was no quantitative or qualitative measure of
microbial function in this study. Thus, while Nüsslein and Tiedje (1999) described
genetic diversity of the soil bacterial community, the inter-relationships between
bacterial community structure and ecosystem functioning were not addressed.
The final study we consider in the Hawaii and Neotropics section used of
phospholipid fatty acid (PLFA) analysis to examine shifts in microbial community
composition in forest, pasture, and sugarcane soils in Hawaii, Brazil, and Ecuador
(Burke et al. 2003). Lipid analysis provides a more general perspective of microbial
community structure; rather than genetic information, it provides information on eco-
logical functional groups, such as Gram-positive bacteria, Gram-negative bacteria,
actinomycetes, and saprophytic fungi (Vestal and White 1989). Generally, Burke et al.
(2003) found that soil type (e.g. Hawaiian Andisols versus Brazilian Oxisols) was most
closely related to the relative abundances of Gram-positive and Gram-negative bacteria.
Conversely, land-use was the major determinant of fungal, actinomycete, and protozoal
abundance. Overall, biomass and more specifically, Gram-negative biomarkers, declined
upon conversion from forest to sugarcane in Hawaii and Brazil. Gram-positive
bacteria were more abundant in agricultural soils, most likely reflecting the less labile
carbon substrate that is available on cropland without continuous additions of fresh
litter. The study by Burke et al. (2003) reaffirmed that land-use conversion affects the
structure of soil microbial communities and introduced the concept that these changes
show some regional variability (i.e. Hawaii versus Ecuador versus Brazil) but can be
broadly applied to a range of tropical soils.
Conclusions: Neotropics
The majority of tropical soil microbiology studies have had sites in the
Neotropics and have concentrated on the effects of land-use conversion on soil
microbial communities. These studies have utilized a wide range of methods for
the study of soil microorganisms, including PLFA, DNA-based methods, and
measures of microbial function. However, we can collectively use the results
from these studies to conclude that the effects of land-use change on vegetative
cover type and plant species diversity changes the structure and function of the
soil microbial communities (Veldkamp et al. 2003; Borneman and Triplett 1997;
Nüsslein and Tiedje 1999; Burke et al. 2003; Cleveland et al. 2003; Carney and
Matson 2005; 2006). There is indication that the conversion to pasture reduces
microbial activity and biomass (Veldkamp et al. 2003; Burke et al. 2003;
Cleveland et al. 2003) and that microbial communities have a less diverse physi-
ological response as aboveground biodiversity decreases (Cleveland et al. 2003;
Carney and Matson 2005). Other studies have indicated there is an immense
diversity of soil microorganisms in the tropics, with many previously unclassi-
fied organisms (Borneman and Triplett 1997; Kim et al. 2007; Nüsslein and
Tiedje 1998). The majority of studies restricted soil sampling to the upper 10 cm
of soil, and given the substantial carbon pools at depth in tropical soils, further
studies of microbial activity in tropical subsoils are much needed (Veldkamp
et al. 2003). In addition, more studies that use both compositional and functional
measures of tropical soil microbial communities are needed to help understand
what changes in ecosystem functioning occur under land-use changes.
Tropics of Africa, Southeast Asia, and Oceania
Africa. In 2000, Africa was estimated to contain approximately 33% of global tropical
forests (FAO 2001). The Congo Basin, located in the heart of Africa, is the second
largest expanse of primary rainforest in the world (FAO 2001; 2005). From 2000 to
2005, six out of the top ten countries with the highest de-forestation rates in the world
were located in Africa (Congo, Nigeria, Sudan, Tanzania, Zambia and Zimbabwe),
making tropical African forests a rapidly diminishing resource. Although it is difficult
to generalize for the whole of Africa, the major force behind de-forestation is often
conversion to small-scale subsistence agriculture (FAO 2001; Rudel 2005). While
much is unknown about the tropics, the tropical forests of Africa are perhaps the most
understudied of all tropical areas, with little known about the structure and function of
these systems and the effects of de-forestation and land-use conversion.
A single study by Bossio et al. (2005) investigated tropical soil microbial
communities in Western Kenya under a variety of land uses and employed a consortium
of microbiological analyses. This study included comparisons among forest, woodlot,
and agriculture land-use types; agriculture practices of traditional maize cropping (two
crops per year), management with improved fallow (alternating maize with a species
of nitrogen-fixing tree, Tephrosia candida), and perennial tea cultivation; and a range
in soil textures from sandy (5% clay) to clay (60% clay). To assess microbial com-
munity activity, Bossio et al. (2005) used BIOLOG and enzyme assays, and used
PLFA and genetic analysis (DGGE analysis of 16S rRNA) to determine microbial
community structure. This is the only study that used such a comprehensive assem-
blage of microbial characterization techniques (Table 1). Molecular methods showed
that soil type (such as sandy, clay) appeared to induce the primary differentiation
among soil microbial communities, while land-use (wooded or agriculture) was the
secondary determinant. The DGGE analyses showed there was no significant differ-
ence in bacterial diversity between wooded and agricultural soils. While PLFA data
showed greater relative abundance of Gram-negative bacterial indicators in wooded
soils than agricultural soils, and agricultural soils had higher proportions of actinomy-
cetes and Gram-positive bacteria. These same shifts in lipid biomarkers under land-use
change were reported in the Neotropics by Cleveland et al. (2003) and are likely to be
linked with varying availabilities of carbon sources under different land-uses. Bossio
et al. (2005) found that soil microbial communities from wooded sites had the most
diverse abilities for substrate utilization, a finding also reported by Cleveland et al.
(2003). Finally, the authors found substantially lower soil carbon, microbial biomass,
and total enzyme activities under traditional maize cultivation, all of which generally
increased under improved fallow management. Using a complex suite of methods and
study sites, Bossio et al. (2005) found that the microbial community shifted with dif-
ferent soil types and land-uses, but overall levels of microbial diversity were similar
among land uses. Shifts in activity or functions of the microbial community were
closely linked to management practices, but showed less specificity than measures of
microbial community structure. This study reiterates the important influences of land-
use change and management practices on soil microbial communities in the tropics.
This study by Bossio et al. (2005) is the only study we are aware of that focused on
soil microbial communities in tropical Africa. Clearly, there is a need for more studies
on this continent with its vast tropical forest reserves and rapid de-forestation.
India. India is unique among the other tropical regions examined in this literature
review in that it has had a high population density exerting pressure on the land
since at least the nineteenth century (Rudel 2005). While pristine tropical forests
are being newly encroached upon and exploited in Amazonia, Africa, and parts
of Southeast Asia, tropical forests in India have been de-aforested and fragmented
in response to poverty and anthropogenic pressures for over a century. As a result,
in the 1980s, only 2% of India’s land area contained primary forest, with much
of the remaining land consisting of cutover and degraded forests. The government
responded by establishing forestry management plans and intensive re-forestation
efforts, resulting in a slight increase (38 ha year−1) in forested area from 1990 to
2000 with 72% of India’s forests under management plans. Not surprisingly, the
majority of soil microbiology studies in the tropical forested regions of India
have examined the effects of land conversion and management practices on soil
microorganism populations. Changes in microbial biomass under different land
uses have been researched intensively in India and studies have generally shown
that microbial biomass declines upon conversion from primary forest (Srivastava
and Singh 1991; Basu and Behera 1993; Singh and Singh 1995; Behera and
Sahani 2003). However, no studies in India were found that explored shifts in
microbial community structure upon land-use changes using culture-independent
methods (PLFA or molecular methods), while one study was found that investi-
gated differences in microbial enzyme activities between pristine forest and land
converted to tea plantations (Venkatesan and Senthurpandian 2006).
In the middle of the nineteenth century, the Western Ghats, a region in Southern
India, was covered with native tropical wet evergreen forests (FAO 2001; Kumara
et al. 2004). In the 1880s, private landowners cleared large tracts of this rainforest
and established tea plantations on the deeply weathered tropical soils, creating a
mosaic of plantations and primary forest (Venkatesan and Senthurpandian 2006;
Kumara et al. 2004). A study by Venkatesan and Senthurpandian (2006) examined
differences in microbial enzyme activities between these two land uses in the
Western Ghats. They sampled every 25 cm down the soil profile to 200 cm depth in
an effort to quantify microbial activities throughout the typical rooting zone of tea.
All enzyme activities (with the exception of protease) were detectable in the deepest
sampling depths, indicating an active microbial community in the subsoil. Veldkamp
et al. (2003) reported a similar result with measurable microbial respiration at 3 m
depth in tropical pasture and forest soils in Costa Rica. Venkatesan and Senthurpandian
(2006) found that acid phosphatase activities were highest in both tea and forest
soils, followed by alkaline phosphatase activities. At any given depth, urease activ-
ity was higher under tea plantations than under forest, and showed no significant
decline with depth under tea crops. The authors attributed this result to the continu-
ous application of urea used in plantation management. Aryl sulfatase activity was
considerably higher in the top 50 cm of the tea plantation than the forest, below
which the forest had consistently higher aryl sulfatase activity. Generally, Venkatesan
and Senthurpandian (2006) summarized their findings of enzyme activities and
attributed observed patterns to organic matter content and beyond this provided
little insight into their results. Thus, data are presented, but lack an ecological con-
text, which is a common shortcoming in soil microbiology. The enzymatic patterns
observed by Venkatesan and Senthurpandian (2006) were generally irregular with
no distinct conclusion to be drawn between types of cover. However, they overlooked
that all enzyme activities were greater below 150 cm in the forest than the tea planta-
tions (with the exception of urease). This may be attributed to the deeper rooting
patterns of tropical wet evergreen forests that extend well below the 2 m zone of
tea plantations. This study is important because of its focus on microbial activity
with depth in the tropics, a relatively unknown realm of study (Veldkamp et al.
2003). However, further studies of depth that include determinations of microbial
community structure along with measures of activity are needed in order to arrive
at ecologically meaningful interpretations of ecosystem functioning.
Pacific Southeast Asia and Oceania. The tropical forests of the Southeast Asian Pacific
and Oceania have some of the highest reported levels of biodiversity in the world (FAO
2001; 2005; Myers and Mittermeier 2000). However, recent estimates have shown
that Pacific Southeast Asia retains only 3–15% of its primary vegetation, while
Oceania, including New Caledonia and Polynesia/Micronesia, preserves 22–28% of its
original vegetation (Myers and Mittermeier 2000). Indonesia lost almost 1.9 million
hectares per year from 2000 to 2005, the second largest annual net loss of forest behind
Brazil (FAO 2005). Conversely, from 1990 to 2000, New Caledonia and French
Polynesia, two countries considered in this review, had no net loss of tropical forests.
De-forestation in Southeast Asia results primarily from logging and the timber trade
(Laurance 1999; FAO 2001). Similar to the tropical forests in Africa, the forests of
Southeast Asia and Oceania have few studies of soil microorganisms and ecosystem
functioning (Waldrop et al. 2000; Amir and Pineau 1998; Krave et al. 2002).
On the island of Java, Indonesia, nearly all primary tropical forests have been
harvested and today approximately 50% of the forest is has been converted to pine
plantations (Krave et al. 2002). A study by Krave et al. (2002) investigated seasonal
influences (wet and dry season) on soil bacterial communities in Javanese Pinus
merkusii plantations. Their objective was to achieve a better understanding of nutri-
ent cycling and so improve plantation management. They sampled litter, duff,1 and
mineral soil samples during successive dry and rainy seasons and then used DGGE
analysis of 16S rRNA to assess bacterial community structure. They found that
each layer of forest floor and mineral soil had significantly different physical and
chemical properties, including organic matter content, and the bacterial communi-
ties were stratified accordingly. Moisture content, pH, and nitrogen levels of the
three forest layers changed significantly with season, but bacterial communities in
the duff and mineral soil were not influenced. Only the litter microbial communities
varied with season. Similarly, Carney and Matson (2006) found that season did not
influence the composition of soil microbial communities in Costa Rica. Krave et al.
(2002) constructed a clone library for one litter sample taken in the wet season and
found that it contained high levels of bacterial diversity. Proteobacteria (specifi-
cally Rhizobium-Agrobacterium) and Gram-positive bacteria with high G+C
content dominated the profiles. Rhizobium-Agrobacterium was reported in the study
of Hawaiian soils by Nüsslein and Tiedje (1998) and Gomez-Alvarez et al. (2007),
1
Duff: the layer of partially and fully decomposed organic materials lying below the forest flar
litter and immediately above the mineral soil.
but was not documented in phylogenetic analyses of tropical soils by Borneman

and Triplett (1997) and Nüsslein and Tiedje (1999). Overall, this study suggests
that microbial communities in duff and mineral soil are better buffered from
seasonal changes than litter microbial communities, and each layer harbours a
distinct community of considerable diversity.
Another study of tropical soil microbial communities in the Southeast Asian Pacific
was conducted by Amir and Pineau (1998) in New Caledonia using culture-dependent
methods to identify microbial community structure and culture-independent meth-
ods to assay microbial activity. Approximately one third of New Caledonia is
underlain by ultramafic outcrops. Soils weathered from ultramafic rock, often also
referred to as ultrabasic or serpentine soils, pose special challenges for plant growth
and survival. These rocks and their resulting soils are characterized by high levels
of metals (e.g. nickel, cobalt); low levels of nitrogen, phosphorus, and potassium;
high levels of magnesium with low calcium; and low soil moisture (Walker 1954;
Proctor 1999; Brady et al. 2005). Ultrabasic outcrops often have poor productivity
and contain many endemic species that are specially adapted to the potentially toxic
levels of magnesium and other metals (Walker 1954). Most ultramafic outcrops are
exposed in orogeny (mountain-building) zones and are rare, occupying approxi-
mately 1% of the Earth’s surface (Proctor 1999). In New Caledonia, many ultrama-
fic outcrops have been mined for nickel and revegetation efforts are underway to
reclaim land degraded by mining activities (Amir and Pineau 1998). Amir and
Pineau (1998) examined soil microorganisms in a series of ultrabasic soils that
varied in vegetative cover (e.g. bare ground, cluster of vegetation and crop cover)
to investigate methods for improving soil fertility on reclaimed lands. Soil micro-
bial populations were estimated using dilution plate counts (a culture dependent
method) and microbial activity was estimated using the fluorescein diacetate (FDA)
hydrolysis test (a culture-independent method). The authors found that microbial
populations and activity in New Caledonian ultrabasic soils were lower than other
estimates reported in studies of nonserpentine soils. These low numbers were attrib-
uted to minimal organic matter contents in the ultrabasic soils. Actinomycetes were
the predominant bacteria, accounting for 70–90% of the total culturable microor-
ganisms, and two very common culturable fungal families, Tuberculariaceae and
Dematiaceae, were absent. The authors suggest these results may be related to the
metal tolerance of these microbial groups. The ultrabasic soils varied widely in
microbial characteristics depending on the level of root colonization of soil, with
the highest biomass and activity under vegetation and the lowest values on bare
uncolonised ground. Cropping appeared to affect microbial communities positively,
as bacterial and fungal diversity increased under cultivation. The authors caution
that their results are difficult to compare with other soils because of the uniquely
extreme conditions of ultramafic outcrops. Amir and Pineau (1998) provided pre-
liminary information about soil microbial communities in tropical ultrabasic soils,
but more comprehensive studies using culture-independent methods are needed
along with further studies of microbial function.
A further study in Oceanic tropical soils used enzyme activity assays and
BIOLOG along with PLFA to investigate responses of the soil microbial community
to land-use conversion from primary forest to pineapple plantations in Tahiti

(Waldrop et al. 2000). Researchers found that conversion to plantation agriculture
increased soil acidity and decreased carbon content, and accordingly soils had
one quarter of the microbial biomass of forest soils. Soil microbial communities
were significantly different between primary forest and plantations, with greater
relative abundances of fungi and actinomycetes in plantation soils. BIOLOG did not
show differences between forest and plantation treatments. This contrasts with the
hypothesis presented by Carney and Matson (2005) in their study of the effects of
plant diversity on soil microbial communities in Costa Rica where they suggested
that under less diverse vegetative cover (e.g. plantation) soils have lower diversity in
their degradative abilities. Waldrop et al. (2000) further found that enzyme activities
were similar between forest and plantation soils, with the exception of b-glucosidase
and sulphatase enzymes. However, the pineapple plantations are managed distinctly
from other crops and every 5 years the entire pineapple crop is cut down and incor-
porated into the soil, ‘resetting’ the system with organic matter input. Thus, pine-
apple plantations may have carbon inputs comparable to natural forests, resulting in
similar microbial community enzyme activities between forest and plantation soils.
Similarly, Venkatesan and Senthurpandian (2006) found no discernible differences
in enzymatic patterns (with the exception of urease) between tea plantations and
tropical forest soils in India. Relating microbial community composition to function,
Waldrop et al. (2000) were able to correlate specific enzyme activities (total activity
divided by microbial biomass) and relative abundance of specific indicator lipids.
They found b-glucosidase and b-xylosidase, enzymes used to degrade simple carbon
compounds, were correlated with Gm-, fast-growing bacteria that metabolize simple
sugars. Thus, the authors successfully deduced the function of a member of the soil
microbial community, lending insight into its role in ecosystem functioning. This
study by Waldrop et al. (2000) confirmed that land-use change in the tropics alters
soil properties and soil microbial communities. Further, it provided a successful
interpretation of microbial structure and function, achieving a major goal of tropical
ecology (Bawa et al. 2004).
Conclusions: Old World Tropics
These studies of soil microbial communities were as varied as their locations. They
examined the effects of soil type and land-use in Africa (Bossio et al. 2005); micro-
bial activity through depth in tea plantations and tropical forests in India; wet and dry
seasons on litter and mineral soil in Java (Krave et al. 2002); vegetative cover in New
Caledonia (Amir and Pineau 1998); and conversion from primary forest to pineapple
plantations in Tahiti (Waldrop et al. 2000). Perhaps the best collective conclusion
from these five studies of the tropics is that little research has been done in this vast
region of the world concerning soil microbial communities and ecosystem function-
ing. Clearly, there is a huge range of diversity of soils and land uses in the tropics, and
many questions concerning ecosystem functioning remain to be answered.
Wetlands
Wetlands are transient or permanently saturated terrestrial habitats. The term

wetland has been applied to freshwater marshes, bogs, peatlands and swamps (EPA
2004). It also encompasses coastal areas such as salt marshes and mangroves (Shaw
and Fredine 1956). This excessively broad definition has long hampered our
understanding of these ecosystems and its inhabitants. Overall wetland ecosystems
comprise a small proportion of the earth surface (at least 9% by some estimates
[Ramsar Convention Secretariat 2007]), yet their contribution to biochemical
cycles, water cycle (as source and filter), and the maintenance of species richness
overshadow that of other larger terrestrial environments (Basu and Behera 1993).
Additionally, wetlands are used by humans for water, flood control, fiber, food, and
recreation. The services delivered by wetlands have been arguably valued at US$14
trillion annually (Ramsar Convention Secretariat 2007). Worldwide, wetlands have
long been underappreciated and drained. Wetland loss and degradation has primar-
ily been driven by land conversion for agriculture, infrastructure development,
water extraction for irrigation or drinking water, eutrophication and pollution and
over-exploitation (Ramsar Convention Secretariat 2007). Climate change is
expected to exacerbate the loss and degradation of wetlands, both through drying
out and resulting shifts in precipitation (Ramsar Convention Secretariat 2007).
Wetland ecosystems link terrestrial, aquatic, and biosphere level processes
through their contribution to biogeochemical cycles and global climate change
(Fig. 2). This includes interactions between environmental, hydrological and biotic
processes. Among these the physical processes, e.g. hydrology, have been studied
in more detail than the biotic processes. A recent review of wetland microbial com-
munities by Gutknecht et al. (2006) notes that research in wetland biogeochemistry
has most often been focused on microbially-mediated processes (e.g., methanogen-
esis, denitrification and methanotrophy), and less often on microbial communities
or on populations of specific microorganisms of interest (Fig. 3). Work to date
indicates an important process level role for hydrology and soil nutrient status. The
impact of plant species composition on processes is potentially critical, but is as yet
poorly understood.
Research on microbial communities in wetland soils has primarily focused on
anaerobic bacteria responsible for methanogenesis (production of methane gas), deni-
trification and sulfate reduction (Gutknecht et al. 2006). There has been less work on
taxonomic groups such as those responsible for nitrogen fixation, or aerobic processes
such as nitrification. While less well known, it is also possible that aerobic organisms
such as nitrifiers and methanotrophs may play an important role in wetland ecosystem
functioning.
Work on general community composition and on wetland mycorrhizal fungi is
particularly sparse. Despite longstanding conventional wisdom that says fungi are not
abundant or important in wet systems, it has now been demonstrated that mycorrhizal
fungi are often abundant in wetlands (Mentzer et al. 2006) and may play a significant
role, and mycorrhizal association was the focus of a recent review (Anupam 2003).
Fig. 2 Wetland structure. Water table height, depth from surface, and distance from plant roots
create oxic to anoxic gradients. The result is a complex interplay between anaerobic and aerobic
conditions that allows for a wide range in processes to occur in wetland soils
While arbuscular mycorrhizas (AMF) can be found at all levels along moisture
gradients from dry to excessive moisture, abundance may decrease under very high
water content (Miller and Bever 1999). In particular, AMF species accustomed to dry
environments may be very sensitive to high water levels, indicating that water level is
an important factor in AMF colonization. Mycorhizzal community composition is
also likely sensitive to plant community structure (Gutknecht et al. 2006).
The most conspicuous effect of alterations to a wetland habitat is perhaps the
drastic change in vegetation composition and structure. Wetlands habitats receive
high levels of pollution (i.e. nitrogen, phosphorus and pesticides) as runoff and
ground water from agricultural activities and human settlements, which has contrib-
uted to their high susceptibility to invasive species (Zedler and Kercher 2004), and
a high proportion of local extinctions. Invasions alone could bring in drastic
changes in vegetation composition and litter chemistry, which ultimately influence
soil processes. Over a span of decade to centuries, changes at plant and microbial
levels would influence ecosystem properties such as geomorphology, hydrology,
biochemistry and disturbance regime (Gordon 1998). Although invasive species are
spreading rapidly in wetland habitats at local and global levels (Zedler and Kercher
2004), their current impact in nutrient cycling and soils processes are not well
Plant
community
Water table
atmosphere
Element release to soil or
Soil type
O2
pH Redox Conditions
Substrate Availability
(e.g. Fe, S, NO3−)
Carbon Microbial
Availability Community
Function
Microbial
Community
Structure ?
Fig. 3 Relationships among controls over wetland ecosystem microbial communities and ele-
ment cycling. Arrows indicate relationships, and width of arrows indicates relative importance of
relationship for ecosystem functioning. Dashed arrows represent interactions that are poorly
understood, even though they may be important
understood (Windham and Ehrenfeld 2003). One study has shown that different
levels of an aggressive wetland invader (reed canary grass, Phalaris arundinacea)
do differentially impact the microbial community, affecting both biomass and the
structure of the biota (Kao-Kniffin and Balser 2007).
In recent years the value of wetlands has been increasingly recognized. The
cost associated with constructing and restoring wetlands contributes to this
appreciation.
Soil Microbial Communities and Changing Agricultural

Management Regimes
Humans have converted one-quarter of the Earth’s surface to cultivated systems,

largely by changing native ecosystems to arable lands within the past 50 years
(Millennium Ecosystem Assessment 2005). Agricultural expansion and intensifica-
tion is likely to accelerate as the world human population grows, and soil functions
at all levels will be affected, including the structure and functions of soil microbial
communities. An important area of study is community-level responses to land-use
changes rather than effects particular species or groups such as arbuscular mycor-
rhizal fungi or nitrogen-fixing bacteria, which are well known to play key roles in
agroecosystems.
It is well established that the conversion of native ecosystems to agricultural uses
can strongly affect microbial community structure, composition and diversity. For
example, conversions of tropical forest to plantations (Waldrop et al. 2000) have been
found to engender distinct soil microbial community structures, and agricultural
intensification has been reported to decrease microbial diversity (Steenwerth et al.
2006). Additionally, the type of land management practices used in agroecosystems
also affects microbial community structure and function through a variety of differ-
ent mechanisms. Numerous studies have documented changes in microbial com-
munity structure resulting from physical disturbance, especially tillage (Frey et al.
1999; Guggenberger et al. 1999). Tillage represents a severe disturbance to fungi by
severing hyphal connections. However, no-till systems favour the development of
fungi as compared with bacterial community components (Minoshima et al. 2007;
Kennedy and Schillinger 2006). Conversions to agriculture and attendant cultivation
practices also alter microbial communities through changes to temperature, soil
moisture (through irrigation and alteration of soil structure), and other physical
parameters.
Land-use changes alter soil microbial community structure through changes in
carbon availability and quality, pH (Cookson et al. 2007), nutrient availability, or
other chemical parameters. For example, studies comparing agroecosystems and
natural systems report that adding nitrogen decreases the relative abundance of
fungi by comparison to bacteria (Bradley et al. 2006; Bardgett et al. 1999). Seghers
et al. (2004) found that nitrogen fertilizers decreased populations of methanotrophs
and root endophytes in the bulk soil microbial community. They also found
differential effects of organic fertilizers versus inorganic, which was consistent with
other studies. For example, Wander et al. (1995) (Cookson et al. 2007) reported that
manure-amended plots showed less diverse populations of microorganisms than
cover cropped soil, but that microbial biomass was more metabolically active
(Wander et al. 1995). Ulrich et al. (2008) found that manure applications led to an
increase in the population densities of cellulytic bacteria within the soil microbial
community (see chapters elsewhere in this book). Fungal-to-bacterial ratios are
commonly measured as indicators of microbial community structure, and the
relative proportions of fungi are increased by no-till practices, crop rotations, and
use of cover crops (Six et al. 2006).
In addition to physical disturbance effects, alterations in vegetation, plant
diversity and species-specific plant traits can cause changes in aboveground litter
quantity and quality, and belowground root dynamics. However, alterations in
vegetation tend to cause idiosyncratic effects of particular plant species or par-
ticular functional traits, and it is difficult to draw more generalized patterns
(Porazinska et al. 2003). The effects on plant litter quality and quantity, in
particular, become limiting factors to microorganisms, and thus species-specific

differences in plant litter can affect microbial community structure and function
(Wardle et al. 2004).
Effects may persist for many years and decades after a given land-use has stopped
(Steenwerth et al. 2006). For example, Fraterrigo et al. (2006) (Ulrich et al. 2008)
found long-term microbial alteration in forest stands that had been cultivated but not
logged. Fungal markers, especially, were lower in previously cultivated sites, suggest-
ing that fungi may need extensive periods of time to recover from agriculture
(Fraterrigo et al. 2006). Spiegelberger et al. (2006) found changes in the microbial
community due to lasting pH changes resulting from liming 70 years after agricul-
tural abandonment (Spiegelberger et al. 2006). Evidence is beginning to accumulate
that the history of land-use can leave significant legacies in the soil microbial com-
munity, ultimately influencing successional dynamics of future plant communities
(Kardol et al. 2007) and hence, providing a mechanism by which changes due to
agricultural management practices may persist.
Conclusion
This chapter has discussed studies of soil microbial communities in the tropics,
highlighted the importance of wetland communities, and reviewed potential
agricultural impacts on soil microbial communities. We have explored general
patterns in microbial communities, and within communities in these systems in
particular. Overall, the complexity of soil microbial communities pose a challenge
that has limited the number and power of studies. The recent emergence of new
methods is beginning to supply much-needed information, but connecting the func-
tion and identity of microbes remains a key research area. Major human impacts
have altered both wetland and tropical ecosystems, which are rapidly diminishing.
Studies have focused on pasture conversion effects in the tropics and reveal a diver-
sity of microbial life underground, and the functional and structural changes
brought with such conversion. Wetland systems experience strong fluctuations in
environmental and biotic conditions. Recent research points to the sensitivity of the
microbial community to both hydrology and plant structure. Finally, agricultural
regimes strongly impact the soil microbial community and its functions, in particu-
lar decreasing fungal biomass and overall microbial diversity. These changes in
belowground community can be persist on the order of decades, even when the
aboveground community shifts.
Acknowledgments The authors would like to acknowledge the members of the Balser laboratory
at the University of Wisconsin-Madison, especially Dr. Harry Read and Kevin Budsberg, for their
untiring support and assistance. We would also like to thank Balser laboratory alumna Drs. Jessica
Gutknecht and Jenny Kao-Kniffin for valuable discussions about these ideas over the years.
Finally, we thank the editors for their valuable comments in strengthening this chapter.
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Chapter 3
Soil Microbiology and Nutrient Cycling
David W. Hopkins and Jennifer A.J. Dungait
Introduction
Soil organisms play a central role in the recycling of nutrients in soils, making them
available to plants, transforming some nutrient elements to gaseous forms which can
be lost from soil, and other transformations which predispose nutrients to loss. In
this chapter we will illustrate these processes in the context of sustainable crop pro-
duction. To do so requires some consideration of what is meant by ‘sustainable crop
production’. To be truly sustainable nutrient supply should correspond to demand,
with the rate of nutrient removal from the fields by crops matched by replacement
into the plant-soil system. There is no escape from the thermodynamic principles
that underlie the law of conservation of mass matter. This implies that for any pro-
cess occurring in a closed system, the mass of the reactants equals the mass of the
products. When viewed from this absolute standpoint, no cropping system would be
truly sustainable unless all the residues of the consumers were returned to the fields.
The use of composts, manures and slurries on fields used for food production is
widely practiced, and in some cases even the collection of human excreta (“night
soil”; Fig. 1) and its application to fields is not unknown. With the increasing urban-
ization of the human population and the necessary development of waste water and
sewerage systems, the return loop for, or connection between, animal (including
human) and food wastes and land used for food production, is broken in many parts
of the world. This effectively leads to the net transfer of vast quantities of nutrients
D.W. Hopkins (*)

Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, UK
J.A.J. Dungait
Rothamsted Research – North Wyke, Okehampton, Devon, EX20 2SB, UK
60 D.W. Hopkins and J.A.J. Dungait
Fig. 1 Human excreta (“night soil”) that has been collected and applied to horticultural plots in
south eastern China
from sites of food production, which are predominantly rural, to cities, usually to be
dissipated to the atmosphere or water courses and the ocean, or locked-up (at least
for the short-term) in waste disposal sites, such as land-fill dumps.
Soil microorganisms almost certainly have a role in the transformation of all the
bioelements (Table 1), but the quantities required by crop plants are usually relatively
small and can be supplied from the trace quantities present in soil atmospheric
deposits and recycled alongside those elements required in larger quantities. The
focus of this chapter is on the major bioelements, carbon and nitrogen, with brief
mention also of phosphorus. We have concentrated on the roles of soil microorgan-
isms in the supply of these elements. In order to improve the sustainability of nutri-
ent cycling in an agricultural system, organic residues and resources need to be both
exploited efficiently and conserved as far as possible in the system. For this reason,
it is essential that the cycling of plant nutrients, such as nitrogen, is considered along-
side the turnover of carbon and the processing of energy in the soil.
3
Table 1 The major bioelements, their main functions in organisms and key contributions made by soil microorganisms to the availability. The elements are
present in approximate decreasing order of typical requirement by crop plants
Principal sources in Contribution made to nutrient supply made by
Element cropping systems Biological roles soil microorganisms
Carbon Atmospheric CO2 Main cellular constituent Decomposition to release C from
organic molecules to CO2
Oxygen Atmospheric O2 Main cellular constituent
H2O Respiratory electron acceptor in
organic molecules CO2 aerobic respiring organisms
Hydrogen H 2O Main cellular constituent
organic molecules NH4+
Nitrogen NH4+ Main cellular constituent Conversion to plant available (often)
NO3− Respiratory electron acceptor by inorganic forms (mineralization)
N2 some bacteria under Conversion from NH4+ to NO3− (nitrification)
Soil Microbiology and Nutrient Cycling
organic molecules anoxic conditions which can predispose N to loss from soils
Atmospheric deposition Conversion from NO3− to gaseous forms
(pollution) N2O and N2 (denitrification) which is a
direct loss mechanism
Conversion from atmospheric N2 to NH3
(N fixation) which is a gain mechanism
Sulphur SO42− Component of several biomolecules Conversion to inorganic forms (mineralization)
organic molecules Structural role in proteins and Dissimilatory reduction to H2S under anoxic
Atmospheric deposition co-enzyme conditions which can lead to gaseous loss
(pollution) Respiratory electron acceptor by
some bacteria under anoxic
conditions
Phosphorus HPO32− Component of several biomolecules Conversion from organic to inorganic forms
Organic molecules Constituent of nucleic acids, ATP/ADP, (mineralization)
and phospholipids Solubilization of inorganic P minerals
Potassium K+ Component of several biomolecules
Co-factor in many enzymes
61
(continued)
Table 1 (continued)
62
Principal sources in Contribution made to nutrient supply made by

Element cropping systems Biological roles soil microorganisms
Calcium Ca2+ Component of several biomolecules
Important regulatory roles
Iron Fe2+ and Fe3+ Component of several biomolecules
Zinc Zn2+ Component of several biomolecules
D.W. Hopkins and J.A.J. Dungait
3 Soil Microbiology and Nutrient Cycling 63
The Global Biogeochemical Cycle of Carbon
Globally, the biogeochemical carbon cycle is quantitatively dominated by massive

pools of carbon in geological deposits as sedimentary carbonates and organic
deposits, which contribute in part to fossil fuel stocks, and marine sediments.
However, these are relatively static compartments compared to the smaller compo-
nents of carbon represented by soil organic matter, atmosphere and biomass.
Globally, the atmosphere contains about 780 Pg C predominantly as carbon diox-
ide, although trace quantities of methane and other hydrocarbons and chlorofluoro-
carbons also have important biogeochemical roles; the global biomass represents a
similar amount, approximately 800 Pg C. The amount of carbon in soil organic
matter (SOM) is approximately the same as the atmosphere and biomass added
together, at around 1,500 Pg (Fig. 2). This pool of carbon is in a permanent state of
flux, undergoing decomposition and subsequent release, returning once again to the
atmosphere. Atmospheric carbon enters the biomass (plants and some soil bacteria)
principally by photosynthesis (110 Pg C year−1), with small contributions from
chemoautotrophic carbon fixation. This primary input is approximately balanced
by carbon dioxide released to the atmosphere as a result of the collective respiration
by consumers and decomposers.
Organic Matter in Soils and Its Turnover
Decomposition is the progressive breakdown of organic materials, ultimately

into inorganic constituents, and is mediated mainly by soil microorganisms,
which derive energy and nutrients from the diverse range of molecules in SOM.
Microorganisms are the major direct contributors to the flux of carbon from soil
to the atmosphere (Hassink et al. 1994; Hopkins and Gregorich 2005). The con-
tribution made by decomposers, i.e. those organisms which utilize dead remains
and waste organic materials from organisms as their main or sole source of
energy and carbon for biosynthesis, to the return of carbon dioxide from the bio-
sphere to atmosphere is between 50 and 60 Pg C year−1, i.e. approximately 50%
of the total respiratory return of carbon dioxide. The process of decomposition is
central to the efficient use and recycling of nutrients in managed, agricultural,
semi-natural and natural ecosystems because it is the route whereby complex
biomolecules are degraded and bioelements, particularly nitrogen, are released in
plant-available forms.
The substrates for decomposition include a wide range of materials, forming a
continuum from recently added plant litter and carbon transferred into the soil by
physical processes such as rainfall, or biological processes via root exudation, the
activity of fungi and bioturbation, to very stable, highly altered organic matter.
Many components of fresh plant litter decompose very quickly, because they are
rich in energy, readily accessible to organisms and, particularly in the case of
Fig. 2 Global biogeochemical cycle of carbon. The units for pools are Pg (= 1015 g = 1012 kg), and
those for fluxes are Pg year-1)
simple sugars and peptides, rapidly assimilated. Consequently, though it represents

only a small proportion of carbon in soil, about half of the carbon dioxide output
from soil on a global basis comes from decomposition of this active pool of biologi-
cally available carbon from annual litter inputs to the soil (Coûteaux et al. 1995) – a
fraction of organic matter that is not typically included in SOM models. The stable
component of organic matter, at the other extreme, decomposes very slowly over
centuries or millennia (Campbell et al. 1967); the size of this pool is comparatively
large (Table 2).
Table 2 Pool sizes and turnover times of carbon in temperate arable soil at equilibrium simulated
using the Rothamsted carbon model with an annual carbon input of 1 t C ha−1 year−1 (Jenkinson
and Rayner 1977). See also Fig. 3
Equilibrium content
Pool (t C ha−1) Turnover time (years)
Decomposable plant residues 0.01 0.2
Resistant plant residues 0.47 3.3
Microbial biomass 0.28 2.4
Physically protected organic matter 11.3 71
Chemically protected organic matter 12.2 2,900
Total 24.2 1,450
CO 2 Volatile
organic
compounds
Decomposer Respiration
RIP
Leaf litter,
faeces, urine, root litter,
exudates
Labile Stable
residues residues
Autochthonous
Zymogenous
decomposer
decomposer
organisms
organisms
Physically Chemically
UPTAKE protected protected
organic organic
matter matter
Dissolved
organic matter
©D.W. Hopkins & E.G. Gregorich, 2003
Inert organic matter
Fig. 3 Decomposition and carbon turnover in soil. A conceptual diagram summarising the main
elements of the initial Rothamsted carbon model (Jenkinson and Rayner 1977). To this we have
added other small, but potentially functionally important compartments: the volatile organic car-
bon and the dissolved organic carbon derived during both decomposition of litter and exudation
from plants. An inert organic matter pool is added as this appears in latter versions of the
Rothamsted model (Reproduced from Hopkins and Gregorich [2005]). See also Table 2
The closest we have come to a unified concept for summarising the interaction of
carbon and organisms in soils is embodied in models of soil carbon turnover. The
conceptual model summarised in Fig. 3 owes much to the Rothamsted carbon model
(Jenkinson and Rayner 1977). Unlike some other models, which perform well in
their individual ways, this model is based on compartments with functional
r elevance. In later versions an inert carbon compartment has been added to improve
the performance of the model. This is not a unique feature of soil carbon models; for
example, the “Century” model has a “passive organic matter” compartment (Parton
et al. 1987) that also functions to slow down the model. Clearly, there cannot be a
completely inert soil carbon fraction in soil (how could it have arisen?), and
modelling simply points to the presence of a very stable organic matter fraction.
Making the link between the presence of this “inert” soil carbon and its biological
role remains elusive, but even soils that have been deprived of fresh organic matter
inputs for prolonged periods (decades) are still biologically active, and activity must
be being sustained by degraded components of the recalcitrant SOM remaining in
these ‘carbon exhausted’ soils (Lawson et al. 2000).
In addition to the compartments represented in the Rothamsted carbon model,
functionally significant compartments have been added such as dissolved and vola-
tile organic carbon (Mackie and Wheatley 1999; Gregorich et al. 2003). Dissolved
organic carbon is important for two main reasons. Firstly, in some high latitude and
altitude ecosystems, organic nitrogenous compounds make an important contribu-
tion to the nitrogen economy of plants (Näsholm et al. 1998). Secondly, because it
is a largely uncharacterised but biologically active component of the soil carbon
which contributes significantly to its export from soils (Grieve 1984), and acts both
as a driver of rhizosphere function and diversity, and of the initial exploitation of
fresh plant litter (Hopkins and Gregorich 2005; Webster et al. 2000).
Factors Controlling Decomposition
The factors controlling decomposition of organic materials are determined by three

sets of interacting factors – substrate quality, organisms and environment (Swift
et al. 1979); thus, decomposition does not occur at uniform rate in either time or
space. In the initial phase of decomposition, which typically lasts less than 1 year,
the majority of readily metabolised components, such as unprotected sugars and
oligosaccharides, amino acids and proteins, are rapidly exploited by decomposer
organisms leaving more recalcitrant components. Recalcitrant components may
resist breakdown because of their biochemistry, or by physical protection within the
soil matrix. The application of modern spectroscopic and stable isotopic approaches
to investigate soil organic matter turnover is revealing the incredible complexity of
organic matter breakdown (Hopkins and Gregorich 2005). In particular, the long-
held view that lignin components and lignin derivatives are inherently more stable
than other components of plant materials is being questioned (Dungait et al. 2008a,
2009). Even simple components of organic substrate may become stabilized for
millenia in the ‘passive’ organic matter pool (Paustian et al. 1992) by interaction
with soil mineral colloids, encapsulation in soil aggregates where the local environ-
mental conditions preclude rapid decomposition (e.g. because of limited oxygen
diffusion), and interaction between organic compounds leading to increased chemi-
cal complexity that is resistant to microbial decomposition.
Substrate Quality
The ability of a soil to supply nutrients, store water, release greenhouse gases,
modify pollutants, resist physical degradation and produce crops is strongly
affected by the quality and quantity of organic matter it contains (Carter 2001). The
physiochemical characteristics of organic material, for example its solubility in
water or hydrophobicity, water content, nitrogen and other nutrient content, bio-
chemical recalcitrance and toxicity, and physical protection strongly influence
decomposition. Thus, the substrate quality of SOM can be regarded as a suite of
combined properties that influence the supply of carbon and energy to heterotrophic
soil organisms. Although this is a simple concept, the ability to assess substrate
quality is not easy. Early studies recognised that different components of plant litter
were decomposed at different rates, which was considered to be a reflection of their
resource value to decomposer organisms (Tenney and Waksman 1929; Minderman
1968). More recently theoretical approaches that regard detritus as a continuum of
biochemical components from the recalcitrant to the highly labile have been devel-
oped (Ågren and Bossatta 1996). Despite substantial progress in the development
and application of increasingly sensistive analytical technques, we are still a long
way from being able to characterise all potential biological substrates in SOM, and
for key groups of organic compounds, most notably those containing nitrogen,
biochemical characterisation remains remarkably sparse. This is because the ana-
lytical chemistry involved in properly characterising and quantifying organic mat-
ter at the molecular level is sometimes difficult and challenging. Indeed, there is a
massive and increasing body of literature on the chemical characterisation of mys-
terious ‘humic substances’ that are obtained from soil by simple extraction with
strong alkalis and acids. The ecological relevance of these substances has been
questioned by soil science “dissidents” (Tate 2001), which include the authors of
this chapter, because it is well documented that such preparations contain artefacts
from sample preparation and are entirely non-selective with respect to biological
entities in soils (Baldock et al. 1991). Nevertheless, deeply cherished terms such as
“humic acid” have, like some of the molecules it purports to represent, proved
remarkably resistant to decay.
The primary source of SOM is plant litter are various stages of degradation.
Plant residues usually enter the soil as litter, including leaves, stems, roots and root
exudates. The typical composition of plant materials is approximately 50% cellu-
lose, 20% hemicellulose (polymers of hexoses, pentoses and uronic acids), 15%
lignin, 5% proteins, 5% amino acids and sugars, 1% pectin and 1% waxes and pig-
ments (Swift et al. 1979; Dungait et al. 2008a; Killham 1994). Excreta from live-
stock is another source of organic matter in soils (Fig. 4). Faeces contain not only
the undigested diet (10–40%), but intestinal-dwelling bacteria (50–85%) and
endogenous wastes from animal metabolism (10–15%) (Van Soest 1994). In agri-
culture, manures and slurries derived from ruminant grazing animals, such as cattle,
are commonly added to soil as a source of nitrogen to promote plant growth. The
organic matter in the manure and slurries is largely composed of biochemically
transformed plant residues from dietary fodder and forage. Simple substrates, such
Fig. 4 Ruminant digestion and the origin of faecal matter, showing major components of dung
(Figure reproduced from Dungait et al. [2010] and adapted from Van Soest [1994])
as proteins, amino acids and sugars are wholly digested, whilst more complex poly-
mers such as cellulose and hemicellulose are partially degraded. Other components,
such as chemically resistant lignin and waxes pass almost unchanged through the
gut. In faecal structures, such as cow pats, decomposition that started in the animal
gut may continue and is later overtaken by the activity of coprophilous flora and
fauna that further modify the organic matter before it is incorporated into the soil.
The modification of plant biomass by passage through the animal gut may contrib-
ute to slower turnover of carbon pools in soil following manure addition due to the
proportional increase in polymers that are resistant to decay (Dungait and Bol
2005) recognised as an associated increase in SOM content in manured soils
(Haynes and Naidu 1998).
Organisms
One to 5% of SOM is the soil microbial biomass, about 90% of which is fungi. These
microorganisms are capable of decomposing the majority of organic material.
Although probably not strictly correct, it is often assumed that all the microorganisms
necessary to complete the decomposition of any natural compounds (and many
anthropogenic compounds) are present in soil and that collectively they are infallible.
This assumption holds for many decomposition processes because of the large func-
tional redundancy in decomposer microbial communities, but it is not safe to assume
that there is redundancy amongst all organisms performing specific functions in soils.
For example, the high degree of specificity in the interaction between plant hosts and
their mycorrhizal symbionts means ubiquity cannot always be assumed.
The Rothamsted model (Fig. 3) distinguishes between organisms which notion-
ally respond to addition of fresh substrate (equivalent to the zymogenous biomass;
sensu Winogradsky [1924]) and those which notionally eek out an existence on the
older, more stable organic matter (equivalent to the autochthonous biomass; sensu
Winogradsky [1924]). The soil community is probably not as sharply divided as
Winogradsky’s definitions would imply. Similarly, the distinction between r-selected
and K-selected organisms, i.e. either those showing rapid proliferation following a
pulse of substrate availability, or maintaining near constant population by efficient
use of low concentrations of organic substrate, is probably not rigid. Therefore
so-called “zymogenous” and “autochthonous” categories of microorganisms are
approximately analogous to r-selected and K-selected organisms, respectively. Both
concepts are useful for understanding soil carbon dynamics, but cannot be related
directly to particular taxa, which may switch strategies (Chapman and Gray 1981),
or applied unreservedly in a complex environment such as soil where many factors
other than carbon supply may affect biological activity and biomass.
As mentioned above, microorganisms are the major drivers of decomposition in
soils, but soil animals also play a major role in facilitating the decomposition pro-
cess by physically fragmenting and mixing organic residues into the soil, thereby
increasing the surface area of substrates and their exposure to microbial activity.
Soil invertebrates, which include mesofauna such as Collembola, consume a wide
range of organic materials which differ in their physiochemical accessibility, spa-
tiotemporal distribution and nutritional value. Macroinvertebrates, such as earth-
worms, are important agents of organic matter decomposition in soils, accelerating
rates of communition and dispersal through feeding activities (Dungait et al.
2008b), which has led them to be described as ‘soil engineers’ (Wolters 2000).
They also influence decomposition by inoculating substrates with microbes, alter-
ing microbial activity (e.g. by deposition of mucus or wastes), altering the composi-
tion of the decomposer community, and the maintaining soil structure. The direct
faunal contribution to soil to atmosphere carbon flux is, typically between 5% and
15% of the total flux (Hassink et al. 1994; Alphei et al. 1996).
The diversity of biochemical reactions involved in decomposition is vast.
Briefly, the main processes involved are predigestion in animals (in some cases),
communition by soil invertebrates, depolymerization of macromolecules, and uptake
and use of the organic molecules by decomposer organisms. These processes do not
occur in sequence because the bodily remains and waste materials from the decom-
poser organisms contribute to the substrate pool (Fig. 3).
Environmental Conditions
The combined physical and chemical conditions in which decomposition occurs

both regulates the rate of decomposition and, in conjunction with the characteristics
of the resources, imposes a selection pressure on the decomposer community
thereby influencing its composition. Soil temperature, water content, acidity or
alkalinity, oxygen supply and texture are amongst the most significant factors con-
trolling the environment.
Like all other biological processes, the rate of decomposition of organic matter
and the other relevant biogeochemical transformations increases with temperature
in line broadly with the van t’Hoff rule that the rate of reaction doubles for a 10°C
rise in the range 10–40°C (Jenkinson 1981; Hartley and Ineson 2008). In temperate
regions, the temperature of the upper surface of the soil may occasionally rise to
about 30°C, particularly if there is no plant cover, and in tropical regions soil
temperatures consistently in the range 20–35°C are common. At greater depths in
the soil temperature is typically in the range 5–20°C, with the size of the daily and
seasonal fluctuations declining with increasing depth (Payne and Gregory 1988).
Consequently biological processes in the sub-soil are typically slower. Recent con-
cern about increasing atmospheric temperature globally has focussed attention on
the effect on soil processes, particularly carbon cycling and the depletion of soil
carbon reserves as a result of increased decomposition. There are a number of feed-
backs which affect the net effect of increased temperature on the soil carbon
reserves which may direct extrapolation difficult (Hartley et al. 2008).
Biological processes require a supply of liquid water, without which they ulti-
mately cease. Even under the driest conditions in soils, there is rarely absolutely no
water, but it is not uniformly distributed and forms a film over soil particles and
colloids leading to a mixture of air- and water-filled pores and voids. Decomposition
slowly proceeds even in naturally dry soil because of the activity of those organ-
isms able to access this water. Griffin (1972) reported that fungi are more active in
dry soils than bacteria because of the ability to translocate water across air-filled
pores and voids. Because of the different range of water contents over which dif-
ferent components of the soil microbial community are active, there is a rather flat
optimum water content for both decomposition and mineralization (Clement and
Williams 1962). Fluctuations in microbial activity accompany rapid changes in
moisture content, for example Birch (1957) showed that rewetting a dry soil
enhances the short-term rate of organic matter decomposition and nitrogen miner-
alization, and that this effect increases with repeated drying and rewetting. These
observations are consistent with the increased turnover of microbial biomass due to
wet/dry cycles in the soil (Jenkinson and Ladd 1981).
Under hot or drying conditions, decomposition and other biological nutrient
transformations may become restricted because of desiccation. This affects both
plant residues and animal wastes including faeces, manure and slurry, and in the
case of animal waste on the soil surface, “capping” may result. Capping occurs as
a hydrophobic crust forms during surface drying and can limit subsequent gas flow
(Dickinson et al. 1981). The presence of faeces, particularly cow pats can have
significant effects on the underlying soil moisture and temperature (Dickinson et al.
1981), as well as acting as point sources of inorganic nitrogen that influence nitrate
leaching (Ryden et al. 1984).
At the wet extreme, biological activity in soil can be affected by lack of or limited
oxygen because of the low solubility and slow diffusion in water. This has important
implication for the nitrification and denitrification discussed below. The decomposi-
tion of organic materials in soils is qualitatively different under long-term anoxic
conditions. This is related to the fact the breakdown of lignin in plant materials is
carried out by a restricted number of organisms which require oxygen (Kirk 1984).
There are many examples of decomposition being slower under acidic soil con-
ditions compared with near neutral soils (Jenkinson 1977). This is in part because
of the reduced overall microbial activity in acidic soils and the smaller abundances
and activity of earthworms in acidic soils.
The Biogeochemical Cycle of Nitrogen
The main components of the nitrogen cycle are shown in Fig. 4. The individual
transformations mediated by microorganisms that are relevant to nutrient cycling in
agricultural systems are nitrogen mineralization which is intimately linked to
decomposition, nitrogen fixation, nitrification and denitrification.
Nitrogen Mineralization
Nitrogen mineralization is the result of a set of processes allied to decomposition

which lead to the conversion of nitrogen in organic compounds to inorganic nitro-
gen, usually ammonium (NH4+), carried out by a wide range of microorganisms
(Harmsen and van Schreven 1955). This is significant because for many plants
inorganic nitrogen is the main form in which nitrogen in taken up from the soil.
This is particularly the case for crop plants.
Nitrogen mineralization operates in effective opposition to nitrogen immobiliza-
tion because, depending on the nitrogen supply in the substrate being utilized by
decomposer organisms, there may be a surplus of nitrogen in relation to carbon, or
a deficit. Jansson and Persson (1982) describe these processes as the mineralization-
immobilization turnover process in which inorganic nitrogen is released into the
soil solution and assimilated from the soil solution. There is more recent evidence
that soil microorganisms assimilate nitrogen from organic sources in the apparently
more efficient (from the microorganisms’ perspective), “direct” immobilization
route with only the nitrogen surplus to the microorganisms’ requirements being
released in to the soil solution (Barraclough 1997):
MINERALIZATION-IMMOBILIZATION TURNOVER
Mineralization
→
Organic nitrogen in organic Inorganic nitrogen
matter and microbial biomass in the soil
←
Immobilization
DIRECT ASSIMILATION
Organic nitrogen in → Organic nitrogen in → Inorganic N

organic matter micro organisms in soil solution
If the substrate offers a nitrogen surplus to the decomposer organisms, inorganic

nitrogen will be released and, if the substrate offers a nitrogen deficit to the decom-
poser organisms, they will uptake nitrogen from the external (soil) environment, i.e.
immobilization. Thus, the carbon-to-nitrogen ratio of the substrate strongly influences
whether inorganic nitrogen is released during decomposition or whether the decom-
poser organisms make a demand on the soil nitrogen pool, whereby nitrogen is assimi-
lated into microbial tissues. Typically a carbon-to-nitrogen ratio of about 20 is the
threshold for nitrogen mineralization above which it is immobilized, because the sub-
strate offers a nitrogen deficit to the decomposers, and below which nitrogen mineral-
ized, because there is a surplus of nitrogen relative to carbon in the substrate (Harmsen
and van Schreven 1955). However, the substrate quality and the composition of the
decomposer community influence the relationship between carbon-to-nitrogen ratio
of the decomposing substrate and mineralization and immobilization (Fig. 5)
Dinitrogen in
the atmosphere
tion
fixa N2
g en
N itro
Ammonium and organic N

(R-NH2) in N fixing bacteria
Nitrous oxide in
NH4+ the atmosphere
N2O
Den
R-NH2
itrific
atio
Host-symbiont transfer
n
Death and decomposition

N assimilation Nitric oxide in
the atmosphere
Organic N
(R-NH2) in microorganisms, NO
plants and animals
R-NH2 Mi
ne
ra
liz Ammonium in Nitrite in Nitrate in
ati
Im on the soil solution the soil solution the soil solution
mo
bil Denitrification
iza
NO2− NO3−
tio
n NH4+ Primary
Secondary
nitrification
nitrification
Potential
nitrate
N uptake
leaching
losses
Fig. 5 The biochemical transformations and major pools of soil nitrogen

50 C in CO2
Higher substrate
assimilation efficiency
Fungal biomass N requirement = 3.3

100 C C-to-N ratio 15-to-1 Threshold C-to-N = 30
in substrate
60 C in CO2
Lower substrate
assimilation efficiency
Bacterial biomass N requirement = 8

100 C C-to-N ratio 5-to-1 Threshold C-to-N=12.5
in substrate
Fig. 6 Relationships between C-to-N ratio and the threshold values for net N mineralization.
Fungi tend to have a larger C-to-N ratio than bacterial because bacteria contain a larger proportion
of structural peptides than bacteria so the threshold for net N mineralization for the substrate is
greater than for bacteria. It should also be noted that the assimilation efficiency of C by fungi
is greater than that for bacteria, so the proportion of C lost as CO2 is less for fungi for bacteria and
this affects amount of C the organisms will assimilate and therefore the amount of N that they will
require from the substrate (Redrawn from Killham 1994)
(Swift et al. 1979; Killham 1994). Wheat straw has a carbon-to-nitrogen ratio typically
in the range 80 to 100:1 and will lead to net nitrogen immobilization, whereas leaf and
stem residues from a nitrogen-fixing legume with a carbon-to-nitrogen ratio in the
range 12 to 15:1 will lead to net mineralization. This provides an opportunity to man-
age the synchrony of nitrogen supply to plants, or to remove inorganic nitrogen from
the soil pool and thus reduce the chance of nitrogen loss by denitrification and leach-
ing (Figs. 6 and 7).
Nitrogen Fixation
Biological nitrogen fixation is the process whereby atmospheric nitrogen or dini-

trogen (N2) is fixed by organisms. This accounts for approximately 140 Mt nitro-
gen per year and is the main route by which nitrogen fixed by natural processes
enters the biosphere, far exceeding nitrogen fixation by oxidation during lightning
discharges (Killham 1994; Sylvia et al. 1995). Biological nitrogen fixation is
carried out exclusively by bacteria with the nitrogenase enzyme complex. In
developed countries the nitrogen input to agricultural systems from biological
High C-to-N substrate
CO2 produced
Inorganic N
concentration in soil
C-to-N ratio of
organic substrate
Time
Inorganic N Low C-to-N substrate

concentration in soil
C-to-N ratio of
CO2 produced organic substrate
Time
Fig. 7 Dynamics of CO2 production, inorganic N concentration in the soil and the C-to-N ratio
of the organic substrate as the substrate decomposes for two contrasting substrates: one with a
high C-to-N ratio (above the threshold for net N mineralization) and one with a low C-to-N ratio
(below the threshold for net N mineralization)
nitrogen fixation is relatively small by comparison with fertilizer nitrogen produced

by the energy-demanding Haber-Bosch and other processes, whereas biological
nitrogen fixation is the main source of additional nitrogen in developing regions
where nitrogen fertilizers are expensive relative to the local economies (Sylvia
et al. 1995).
Biological nitrogen fixation is carried out by: (i) free-living bacteria such as
members of the genera Bacillus, Klebsiella and Clostridium, (ii) bacteria of the
genus Rhizobium and close relatives which live symbiotically in nodules on the roots
of leguminous plants such as beans, alfalfa (lucerne) and clover, (iii) actinobacteria
(actinomycetes) in the genus Frankia which fix nitrogen in root nodules of some
non-leguminous plants such as alder, free-living cyanobacteria such as Nostoc
which are photosynthetic and live on or close to the soil surface, and (iv) nitrogen
fixation by bacteria such as members of the genera Azotobacter and Azospirillium
living in association with plant roots (though not in symbiotic association), described
as ‘associative nitrogen fixation’. The N2 molecule is very stable and the reaction to
reduce it to biologically available NH4+ requires a source of energy. In the case of
nitrogen fixation by cyanobacteria this is provided by organic carbon from photo-
synthesis, and in the case of other bacteria from organic substrates in the soil-plant
system. The symbiotic nitrogen fixers receive organic carbon from plants and the
associative nitrogen fixers receive some organic carbon from rhizodeposits.
Chemical nitrogen fixation: N2 + 3H2 → 2NH3 ∆G = 53 kJ
(Haber-Bosch): 20 MPa (200 atmospheres) pressure and 400-500°C
Biological nitrogen fixation: N2 + 8H+ = 8e- + 16ATP → 2NH3 + H2 + 16ADP + 16Pi

Atmospheric pressure and environmental temperature
Denitrification
Denitrification is the dissimilatory reduction of nitrate (NO3−), to N2 that occurs under

anoxic conditions by a wide range of facultatively anaerobic bacteria which use nitro-
gen species as an alternative to oxygen as a respiratory electron acceptor (Sylvia et al.
1995). The reaction proceeds via a series of intermediates catalyses by separate
enzymes, not all of which are necessarily expressed in all denitrifying bacteria or
induced simultaneously. In many cases the enzymes are induced in a cascade so that
as anoxic conditions develop the further stages of the process occur (Cooper and
Smith 1963). However, as all steps are not induced simultaneously, and because not
all denitrifiers are capable of the full reduction pathway from NO3− to N2, intermedi-
ates can accumulate. The main product, gaseous nitrogen diffuses from the soil in to
the atmosphere and is, thus, a route by which nitrogen is lost from the soil. Smaller
quantities of nitrous oxide (N2O) and nitric oxide (NO) are also produced because the
coupling between the different steps in the overall pathway is complete (Firestone
and Davidson 1989). The production of nitrous oxide is of particular interest because
it is both a greenhouse gas and involved in the depletion of stratospheric ozone.
NO3 − → NO2 − → NO → N 2 O → N 2
Nitrate Nitrite Nitric Nitrous Dinitrogen
oxide oxide
Denitrification is promoted by any factor that reduces oxygen concentration in

the soil, such as the water-logging or the supply of labile organic substrate, produc-
ing conditions where microorganisms have the potential to consume oxygen faster
than it can diffuse to the site of decomposition in the soil. Managing denitrification
losses from soil is an important objective in a sustainable agricultural system, but because
it is driven in part at least anoxic conditions, the weather is main controlling factor.
Management by ensuring that the nitrate concentration in the soil is minimised

during wet season is, in principle, possible using high C-to-N ratio organic
amendments.
Nitrification
Nitrification is the oxidation of NH4+ to NO3− performed mainly by chemoauto-

trophic bacteria under oxic conditions which couples the reducing power of
oxidation to the reduction of carbon dioxide from the atmosphere into organic
carbon (Sylvia et al. 1995). The reaction typically occurs in two stages carried out
by different genera of nitrifying bacteria, but because of the high affinity of the
organisms carrying out the second stage for nitrite (NO2−), the intermediates rarely
accumulate. Indeed, because the energetic yield from either NH4+ or NO2− oxidation
is very small, provided environmental conditions permit (oxic and near neutral pH)
and the pertinent organisms are present, free ammonium ions rarely persist in soils
and are rapidly oxidized to nitrate ions by chemoautotrophic nitrifiers for which
ammonium is the only source of energy. Chemoautrophic nitrification is restricted
to a few genera of bacteria, termed the “nitroso” genera, Nitrosomonas, Nitrosovibrio,
Nitrosolobus and Nitrosococcus, that carry out oxidation of ammonium to nitrite and
the “nitro” bacteria, Nitrobacter, Nitrospira, Nitrococcus and Nitrospina, that carry
out the oxidation of nitrite to nitrate. There is increasing evidence that fungi may
also oxidize ammonium, but this is believed to be restricted to acidic forest soils.
Primary nitrification Secondary nitrification

NH 4 + → NO2 − → NO3 −
Nitroso − bacteria Nitro − bacteria
Nitrification does not directly influence the availability of nitrogen to plants, as

most crop plants are able to take up both NH4+ or NO3−, but it does indirectly
influence nitrogen supply to plants because it predisposes the nitrogen to loss from
the soil. Nitrate is an anion so it is poorly held at exchange sites in soils by com-
parison with the cationic NH4+; nitrification therefore predisposes to nitrogen loss
by leaching. As mentioned above, nitrate is the starting point for denitrification, so
nitrification can promote denitrification even though the two processes are pro-
moted by contrasting environmental conditions (denitrification is an anoxic process
whereas nitrification is an oxic process). This can occur because nitrate may be
produced at oxic microsites in the soil and, being mobile, diffuse to anoxic micro-
sites. In addition, it may be produced under oxic conditions by nitrification and be
reduced by denitrification when environmental conditions become anoxic.
The potential to manage nitrification in soils exists through the manipulation of
ammonium as outlined above or by using nitrification inhibitors. However, nitrifi-
cation inhibitors have not been widely adopted because of expense, and the fact that
they are often unreliable because they cannot be always be applied at the precise
sites in the soil where nitrification is occurring.
Microbial Contributions to Phosphorus Cycling in Soil
In developed countries, there has been considerable attention to the cycling of

phosphorus though soil because losses to surface water contribute to aquatic pollu-
tion. However, in terms of sustainable agriculture, attention is turning increasingly
to sources of phosphorus to sustain agriculture because the exploitable phosphorus
rock sources are now seriously depleted and at the current rate of consumption are
likely to be exhausted within decades. This has focussed attention on exploiting
sources of phosphorus in the soil. This includes manipulating the soil-plant system
to solubilize phosphorus from inorganic sources and promoting the mineralization
of phosphorus from organic sources.
Solubilization of inorganic phosphorus by soil microorganisms is the result of
acidification, chelation and exchange reactions. However, there is relatively little
understanding of how these processes actually operate and interact, and which spe-
cific free-living organisms are involved, although both bacteria and fungi are impli-
cated. Mycorrhizal fungi are however, known to promote the solubilisation and
uptake of phosphorus. These are considered in a separate chapter in this book.
Concluding Remarks
At the beginning of this chapter we presented the argument that cropping systems
are inherently unsustainable if one uses an absolute definition because they result
in the export of nutrients from the plant-soil system. However, rather than
consideringsustainable nutrient supply in cropping systems in impossibly absolute
terms, it is more helpful to adopt a more practical stand point and incorporate the
following processes in to nutrient management:
1. Recycling of nutrients in crop residues
2. Using of green manuring crops which fix atmospheric nitrogen
3. Minimising nutrient losses
4. Returning of animal wastes (manures and slurries) to fields
5. Relying on nutrient release by weathering
6. Adding nutrients as atmospheric “pollutants”
Soil microorganisms play important roles in these processes either directly or
indirectly.
Acknowledgements SCRI (Scottish Crop Research Institute, Mylnfield, Dundee) is funded in

part by the Scottish Government’s Rural Environment Research and Analysis Directorate
(RERAD). Rothamsted Research – North Wyke, Devon is funded in part by the UK Biotechnology
and Biological Sciences Research Council (BBSRC). This article is an output of the RERAD
programme on Sustainable and Profitable Agriculture Plants and the BBSRC Cross-Institute pro-
gramme on Sustainable Soil Function. Both funders are gratefully acknowledged.
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Chapter 4
The Role of Microbial Communities
in the Formation and Decomposition
of Soil Organic Matter
Leo Condron, Christine Stark, Maureen O’Callaghan,

Peter Clinton, and Zhiqun Huang
Introduction
Organic matter is mainly present in the top 20–30 cm of most soil profiles and is
essentially an array of organic macromolecules consisting principally of combina-
tions of carbon, oxygen, hydrogen, nitrogen, phosphorus and sulphur. Soil organic
matter is commonly measured as the quantity of organic carbon. The global pool of
organic carbon in soil to a depth of 1 m has been estimated at 1,200–1,550 Pg (2 m:
2,370–2,450 Pg), and as such is significantly greater than either the biological-biota
(560 Pg) or atmospheric (760 Pg) carbon pools (Baldock 2007). Almost all organic
matter in soil is directly and indirectly derived from plants via photosynthesis. Thus
atmospheric carbon dioxide is transformed by reduction into simple and complex
organic carbon compounds, which in combination with key nutrients enable the
plant to function and grow. Carbon dioxide is released directly from plants by respi-
ration, but most of the fixed carbon is retained and ultimately transferred to the soil
ecosystem via a combination of spatially distinct pathways over a variety of times-
cales. The most important pathways are the direct addition of senes-cent material as
L. Condron (*)
Agriculture and Life Sciences, Lincoln University, PO Box 84, Lincoln 7647,
Christchurch, New Zealand
C. Stark
Environmental and Geographical Sciences, Manchester Metropolitan University,
Manchester, UK
M. O’Callaghan
e-mail: Maureen.o’[email protected]
P. Clinton and Z. Huang
Scion, PO Box 29-237, Fendalton, Christchurch 8041, New Zealand
e-mail: [email protected]; [email protected]
82 L. Condron et al.
above-ground and below-ground detritus, return of ingested plant matter in animal

faeces, and exudation of soluble organic compounds from roots (Howarth 2007).
Plant and animal detritus and root exudates represent essential sources of energy
and nutrients for soil microbial and faunal communities. Bacteria and fungi repre-
sent 95%+ of the biomass present in most soils, where they interact with a combina-
tion of micro-fauna (nematodes, protozoa), meso-fauna (acari, Collembola, mites)
and macro-fauna (earthworms, termites, molluscs) in complex soil food-web sys-
tems that determine the turnover of organic matter and associated nutrients in the
soil environment (Wardle 2002; Coleman and Wall 2007). Decomposition of
organic carbon in soil is driven primarily by the activities of bacteria and fungi,
while only 10–15% of soil carbon flux can be directly attributed to the actions of
fauna (Hopkins and Gregorich 2005). The vast majority of soil microorganisms are
heterotrophs that rely on organic matter for energy and nutrients. These can be
divided into microorganisms that respond primarily to the addition of fresh carbon
substrates (zymogenous or r-selected biomass) and those that derive their energy
mainly from the decomposition of older, more recalcitrant forms of organic carbon
(autochthonous or K-selected biomass) (Hopkins and Gregorich 2005).
Soluble plant root exudates account for 10–40% of the total carbon fixed by pho-
tosynthesis and are composed mainly of mixtures of sugars, amino acids, sugar
alcohols, organic acids and secondary metabolites (Bais et al. 2006). Root exudates
are particularly important drivers of microbial and faunal activity in soil due to a
combination of their relatively high bioavailability compared with senescent plant
detritus, their role in controlling the bioavailability of nutrients (e.g., phosphorus)
and phytotoxic elements (e.g., aluminium), together with the fact that they are added
to soil on a regular/semi-continuous basis (Singh and Mukerji 2006; Neumann
2007). Thus microbial numbers and activity in the immediate vicinity of growing
roots (1–3 mm – rhizosphere) are commonly orders of magnitude greater than in
non-rhizosphere soil. Ratios of microbial communities in rhizosphere and adjacent
‘bulk’ soil vary with plant species and environmental conditions, although 12–25-
fold differences in bacterial and fungal populations have been observed (Kennedy
2005). Plants benefit directly from exudate enhanced biological activity in the rhizo-
sphere, mainly via improved acquisition of sparingly soluble and organic soil nutri-
ents mobilised by microorganisms in response to the provision of energy-rich carbon
substrate. This includes the specific symbiotic relationship between plant roots and
mycorrhizal fungi, whereby fungi living in close association with plant root cells
obtain a supply of soluble carbon from the plant (up to 20% of assimilated carbon)
in exchange for improved access to and mobilisation of sparingly-soluble mineral
and organic forms of soil nutrients (Sylvia 2005). This is mainly attributed to the
proliferation of fungal hyphae and associated bacteria into surrounding soil which
markedly increases overall root surface area and the potential depletion zone for key
nutrients (principally phosphorus and nitrogen) compared with non-mycorrhizal
plant roots (Timonen and Marschner 2006; Powell and Klironomos 2007).
In most natural and managed ecosystems up to half of the organic carbon added to
soil on an annual basis in plant detritus and root exudates is rapidly consumed by micro-
bial and faunal activity and released as carbon dioxide (Hopkins and Gregorich 2005).
4 The Role of Microbial Communities in the Formation and Decomposition 83
It has been shown that 64–86% of root exudates are rapidly respired by rhizosphere
microorganisms (Hutsch et al. 2002). However, the remainder of the added organic
matter, together with organic compounds synthesised by soil microorganisms and fauna
during decomposition and released mainly as detritus, persist in the soil for an extended
period. It is important to note that all the organic carbon added to soil is eventually
mineralised and released as carbon dioxide by the combined actions of microorganisms
and fauna (Hopkins and Gregorich 2005; Wolf and Wagner 2005). The process of plant
residue decomposition is comprised of a number of stages, which occur over periods
ranging from days to centuries (Fig. 1).
The quantity, form and spatial and temporal distribution of organic substrate
addition to soil and its subsequent decomposition are primarily influenced by the
nature and productivity of different ecosystems. Ecosystem productivity is deter-
mined by a combination of factors such as climate and soil type, which in turn are
closely linked to human disturbance, including changes in land-use and manage-
ment, together with inputs of nutrients in fertilisers (Baldock 2007; Janzen 2004;
Six et al. 2002). For example, it has been found that biological activity, moisture
content and aggregate stability are greater in soil managed under no-till compared
with conventional till, which in turn results in increased levels of organic carbon in
no-till soils (Frey et al. 1999; Six et al. 2000; White and Rice 2009).
The study of organic matter dynamics in soil is widely acknowledged as being
extremely challenging given the diversity of biological, chemical and physical
properties and processes involved. An often-bewildering array of methods has
been developed to define different pools of soil organic matter (Baldock 2007;
Hopkins and Gregorich 2005). These include separating living from non-living
organic matter components, which can then be divided into more specific fractions
based on chemical (alkali-acid solubility), physical (size fractions), and kinetic-
functional (susceptibility to decomposition) variables (Fig. 2). The non-living soil
organic matter can be divided into a number of fractions based on a combination
of physical size and chemical form. Identifiable plant and faunal detritus ranging
in size from over 2 mm down to 50 mm can be physically separated from soil by
sieving and density floatation, although these fractions are often not considered
part of soil organic matter (Hopkins and Gregorich 2005). The remainder of the
non-living soil organic matter is comprised of organic macromolecules collectively
known as ‘humus’. Humus can be further divided into two broad categories of
compound, namely ‘non-humic substances’ (ca. 30%) and ‘humic substances’ (ca.
70%). Non-humic substances are defined as being chemically identifiable plant,
microbial, and faunal constituents, which includes nucleic acids, peptides and
amino acids, sugars and polysaccharides, lipids and lignin. On the other hand,
humic substance is a collective term for unidentified polydispersed organic mac-
romolecules that are produced during decomposition of organic substrates in the
soil environment. Humic substances in soil are characterised according to extract-
ability and subsequent solubility as fulvic acid (alkali extractable – acid soluble),
humic acid (alkali extractable – acid insoluble), and humin (non-extractable)
(Stevenson 1994). Non-humic substances are generally regarded as being more
readily decomposed in the soil environment compared with humic substances.
Fig. 1 The stages of crop residue decomposition in soil (changing box sizes indicate the relative
quantities of carbon in the various fractions as decomposition proceeds to completion) (Wolf and
Wagner 2005) (Reproduced with permission of Pearson Prentice Hall, USA)
The techniques described above have been used extensively over many years to
investigate the processes involved in the formation and decomposition of organic
matter in soil, with an emphasis on elucidating the chemical nature and synthesis of
non-humic and humic substances (Baldock 2007; Howarth 2007; Hopkins and
Gregorich 2005; Six et al. 2002; Stevenson 1985, 1994). There have been many
mechanisms and pathways proposed which encompass the biological-biochemical and
4
The Role of Microbial Communities in the Formation and Decomposition
Fig. 2 Summary of soil organic matter fractions (This figure was published in Biological Diversity and Function in Soils (Editors RD Bardgett, MB Usher,
DW Hopkins), 2005, Chapter 4 – Carbon as substrate for soil organisms, DW Hopkins and EG Gregorich, p. 60, copyright Cambridge University Press –
85
reprinted by permission of the publisher)

physicochemical processes responsible for organic matter dynamics in soil. The most
commonly hypothesized mechanisms for the formation of humic substances in soil are
based around condensation of cellular metabolites, organic nitrogen compounds
(amino acids, amino sugars), and lignin and phenolic substrates of microbial origin,
which in turn are stabilised by a combination of chemical interaction with mineral
colloids and physical incorporation into soil aggregates (Fig. 3). The presence and/or
addition of relatively inert charcoal carbon may also influence the stability and turn-
over of organic carbon in soil due to its effect on microbial activity and nutrient avail-
ability (Hopkins and Gregorich 2005; Lehmann 2007; Wardle et al. 2008).
The ongoing formation and decomposition of organic matter in soil plays a
major role in determining plant and ecosystem productivity, via a combination of
its role in the storage and provision of nutrients and water, together with the devel-
opment and maintenance of physical structure. In most soils over 90% of total
nitrogen and sulphur, together with over 50% of total phosphorus, are associated
with the microbial biomass and organic matter, and so the cycling and bioavail-
ability of these key nutrients in soil are primarily controlled by organic matter
Fig. 3 Hypothesized mechanism for formation and stabilisation of humic substances in soil (This
figure was published in Soil Microbiology, Ecology, and Biochemistry, Third edition (Editor EA
Paul), 2007, Chapter 12 – Carbon cycling and formation of soil organic matter, W Horwath, p. 330,
copyright Elsevier – reprinted by permission of the publisher)
transformations linked to microbial and faunal activity (McNeill and Unkovich

2007; Bünemann and Condron 2007). Soil organic matter is the major source
of negative charge in most soils and therefore determines the retention
and bioavailability of nutrient cations such as calcium, potassium, magnesium and
ammonium. Organic matter also plays a vital role in the retention and availability
of water in soil, and the association of soil organic matter with secondary minerals
such as clay to form aggregates underpins soil structure, promoting aeration and
water infiltrationnecessary for plant growth. In addition, the formation and decom-
position of soil organic matter regulates production of carbon dioxide and nitrous
oxide and consumption of methane (Howarth 2007). As soil organic matter may act
as both a sink and source of carbon during global environmental change (Johnston
et al. 2009), effective management of soil organic matter has become a key objective
of research aimed at protecting the environment.
The remainder of this chapter will focus on description and discussion of
selected key topics that relate to the role and function of microbial communities in
the dynamics of organic matter in the soil environment, including microbial diver-
sity, microbial-faunal interactions, the influence on nutrients on organic matter
mineralization, and the microbial origin of organic matter.
Importance of Soil Microbial Community Composition

and Diversity
The ability of soils to provide long-term carbon sequestration and the nutrients neces-
sary for plant productivity are largely dependent on soil organic matter dynamics
(Billings and Ziegler 2005). Thus, in the view of increasing sustainability of agricultural
production systems, soil organic matter, which is at once part and driver of ecosystem
processes such as primary productivity (= the total sum of organic matter production
through assimilation of atmospheric carbon dioxide primarily by photosynthesis) and
decomposition (= breakdown of soil inherent or added organic matter) demands to be
continuously maintained and improved. Carbon accumulates in soils when productivity,
i.e., the input of carbon substrates, exceeds decomposition, which leads to the build up
of organic matter. In turn, decomposition entails a reduction in the soil carbon stock.
Typically, the decomposition of inherent soil or added organic materials releases essen-
tial nutrients for uptake by plant and soil communities, whereas decomposer organisms,
mainly bacteria and fungi, use carbon sources that are supplied mainly by primary
producers, like plants. Soil organic matter cycling is therefore closely linked to the
decomposer activity, which mineralise organic compounds and make essential nutrients
plant available. It is therefore reasonable to assume that the abundance, composition
and/or diversity of microorganisms in soils matter to ecosystem functioning.
Although changes in microbial community composition are increasingly seen as
the driving factor behind functional capabilities in soils, there is only limited direct and
specific evidence with regard to the relevance of microbial diversity in soil processes
(Schimel and Gulledge 1998; Griffiths et al. 2000, 2001; Nannipieri et al. 2003).
This is especially true with regard to ecosystem processes related to the carbon
cycle, such as decomposition and carbon sequestration. It is widely believed that
these processes are too universal to be affected by changes in microbial diversity
and that in functionally very diverse communities these processes are taken over by
other members of the community if a microbial group is lost (cf. functional redun-
dancy hypothesis) (Nannipieri et al. 2003). Moreover, microbial community com-
position, diversity and abundance are often deemed entirely unimportant to
ecosystem processes and are generally not considered in ecosystem models, esp. in
terrestrial environments. Most models, including CENTURY (Parton et al. 1987)
and TEM (terrestrial ecosystem model) (McGuire et al. 1993), work on the assump-
tion that changes within the microbial community only have limited effects on soil
processes. This idea only holds true if the microbial community in question is
resistant1, resilient2 and/or functionally redundant3 (Allison and Martiny 2008). In
their review, Allison and Martiny (2008) demonstrate the increasing recognition of
the importance of microbial community composition to ecosystem processes. The
authors note that the composition of soil microbial communities is generally not
resistant to change as it is often sensitive to perturbations, including climate change
scenarios such as increases in temperature and carbon dioxide, or agricultural
management, such as mineral fertilisation or organic matter amendments. They
also suggest that over the medium term time-scale of a few years disturbed com-
munities still differ in composition from undisturbed ones. While the degree of
functional redundancy is not easily quantified due to methodological and experi-
mental difficulties, the authors conclude that disturbance induced changes to
microbial composition often persist over time and have an effect on ecosystem
process rates (also cf. Griffiths et al. 2000; Ekschmitt and Griffiths 1998; Balser
and Firestone 2005; Wertz et al. 2006). This suggests that at least some groups
within soil microbial communities are functionally dissimilar and not functionally
redundant.
Links and interactions of above-ground (i.e., plant) diversity with primary produc-
tivity and their impact on below-ground communities have been studied extensively
and are firmly established (Schulze and Mooney 1993; Loreau et al. 2001; Carney
and Matson 2005; Catovsky et al. 2002; McGrady-Steed et al. 1997). Carbon avail-
ability is a key determinant of microbial growth and activity in soils, which links them
closely to primary production, rhizosphere activity and litter substrate quality (Smith
and Paul 1990). As root exudation and rhizodeposition vary in amount, quality and
quantity with plant species type and abundance as well as in space and time, plant
identity and plant community composition have significant bearing on the composi-
tion, diversity and activity of soil microbial communities, which affects the rate of
1
Resistance, the degree to which microbial composition remains unchanged following
disturbance.
2
Resilience, the rate at which microbial composition returns to its original composition after the
disturbance.
3
Functional redundancy, the ability of one microbial group to carry out a process at the same rate
as another under the same environmental conditions (according to Allison and Martiny 2008).
decomposition of the added carbon compounds (Catovsky et al. 2002; Marschner

et al. 2001; Johnson et al. 2003; Brant et al. 2006; Hamilton and Frank 2001; Grayston
et al. 1996). There is evidence that through rhizodeposition plants actively select for
microbial communities to promote the supply of limited soil resources (Hamilton and
Frank 2001). Similarly, a microbial community with a specific composition and
functional diversity might select for a plant community that ensures the supply of
favoured carbon compounds (Rillig 2004; Wardle 2005). Generally, more diverse
plant communities exhibit greater primary productivity, resulting in higher carbon
assimilation from the atmosphere. This adds to soil carbon storage, nutrient retention
and energy turnover in soils, resulting from higher microbial diversity, and leading,
yet again, to changes in the quality and quantity of rhizodeposition and stimulates
decomposition (Dang et al. 2005; Broughton and Gross 2000; Ekschmitt et al. 2001).
This clearly shows how primary productivity is not only directly affected by plant-
related factors, but equally stimulated from below-ground, by decomposition rates,
root processes, microbial community composition and soil organic matter content
(Bausenwein et al. 2008; Tiunov and Scheu 2005).
Although no less crucial in ecosystem functioning, the complementary process in
the carbon cycle – the decomposition or mineralisation of organic matter – and its
interactions with soil microbial diversity have received significantly less attention.
Decomposition is a mostly microbially mediated process, although its actual rate
and extend are influenced by environmental variables, including soil temperature,
moisture, oxygen, nitrogen content, the quality and quantity of available carbon
substrates as well as soil management (physical disturbance, for example, might
expose previously protected organic matter) and presence/absence of microbivorous
soil fauna (Janzen 2004; Johnston et al. 2009; Swift et al. 1979). The implications
soil microbial species composition and diversity have on carbon cycle related eco-
system processes are not entirely clear, and determining microbial diversity effects
on soil processes while controlling for other environmental parameters is a huge
challenge, not least as a result of the interactions between microbial communities,
environment variables and ecosystem processes (Reed and Martiny 2007) (Fig. 4).
It is necessary to consider various levels of diversity, which might be differently
affected by, or have different effects on, decomposition rates. We differentiate
between species or microbial abundance (total number of individuals of a species or
total size of the microbial biomass, respectively), species richness (total number of
species), species evenness (relative number of species), species identity or commu-
nity composition (types of species/taxa present) and species or ecosystem diversity,
which accounts for number as well as frequency of species. For example, changes in
microbial abundance have been identified to be more important to soil carbon
mineralisation than differences in microbial community composition (Carney and
Matson 2005). A basic, positive relationship can be observed between organic mat-
ter quantity and microbial biomass size. Higher soil organic matter content is gener-
ally associated with a larger microbial biomass, while organic matter amendments
lead to increases in biomass (e.g., Nannipieri et al. 2003; Plassart et al. 2008; Bastida
et al. 2008; Gunapala and Scow 1998). Similarly, the differential effects of substrate
quantity and quality on the size and composition of soil microbial communities need
Fig. 4 Interactions and feedback effects of below- and above-ground communities, environmental
conditions and soil carbon cycling
to be emphasised. Generally, carbon compounds of higher quality are characterised

by a lower carbon-to-nitrogen ratio, which makes the substrate more readily avail-
able to decomposer organisms, in particular bacteria, while low quality substrates
with high carbon-to-nitrogen ratio are preferentially used by fungi. Higher carbon
substrate quality, i.e., more readily available and easily decomposable organic
matter, has been found to lead to an increase in microbial biomass size whereas
functional diversity decreased when studying the decomposition of different plant
leaf components(Webster et al. 2000; Paterson et al. 2007) and increases in organic
matter amendment had little influence on the diversity of rhizosphere communities
(Ros et al. 2006). In contrast, Kubartová et al. (2007) showed that differences in
carbon substrate supply resulting from changes in plant community composition
(i.e., a change in substrate quality as well as quantity) impacted on microbial com-
munity composition while microbial abundance remained uninfluenced. More
importantly, positive links have been established between microbial biomass and
carbon mineralisation rates (Garcia-Pausas et al. 2008) (also cf. Fig. 4).
Due to experimental and methodological limitations, the effects of species rich-
ness and species diversity are often blurred and difficult to distinguish from each
other. Generally richness effects on organic matter processes are less unequivocal
than those of microbial abundance or species identity and composition of decom-
posers (Tiunov and Scheu 2005), and most current evidence suggests that decom-
poser community composition is of greater importance on ecosystem processes
than species richness or diversity. As species richness is closely correlated with
substrate quality of organic compounds, it is feasible that a community higher in

species richness is also more diverse with regard to its decomposition capabilities
(Wardle 2005; Bausenwein et al. 2008; Cookson et al. 2005; Orwin et al. 2006).
Most research findings, however, suggest that decomposition activity is unaffected
by increases, or decreases, in species richness (Nannipieri et al. 2003; Ekschmitt
and Griffiths 1998; Wardle 2005; Ekschmitt et al. 2001) and it has been observed
that microbial communities of different size or composition have similar or even
identical carbon utilisation patterns (Buyer and Drinkwater 1997; Waldrop et al.
2000; Kemmitt et al. 2008; Chander et al. 2002). For example, mixtures of two to
eight fungal species performed similar to monocultures when assessing respiration
following wheat straw addition (Hedlund and Öhrn 2000), leaf litter mass loss
(Dang et al. 2005; Duarte et al. 2006) or oxygen consumption (Tiunov and Scheu
2005) (Table 1). This lack of effect was often independent of variation in environ-
mental conditions (e.g., Dang et al. 2005). Studying overall microbial diversity and
its effects on soil processes before and after fumigation, Griffiths et al. (2000)
found that a reduction in microbial diversity in some cases increased decomposition
rates of plant residues. Similarly, Degens (1998) observed higher straw decomposi-
tion rates in soils with reduced microbial functional diversity.
Contrary to these conclusions, some positive links between species richness/
diversity and measures of decomposition have also been observed. Higher diversity
of saprotrophic fungi has been shown to result in an increase in decomposition rates
when assessing mass leaf mass loss (Bärlocher and Corkum 2003) or respiration
(Robinson et al. 1993; Setälä and McLean 2004). Similarly, Tiunov and Scheu
(2005) found that the rate of organic matter decomposition was positively associated
with fungal species richness in sterile forest soils and even more so in a single-
resource substrate (powdered cellulose). While competitive interactions can limit as
well as increase decomposition activity (Jiang et al. 2008; Hättenschwiler et al. 2005),
impacts of species richness and diversity on decomposition rates are generally
believed to be more pronounced in species poor communities as species rich
environments are characterised by overlaps of functional niches and higher func-
tional redundancy (Ekschmitt et al. 2001; Tiunov and Scheu 2005; Setälä and
McLean 2004).
Although biodiversity per se seems to be unaffected by decomposition, there are
clear differences in decomposition patterns when the community shifts from fungal
to bacterial dominated and vice versa depending on the age and complexity of the
carbon compound (Wardle 2005; Six et al. 2006; Paterson et al. 2008a). Bacteria
are generally more dominant in fertile soils that receive more labile organic matter
with a low carbon-to-nitrogen ratio, under and fast growing plants with high leaf
litter quality and in tilled soils that are characterised by the incorporation of resi-
dues into the soil profile (Wardle 2005; Scow 1997). In general, early stages of
decomposition (14–28 days after substrate addition) are governed by bacterial
activity (Bastian et al. 2009). Fungi, on the other hand, prefer more recalcitrant
residues with a higher carbon-to-nitrogen ratio and dominate the later stages of
decomposition (from 56 to 168 days) (Bastian et al. 2009). They are dominant in
infertile soils that show slower plant growth and often low leaf litter quality known
92
Table 1 Relationship between fungal or bacterial decomposer diversity and organic matter decomposition based on experimental studies using single
(cellulose) or complex (several compounds) carbon sources. Janzen et al. (1995) reported experiments with a neutral and a negative relationship, respectively.
Table based on Jiang et al. (2008)
Decomposer type Number of species tested Substrate type added Process measured Process-diversity relationship Source
Fungi 1–2 species Cellulose Cellulose mass Positive Deacon 1985
loss
Fungi 1–5 species Cellulose O2 consumption Strong positive Tiunov and Scheu 2005
Bacteria 1–8 species Cellulose Cellulose mass loss Positive Wohl et al. 2004
Bacteria 1–72 species Leaf disk CO2 production Positive Bell et al. 2005
Fungi 1–5 species Oak leaf Leaf mass loss Positive Bärlocher and Corkum
2003
Fungi 1–2 species Wheat straw CO2 production Positive Robinson et al. 1993
Fungi 1–43 species Soil CO2 production Positive Setälä and McLean 2004
Fungi 1–5 species Soil O2 consumption Weak positive Tiunov and Scheu 2005
Fungi 1–2 species Alder leaf Leaf mass loss Positive Treton et al. 2004
Fungi 1–8 species Alder leaf Leaf mass loss Neutral Dang et al. 2005
Fungi 1–4 species Leaf Leaf mass loss Neutral Duarte et al. 2006
Fungi 1–3 species Soil + wheat straw CO2 production Neutral Hedlund and Öhrn 2000
Bacteria 1–4 species Wheat seed Wheat seed mass loss Neutral Jiang 2007
Fungi 1–2 species Barley straw CO2 production Neutral/negative Janzen et al. 1995
Fungi 1–16 species Wood Wood mass loss Negative Toljander et al. 2006
L. Condron et al.
to better utilise carbon sources with patchy distribution patterns, as is the case in
no-till systems, where undecomposed residues are left on the soil surface.
Consequently, more fungi are found in surface soils, while bacteria dominate in
subsoils. Fungi thus mediate the translocation of nutrients between the surface and
the underlying soil profile (Hendrix et al. 1986) and have a higher carbon storage
capacity compared to bacteria.
The relationship between microbial diversity and decomposition rates is
ambiguous at best. Many experimental studies have demonstrated that whenever
diversity effects on the speed and extent of decomposition exist they are highly
context specific and other, external, factors have proven to be equally, if not more
important in governing soil processes or driving changes in microbial community
composition (Loreau et al. 2001; Wardle 2005). The influencing factors range
from environmental conditions, such as precipitation, nutrient availability or eco-
system properties (Loreau et al. 2001; Bärlocher and Corkum 2003; Schwartz
et al. 2007), to seasonal effects (Kubartová et al. 2007) and experimental treat-
ment (Jiang et al. 2008). According to Fierer et al. (2005), the physiological
response observed in their study determining litter quality and temperature effects
on decomposition was more strongly related to changes in temperature than in
community composition. This effect increased with decreasing litter quality. Jiang
et al. (2008) identified distinct differences in relationships between decomposer
diversity and decomposition activity depending on the type and diversity of carbon
sources used. They found primarily positive links in experiments using a single
carbon source and more diverse links with relationships ranging from positive to
neutral to negative in experiments using multiple carbon sources (Table 1).
Moreover, specific traits of certain species rather than species number have been
identified to exert greater influence on ecosystem functions and processes (Duarte
et al. 2006; Jiang et al. 2008).
It is easy to see that interactions of above- with below-ground communities and
the relationships between soil biodiversity and ecosystem processes are complex and
diverse. A wide range of positive and negative feedback loops exists (Fig. 4). This
complicates the interpretation of results and makes it difficult to test and understand
cause-effect relationships, which makes it a “chicken and egg” situation. Whether
higher biodiversity maintains soil functionality and sustainability or whether a health-
ier, more functional soil environment supports a more diverse microbial community
cannot unambiguously be determined. Nonetheless, the very existence of feedbacks
and co-dependencies strongly suggests that decomposer biodiversity is of similar
significance to ecosystem processes as plant diversity. Overall, species diversity
effects are most likely to occur at local scales and are greatest at low to intermediate
diversity levels. That in many experimental situations microbial diversity changes
are not reflected in changes in ecosystem process rates might be explained by the
phenomenon of functional redundancy, whereby different species may takeover
functions of lost microbial groups and a loss in diversity is thus compensated for.
Alternatively, it is feasible that a microbial community expresses different function-
ality following perturbation, while its activity results in the same, pre-disturbance
process rate, i.e., soil function and microbial activity do not closely correlate.
The exact nature and extent of the relationship between microbial diversity
and carbon turnover remains opaque. It is obvious that microbial diversity and/
or community composition have some effect on soil functionality, be it by chang-
ing ecosystem processes directly, or via interactions with above-ground com-
munities, which impact on the availability of carbon and other essential nutrients
and affect microbial metabolic activity. Improved knowledge on microbial com-
munity composition and function and their links with nutrient turnover will
help achieve understanding of decomposition and other nutrient cycles at the
process-level, and possibly pave the way to a more accurate and more sustain-
able management of agroecosystems.
Interactions Between Microorganisms and Soil Fauna
The role of microorganisms in formation and decomposition of soil organic matter

cannot be discussed without reference to the interactions of microorganisms with the
soil micro- and macrofauna. A diverse range of biota is present in soil, including
micro-fauna (e.g., nematodes and protozoa), meso-fauna (e.g., Collembola, mites and
acari) and macro-fauna (e.g., earthworms, molluscs and termites). The impact of inver-
tebrates on soil organic matter turnover has been studied since Darwin’s pioneering
work (Darwin 1881). However, despite the importance of soil fauna in organic matter
breakdown being well recognised, the complex interactions between soil fauna and
microorganisms and the indirect effect on microfauna and microbial communities are
less well understood. While soil invertebrates mediate about 15% of the carbon and
30% of the nitrogen turnover in a range of ecosystems (Anderson 1995), their indirect
effect through activation of microflora is likely to be much greater. Microorganisms
are the main agents of biochemical decomposition and turnover of organic matter but
soil fauna enhance the fragmentation of coarse particulate matter into finer fractions
and influence the distribution of organic matter in soil. Through comminution of
organic matter, which increases the surface area available for attack by microbial
decomposers, soil fauna directly stimulate microbial populations and activity.
The interactions between soil microflora and fauna have been most well studied
in earthworms. However, other groups of soil-inhabiting invertebrates undoubtedly
play a significant role in formation and turnover of organic matter, in particular
mesoarthropods such as mites and Collembola. The extent to which these various
groups of soil fauna control decomposition may depend on the ecosystem. For
example, microarthropods that feed on fungi, especially Collembola, can play a key
role in no-tillage agroecosystems, but are less important in conventionally tilled
systems (Hendrix et al. 1986). Few studies have been carried out but it seems likely
that, like the earthworms, microarthropods can play key roles in influencing the
activities of microorganisms in organic matter processing in soil (Edwards 2000).
Although not numerically dominant, earthworms are major contributors to
invertebrate biomass in soil because of their size. Earthworms are divided into
three groups based on their feeding preferences and habitat. Epigeic earthworm
species may feed directly on microorganisms or litter material, and dwell in the
organic layer of soil. They have been shown to strongly affect decomposition
processes (Sampedro and Dominguez 2008) and have been demonstrated to modify
the fungal composition of forest soils (McLean and Parkinson 2000). Endogeic
earthworms feed their way horizontally through the upper mineral layer of soil
while aneic earthworms generally inhabit one single vertical burrow and can trans-
port fresh organic detritus from the soil surface into burrows while mixing it with
mineral soil. It is clear that all detritivorous earthworms can accelerate organic
matter decomposition and nutrient release in soil. They exert a controlling activity
through their strong interactions with microorganisms in the decomposition pro-
cess; they strongly modify the physical, biochemical and biological properties of
the substrates in which they live, including the structure and function of soil micro-
bial communities.
The interactions between earthworms and microorganisms occur on several spatial
scales but most research has been directed at microscale interactions which occur in
the drilosphere (Brown and Doube 2004). The “drilosphere”, as defined by Brown
et al. (2000), includes the internal microenvironment of the earthworm gut; the earth-
worm surface in contact with the soil; surface and belowground casts; middens; and
burrows and chambers made by the earthworm, all of which are microhabitats for a
range of bacteria and fungi. Up to 60% of the carbon losses from earthworms during
their life span can be in the form of mucous secretions, and this soluble organic carbon
is an important microbial stimulant in the drilosphere (Brown and Doube 2004).
Different species and ecological categories of earthworms differ in their ability
to digest various organic residues and assimilate nutrients from ingested organic
matter (Lattaud et al. 1998, 1999). Many earthworm species consume a mixture of
soil and organic matter, often choosing to feed in patches of soil that are relatively
rich in organic matter, or microsites in soil that are enriched with bacteria and fungi
(Wolter and Scheu 1999). Selective feeding on these sites may provide earthworms
with additional soluble carbon sources such as those derived from microbial metab-
olism. Microorganisms themselves may be a source of food for earthworms but the
amounts consumed and the ability of earthworms to digest and assimilate microbial
biomass varies with earthworm species (Brown and Doube 2004). Based on limited
data, it seems likely that at least some earthworm species have an indigenous,
autochthonous gut flora. Lavelle and Gilot (1994) showed that several temperate
earthworm species had a mutualistic digestive system in which the mixture of solu-
ble organic carbon in the form of low molecular weight mucus with ingested organic
matter, together with the moist conditions and neutral pH in the foregut, promoted
the development of a microbial community that could digest cellulose and other
substances that earthworms cannot typically digest. Essentially, the earthworm gut
can act like a bioreactor where microbial activity and biomass are increased due to
favourable conditions with readily available carbon of mucus and water.
All earthworms have high rates of consumption of soil and/or litter but their
assimilation efficiencies vary widely depending on ecological grouping – 1% of
ingested carbon for endogeic species Aporrectodea rosea through to 30–75%
assimilation efficiencies in litter feeding species such as Lumbricus rubellus or
L. terrestris (Brown and Doube 2004). Hence, earthworm casts may contain large
amounts of organic matter that has not been assimilated but has been modified both
physically and chemically during passage through the earthworm gut.

Microorganisms that can survive passage through the earthworm gut (mainly fungal
and protozoan spores and some resistant bacteria) provide inocula for microbial
colonisation of newly formed earthworm casts (Brown 1995). These newly depos-
ited casts are usually rich in ammonium-nitrogen and partially digested organic matter,
providing a good substrate for growth of microorganisms. There is evidence to suggest
that microorganisms may become activated or “primed” in the gut because of the
abundance of soluble carbon and other nutrient resources (Lavelle and Gilot 1994)
and this effect may also continue for a short time in the casts. It is well established
that there are larger populations of fungi, bacteria and actinomycetes (Shaw and
Pawluk 1986), and higher enzymatic activity in earthworm casts than in bulk soil.
Higher proportions of cellulolytic, hemicellulolytic, nitrifying and denitrifying bac-
teria (Edwards et al. 2004) and larger, more diverse fungal populations have been
recovered from casts than surrounding soil (Tiwari and Mishra 1993). Similarly, the
walls of earthworm burrows typically support more diverse and larger microbial
populations than surrounding uningested soil (Sampedro and Whalon 2007).
Given that increased microbial biomass, activity and diversity is often measured
in earthworm casts, higher rates of organic matter decomposition might also be
expected. However, balanced against the short term priming effects is the protection
of the organic matter from microbial attack in compact, water stable castings of
endogeic species, which may lower the organic matter decomposition rate
(Marinissen and Hillenaar 1997). In addition, earthworms assimilate the more labile
soil organic matter fractions preferentially, so that the more recalcitrant organic mat-
ter, with slower mineralisation kinetics, remains in the casts. However, it appears that
in the drilosphere at least, the earthworm’s priming of microorganisms leads to
increased mineralization of nutrients. Some studies have demonstrated a short-term
increase in carbon turnover in earthworm casts. For example, Coq et al. (2007)
showed that casts extracted from mesocosms containing earthworms were slightly
enriched in carbon and showed significantly higher mineralisation than soil which
had not been ingested by earthworms, indicating that the carbon contained in the
casts was not protected against mineralisation. Aira et al. (2008) characterized
changes in the microfauna, microflora and biochemical properties of an organic
substrate over a short (72 h) exposure to four densities of the epigeic earthworm
Eisinia fetida. Carbon and nitrogen mineralisation increased with increasing earth-
worm density, as did microbial metabolic activity. The presence of earthworms
enhanced the fungal populations and reduced the numbers of bacterivorous
nematodes. The authors suggested that rapid changes in microflora and microfaunal
communities and subsequent alterations in the decomposition rate of organic matter
can be attributed to earthworm gut-associated processes.
The role of earthworms in soil carbon dynamics has studied in more detail in
recent studies. Aneic earthworms, which inhabit a single vertical burrow for their
lifespan, transport fresh organic detritus from the soil surface deep into their bur-
rows while mixing it with mineral soil. Don et al. (2008) used measurement of
enzyme activity, stable isotopes, carbon form (nuclear magnetic resonance spec-
troscopy) and carbon-14 ageing of burrow linings to test the hypothesis that persistent
burrow structures provide space for additional carbon in soils and that the carbon
turnover in the burrows should be decreased, due to physical stabilisation of
organic carbon. They found that carbon stocks increased rapidly in burrow linings,
but accumulated carbon could be mineralised rapidly, with turnover times of 3–5
years. Carbon stocks depended on earthworm activity within the burrow to main-
tain continuous carbon input. They concluded that earthworm activity does not
substantially increase subsoil carbon stocks but the burrows facilitated movement
of carbon into deep soil horizons.
Links between soil biota (in particular earthworms), soil aggregate dynamics,
and soil organic matter decomposition and stabilisation are well-established (Six
et al. 2004), although the precise mechanisms responsible are not fully understood.
Earthworms are thought to enhance aggregation by stimulating formation of
organo-mineral complexes through the fragmentation of organic residues and min-
eral material within the gut. Soil organic matter stored within aggregates often
represents the vast majority of carbon within soils (Fonte et al. 2009). Aggregates
are thought to protect soil organic matter physically by rendering it inaccessible to
further decomposition. Differences in the binding mechanisms between aggre-
gates of different sizes results in varying levels of aggregate, and hence soil
organic matter, stability. Macroaggregates (>250 µm) are formed around fresh
organic matter, using binding agents such as polysaccharides. More stable micro-
aggregates (50–250 µm), consisting of mineral particles bound together by persis-
tent binding agents such as complexes of clay and organic matter, are found within
the macroaggregates. Microaggregates are thought to decrease the rate of soil
organic matter turnover and protect carbon more effectively than macroaggregates
(Fonte et al. 2009). By tracing carbon-13 labelled plant residue during formation
of aggregates in soil with and without earthworms, Bossuyt et al. (2004) showed
that earthworms helped to form large macroaggregates, which contained four
times more stable microaggregates than those recovered from samples without
earthworms. Larger amounts of organic matter were found within stable microag-
gregates in casts than in bulk soil after just 12 days, indicating these microaggre-
gates are formed rapidly around freshly incorporated residues in casts.
While soil microorganisms are thought to be central to earthworm-facilitated
aggregate formation, less is known about mechanisms by which earthworms drive
microbial community structure and soil aggregation. As discussed in the previous
section, soil organic matter decomposition rates can be strongly affected by the
microbial community structure. It is now understood that bacteria are not randomly
distributed throughout soil; there are variations in biomass and differential coloni-
sation among different sizes of aggregates (Nunan et al. 2003; Schutter and Dick
2002). Recent studies have begun to examine the interactions between aggregate
dynamics, differentiated microbial communities and activity and soil organic matter
turnover (Noguez et al. 2008).
Differences in the distribution of bacterial species in various aggregate sizes
have been revealed using molecular methods. For example, Mummey et al. (2006)
used 16S rRNA gene-based terminal restriction fragment polymorphism (T-RFLP)
analyses to examine bacterial communities associated with different aggregate size
fractions in earthworm-worked soil relative to soil receiving only plant litter.

Earthworms altered the bacterial community composition in all soil fractions analy-
sed, with the greater changes seen in the macroaggregate than in the microaggre
gate communities. It appears that after earthworm ingestion, microbial substrates
may rapidly become limited in microaggregates. For example, Bossuyt et al. (2005)
found that activities of Aporrectodea caliginosa resulted in newly added plant
residue carbon being protected in microaggregates, but there was almost no
protection of recently added carbon in macroaggregates, where it was available for
microbial decomposition and proliferation of the bacterial community.
The extent to which microbial activity and diversity vary between different
soil aggregates and fractions appears to differ with soil type and management
practice. For example, Kim et al. (2008) found that bacterial community structure
and species composition for inner and outer-aggregate fractions of microaggre-
gates of a desert agricultural soil were nearly identical; they suggested that much
of the organic matter present was unprotected by association with clay surfaces
within microaggregates. Thus, sandy desert agricultural soils with low organic
matter content and high aggregate turnover are less likely to manifest distinct
microbial communities in various soil fractions than is seen in soil types containing
more stable aggregates.
Improved understanding of the complex linkages between soil organic matter
turnover, microbial activity in soil aggregates, and soil biota is slowly developing.
Progress in this area will require greater use of developing molecular techniques to
characterise not just microbial diversity but also function and activity of microbial
communities, including expression of genes involved in organic matter turnover.
Coupled with these studies, further chemical analysis of soil organic matter fractions
in micro- and macroaggregates across a range of soil types and in response in biota
other than earthworms is warranted.
Impacts of Nutrient Inputs on Microbial Mineralisation
Over the last half century, human activity has dramatically altered the global nutrient
cycle, and many ecosystems are undergoing increases in the input of anthropogenically
derived nitrogen and phosphorus (Galloway et al. 2004; Vitousek 2004). One major
consequence of increasing nutrient input into terrestrial ecosystems is associated
changes in ecosystem functioning because ecosystem processes, such as decomposition
of soil organic matter, likely depend on the soil nutrient concentrations (Vitousek 2004).
Soils in terrestrial ecosystems are major carbon sinks. This pool of organic carbon is of
particular interest because even small changes in flux rates into or out of pools of soil
organic matter could have a strong influence on atmospheric carbon dioxide concentra-
tions and associated climate change. The impacts of nutrient additions on the decompo-
sition of soil organic matter continue to receive attention, but reports on changes in
decomposition due to nutrient additions often lead to divergent conclusions, making it
difficult to formulate a generalised model about the lasting effects of continued nutrient
input on the decomposition of soil organic matter. Thus, the intention of this section is
to synthesize present literature and to extract a possible common pattern describing the
impact of nutrient additions on microbial mineralization of soil organic matter.
Microorganism-Nutrient Relationships
Microorganisms control the decomposition of soil organic matter, and it is conceiv-

able that the biomass of fungi, bacteria and/or the microbial community composition
would change following continued nutrient additions (Vitousek and Howarth 1991)
(Fig. 4). For example, nitrogen additions may decrease soil pH leading to the leaching
of micro-nutrients and organic carbon (Vitousek et al. 1997). Accordingly, microor-
ganisms may become micronutrient- or carbon limited (Vitousek and Howarth 1991;
Fontaine et al. 2007). Other mechanisms, which could elicit indirect effects of nutri-
ent additions on soil microorganisms, include the decrease in soil carbon to nutrient
ratios and an increase in melanoidins. The increase in melanoidins has been reported
to accentuate carbon limitation of soil microorganisms (Fog 1988). Treseder (2008)
synthesized responses of microbial biomass to nitrogen additions from 82 published
field studies and found that microbial biomass decreased by an average of 15% under
nitrogen fertilization. The extent of the decline depended on the loads and duration of
nitrogen fertilisation as well as on the ecosystem itself. In addition to changes in
biomass, a shift in soil microbial community composition from fungal to bacterial
dominance has been observed under nitrogen addition resulting from decreased
carbon-to-nitrogen ratio (Moore et al. 2003). Bacterial community composition
changed in the nitrogen fertilized soil, with increases in the relative abundance of
sequences related to the bacteroidetes and gemmatimonadetes, and decreases in the
relative abundance of the verrucomicrobia (Nemergut et al. 2008). Decreases in
abundance and diversity of soil archaea have also been reported in response to nitro-
gen amendments (He et al. 2007; Gattinger et al. 2007). Detailed reviews about
microorganism-nitrogen relationships can also be found elsewhere (Treseder 2008).
Relative to nitrogen, the influence of phosphorus on microbial populations has been
less extensively studied. In most studies, phosphorus fertilizer was applied in combina-
tion with nitrogen fertilizer or other nutrients (Cruz et al. 2009; He et al. 2008), which
may obscure the phosphorus effect. In spite of this, studies imply that phosphorus
additions could increase soil microbial biomass (Chu et al. 2007; Zhong and Cai 2007;
Bünemann et al. 2004). Soil bacteria and actinomyces might have more evident
responses to phosphorus fertilizer than fungal communities (Zhong and Cai 2007).
Mechanisms for Nutrient Effect on the Decomposition

of Soil Organic Matter
Stoichiometric decomposition theory: Conceptual and analytical models developed

from a long history of research suggest that decomposition is driven by the stoichi-
ometry of substrates and microbial demands for resources, with maximal rates of
decomposition observed when the supplies of carbon, nitrogen and phosphorus

match the microbial demand (Hessen et al. 2004; Enríquez et al. 1993; Melillo et al.
1982). The theory predicts that microbial decomposition of soil organic carbon is
constrained by the need of microorganisms to maintain their carbon to nutrient bal-
ance, and the soil will store more carbon in response to extra supply (Hessen et al.
2004; Dukes and Field 2000). Conversely, increasing nutrient availability would
decrease soil carbon storage (Cleveland and Townsend 2006; Mack et al. 2004).
The nutrient stimulation of decomposer activity often occurs in ecosystems where
litter or substrate quality is low and carbon-to-nitrogen ratios are high (Berg and
Matzner 1997). Indeed, increased carbon mineralization due to nutrient addition
has been observed in a number of litterbag decomposition experiments (Conn and
Day 1996; Downs et al. 1996; Prescott 1995), in the light fraction of soil organic
matter (Neff et al. 2002) as well as in the whole organic matter (Cleveland and
Townsend 2006; Mack et al. 2004; Bragazza et al. 2006; Paterson et al. 2008b).
Although there is a general ignorance regarding phosphorus stoichiometry, in a
highly weathered Hawaiian soil, where phosphorus availability limits net primary
production, phosphorus additions have been reported to increase the rates of litter-
mass loss (Hobbie and Vitousek 2000). It should be noted that in several situations,
the decomposition of plant litter may be decoupled from nutrient additions. For
example, in some tropical rain forests the decomposition or mass loss of plant litter
is primarily attributed to leaching, not microbial carbon mineralization (Hobbie and
Vitousek 2000). In this circumstance, the mass loss of plant litter may be more a
product of litter solubility and rainfall. In addition, added nutrients may interact
with aspects of litter carbon chemistry (Hobbie and Vitousek 2000). Decomposers
may be unable to use additional nutrients due to low availability of labile carbon
substrates (Fontaine et al. 2007). In the later stages of litter decomposition, where
mass loss is dominated by lignin-degradation, other added nutrients may have a
neutral or even retarding effect on decomposition (Berg and Matzner 1997).
Microbial nitrogen mining hypothesis: In sharp contrast to the basic stoichiometric
decomposition theory, the microbial nitrogen mining hypothesis predicts that when
mineral nitrogen becomes scarce in the soil, the population size of decomposers
and the net decomposition rates of soil organic matter will increase (Fontaine and
Barot 2005; Moorhead and Sinsabaugh 2006). Microbial nitrogen mining refers to
the process whereby some microorganisms use energy-rich substrates (e.g., labile
carbon) to actively degrade recalcitrant carbon compounds with their extracellular
enzymes in order to acquire nitrogen (Moorhead and Sinsabaugh 2006). Based on
the theory, soil carbon storage will increase with greater nitrogen availability as
mining of recalcitrant carbon compounds for nitrogen is suppressed. Since the
decomposition of recalcitrant carbon compounds yields little to no energy (Couteaux
et al. 1995), the supply of fresh or labile carbon is important for various microorgan-
isms to access recalcitrant nitrogen (Fontaine et al. 2004, 2007). This hypothesis
might explain observed declines in the decomposition of soil humus (Berg and
Matzner 1997; Michel and Matzner 2002) and increases in formation of refractory
soil carbon (Neff et al. 2002; Resh et al. 2002; Dijkstra et al. 2004; Hagedorn et al.
2003) under conditions of high nitrogen deposition, fertilization, or fixation in
terrestrial ecosystems. The increased in old and stable humus may significantly
improve soil carbon storage in the long run (Hagedorn et al. 2003; Franklin et al.
2003) and decrease soil respiration in terrestrial ecosystems (Olsson et al. 2005;
Billings and Ziegler 2008). However, there is no evidence for phosphorus mining as
previous studies suggest that phosphorus additions would either increase short- and
long-term mineralization of soil organic matter (Hobbie and Vitousek 2000; Craine
et al. 2007) or have no effect (Cleveland and Townsend 2006; McGroddy et al. 2004)
depending on the initial phosphorus availability in the substrate and soil.
Effects of Nutrient Addition on Decomposition

of Soil Organic Matter Components
Lignin: Lignin has long been suspected to contribute substantially to the stable car-
bon pool in soils due to its chemical recalcitrance (Bahri et al. 2006). Studies have
shown that long-term nitrogen additions would reduce decomposition rates of lignin
components in soil organic matter (Fog 1988; Berg and Matzner 1997). The
decreased rate of lignin decomposition by nitrogen addition could be attributed to a
number of factors. Firstly, lignin degradation is carried out by a subset of the soil
microbial community, which primarily consists of fungi. Nitrogen additions would
cause a shift in fungal community composition. For example, decreases in the rela-
tive abundance of a well studied soil lignolytic fungus of the phylum basidiomycota
have been observed in response to nitrogen amendments in both alpine tundra
(Nemergut et al. 2008) and forest ecosystems (Edwards et al. 2004; Allison et al.
2007a). Basidiomycetes are key drivers of decomposition and soil carbon mineraliza-
tion and responsible for the release of several enzymes that mediate lignin degrada-
tion, including laccase, manganese peroxidase and lignin peroxidase. Secondly,
nitrogen fertilization can inhibit the formation of enzymes that break down lignin.
Phenol oxidase, an important ligninolytic enzyme, has been shown to be greatly
reduced by increased nitrogen availability during the decomposition of plant litter
(Carreiro et al. 2000) and soil organic matter (Gallo et al. 2004; Saiya-Cork et al.
2002). However, it is still unclear how the nitrogen additions affect white rot fungi
(Phanerochaete chrysosporium), the only microorganism known to produce phenol
oxidase. Reduced phenol oxidase may contribute to either the suppressed production
of this enzyme and/or to reduced competitiveness and hence decreased abundance of
white rot fungi relative to other fungi in nitrogen-enriched plant litter and soil organic
matter. Thirdly, nitrogen can react with lignin compounds in plant litter or soil
organic matter to form more recalcitrant complexes. Reactions between nitrogen and
phenolic compounds have been shown to occur (Haider et al. 1965; Zech and Kögel-
Knabner 1994). For example, ammonium-nitrogen (Nömmik and Vahtras 1982) and
amino acids (Kelley and Stevenson 1996) have been reported to be bound to aromatic
ring structures. However, more recent studies have not detected such phenolic com-
pounds in soil organic matter using nitrogen-15 nuclear magnetic resonance spectros-
copy (Knicker et al. 1996; Knicker and Lüdemann 1995; Sjöberg et al. 2004).
Carbohydrates: The largest fraction of organic carbon entering the soil is that
contributed by plant residues. Cellulose, the most abundant constituent of plant
residues, is composed of glucose units with b(1→4) linkages. It has been reported
that cellulose decay would be enhanced by nitrogen addition due to the increased
cellobiases (Carreiro et al. 2000; Sjöberg et al. 2004). Serpula lacrimans, a cellu-
lose decay fungus, will produce more mycelium and decay cellulose faster under
nitrogen fertilization (Watkinson et al. 1981). Neff et al. (2002) determined the
concentrations of two plant polysaccharide markers in soil samples (5-methyl-2-
furanone and 2-hydroxy-3-methyl-2-cyclopentenone) and found that these two
chemicals are substantially lower in the light fraction of fertilized plots than in that
of unfertilized plots. The authors concluded that nitrogen addition led to signifi-
cantly faster decomposition of polysaccharide markers in the light fraction of soil
(Neff et al. 2002). Another experiment in a grassland suggested that decomposition
of cellulose is much more rapid in fertilized plots, compared with unfertilized plots
(Heal et al. 1981). Addition of nutrients produced no significant change in the rate
of cellulose decomposition in the surface layer of soil, but at 4–8 cm, phosphorus
fertilizer caused a threefold increase in decomposition rate compared with a two-
fold increase caused by nitrogen addition (Heal et al. 1981).
Nitrogenous components: The majority of identifiable soil nitrogenous compo-
nents occur as amino compounds, which include two main categories, the intact
proteins released for various extracellular functions and detrital proteins – plant and
microbial-derived residues in various stages of transformation. The protein content
of soil organic matter may vary between 15% and 45% (Stevenson 1994). Recently,
proteins are recognized increasingly as playing important roles in stabilization and
destabilization of soil organic matter (Schindler et al. 2007). In soil ecosystems,
most proteins are easily degraded although a significant amount of protein is stabi-
lized for some time against microbial attack through interaction with other soil
organic matter and/or physical protection (Rillig et al. 2007). Considerable effort
has been invested in defining how increased nutrient input can influence decompo-
sition of lignin and cellulose in litter and soil, but knowledge on decomposition of
proteins is very limited. Antibus et al. (2006) investigated the decomposition pro-
cesses of glomalin, a unique glycoprotein produced by arbuscular mycorrhizal
fungi in three forest soils, and found a significant difference related to forest type
but failed to detect significant effects of nitrogen amendment.
Lipids: Soil lipids, a complex series of 500 different fatty acids, are mostly derived
from plants and microorganisms. The lipid content of soil organic matter ranges
from 2% to 20% (Allison et al. 2007b). Assays of specific lipids such as ergosterol
and the phospholipids are proving useful for quantitative evaluation of soil fungal
biomass and for qualitative evaluation of microbial diversity (Bünemann et al.
2004; Allison et al. 2007b). In addition, characterization of the decomposition pro-
cess of soil lipids often provides valuable biogeochemical information about the
impact of vegetation, microorganisms, and abiotic factors on soil carbon sequestra-
tion (Almendros et al. 2001). Cutin-derived compounds, which originate from the
waxy coating of leaves and roots are expected to remain stable on decadal to
centennial timescales (Feng et al. 2008; Simpson et al. 2008; Otto et al. 2005).
This part of soil lipids is especially interesting because they are important
components of recalcitrant alkyl carbon structures in soil organic matter and can
potentially enhance carbon sequestration in the soil in the long-term (Lorenz et al.
2007; González-Pérez et al. 2008). However, like soil protein, limited studies on
decomposition of soil lipids have been reported, and there is no literature on how
nutrient additions affect the decomposition of soil lipids.
In summary, it has been shown that nitrogen addition can alter microbial decom-
position of soil organic matter, but the direction and degree of response depend on
addition rates, chemical composition of soil organic matter and supply of fresh
carbon. While the responses of lipid and protein components of soil organic matter
to nutrient amendment remains opaque, it is evident that in most ecosystems, long-
term nitrogen addition would lead to a decline in lignin decomposition and an
increase in cellulose decay. These trends, coupled with further studies on decom-
position of soil lipids and proteins, may provide insight into the biogeochemical
processes that create global patterns in soil organic matter storage.
Microbial Origin of Soil Organic Matter
There exists a considerable body of literature concerned with the nature of soil
organic matter, its origin, composition and fate. Clearly, soil microorganisms and
their interactions with soil fauna and responses to common management activities
play an important role in the formation of soil organic matter. In addition, soil
microorganisms can be significantly involved directly in terms of soil borne
plant pathogens contributing to plant death and the subsequent inputs of organic
matter of various qualities from above and belowground plant organs (see chap-
ters elsewhere in this book). Soil microorganisms may also play a significant
part in plant growth promotion either directly or indirectly through the production
of plant growth promoting substances, nitrogen fixation, biological control of
plant pathogens, improving soil structure, and increasing nutrient uptake (Alami
et al. 2000; Barea 1997; Barea et al. 1998, 2002; Dakora and Tsavkelova et al.
2006; Vessey 2003; Postgate 1998) (see chapters elsewhere in this book).
This section will further examine the role of soil microorganisms as soil organic
matter itself and examine the importance of the turnover of microbial biomass and the
fate of the constituents of that same microbial biomass (Badalucco et al. 1992) together
with the production of microbial metabolites that assist in the function and defence of
soil microorganisms. This more passive activity represents the outcome of the normal
function of soil microorganisms in contributing to carbon and nutrient cycling.
Our understanding of the carbon cycle has been dominated by several conceptual
processes that have helped overshadow the role of role of microorganisms in the
formation of soil organic matter. Firstly, it is commonly assumed that most organic
matter enters the soil as fresh plant material (senescent or recently detached) or as
dissolved organic carbon from root exudates and canopy leaching. This plant
material or dissolved organic carbon then undergoes decomposition and stabilisation
which can be defined as a decrease in the potential for soil organic matter loss by
respiration, erosion or leaching (Sollins et al. 1996). Degradation and stabilisation of

this material occurs by both biotic and abiotic processes. These processes result in
the original plant material and dissolved organic carbon being decomposed to sim-
pler organic molecules and ultimately carbon dioxide. Simultaneously, these break-
down products can be synthesised into a range of new larger molecules both within
the original plant cell and outside it. Clearly at the heart of the release of carbon
dioxide from the original material is the conversion of energy into microbial biomass
(Sollins et al. 1996). What then happens to this microbial biomass is of some conse-
quence for the role that soil microorganisms play in developing and increasing soil
organic matter. That role is likely to be larger than previously considered and in fact
soil microorganisms may be playing a controlling role in this globally important
function of soil carbon storage (e.g., Simpson et al. 2007a; Kindler et al. 2009).
There is an increasing body of evidence supporting the notion that soil microorgan-
isms are important in forming and increasing soil organic matter.
Another reason that the role of soil microorganisms in soil organic matter forma-
tion has not been widely considered may be due a research focus on the fraction-
ation of soil organic matter for the purposes of understanding its origin. There are
many possible fractionation systems for soil organic matter, and some have been
used to describe the nature of soil organic matter (von Lützow et al. 2007).
However, relationships between operationally defined fractions and functional
pools that determine the turnover of soil organic matter are not well understood.
Plant material on or beneath the soil surface is invaded and used by microorganisms
and fauna, subsequently transformed by the physical and chemical environment
and other organisms and translocated in the soil profile by leaching, animal move-
ment and fungal hyphae. The fate and transformation of plant derived carbon has
been the primary focus of research into soil organic matter dynamics (Kögel-
Knabner 2002). This may have led to a greater focus on soil organic matter as a
source for microbial growth (Kramer and Gleixner 2008) rather than on what
happens to microbial biomass upon its death.
Clearly carbon inputs to soils are used by soil microorganisms, which acquire
energy and nutrients from them to support growth and maintenance of metabo-
lism. Estimates of microbial biomass size range from only 1% to 5% of total soil
organic carbon that may have distracted from the importance of soil microorgan-
isms in organic matter dynamics. As a result of predation by soil fauna, changes
in soil conditions or natural death, microbially derived carbon will re-enter the
soil organic matter pool. From this simple model, it can be concluded that the
major sources of organic matter in soils are plants and microorganisms. Kindler
et al. (2006, 2009) provide examples of how constituents of microbial cells can
enter the soil organic matter pool, while the conclusions of Kelleher and Simpson
(2006) support such a simple model of the origin of soil organic matter. They
identified that nearly all of the nuclear magnetic resonance spectroscopy signals
observed in traditional humic substances could be assigned to intact and degrading
biopolymers from plants and microorganisms. This new insight should provide
the starting point for any fractionation system of what will be a mixture of plant
and microbially derived products reflecting the great diversity of these organisms
and degrees of microbial utilization.
Along with fractionation, considerable attention has been given to researching
the stabilization of organic matter in soils and the importance of this process to
global carbon budgets. Recently, Ekschmitt et al. (2005) extended our understand-
ing of the various processes involved in the stabilization of soil organic matter by
suggesting that it is not only substrate quality or soil conditions that constrain
decomposition rates but that the biology of the decay organisms also played an
important part in determining decay rates. Clearly, soil microorganisms are not only
an important source of carbon but they also have an important role in determining
turnover rates, as directed by their physiological requirements and activities.
When bringing attention to microbial biomass in soils, we should not overlook
that while carrying out their widely accepted role of organic matter decomposition,
some microorganisms produce compounds that can fulfil a large range of roles from
defence to energy storage (e.g., fatty acids, amino acids). The latter has received
considerable attention due to the interest in the potential production of bioplastics
(Luengo et al. 2003), but little attention has been paid to the ecological point of
view of how this activity contributes to the formation of soil organic matter. Among
the energy storage compounds, polyhydroxyalkanoic acids are synthesised by a
wide variety of microorganisms, which use a variety of carbon sources and have
been isolated from a wide range of soils. Microbial communities are diverse, so any
activity to store energy, resist other microorganisms or carry out their function will
result in a large mix of compounds produced which is clearly the case. Focusing on
these compounds or biomarkers should provide new knowledge on the contribution
of soil microorganisms to the development of soil organic matter (Badalucco et al.
1992; Kindler et al. 2006, 2009; Liang et al. 2007). The fact that a range of micro-
bial compounds is found in soils despite their unstable nature suggests that consid-
erable amounts are produced and subsequently protected within the soil matrix.
Considerable effort has been expended on the development and application of
techniques to determine the ratio of fungi to bacteria in soil (Joergensen and
Wichern 2008) so that the importance of the different functional groups under dif-
ferent land use and management practices can be determined. The use of certain
compounds such as amino sugars or phospholipid fatty acids for this purpose has
highlighted the degree to which some of these materials accumulate within soil
organic matter. These observations suggest that there may be potential to manipu-
late and possibly increase the contribution of certain soil microbial assemblages to
soil carbon sequestration (Bailey et al. 2002; Jastrow et al. 2007), which if success-
fully applied may add weight to the importance of soil microorganisms in
controllingthe formation of soil organic matter.
We should assume that the processes affecting the decomposition or stability of
plant-derived soil organic matter will have similar affects on microbial derived
carbon. Experiments that manipulate the conditions under which these processes
operate will ultimately show if these two sources of soil organic matter are funda-
mentally different in their nature in terms of composition and turnover.
The simple question of what happens to the microbial biomass when it dies has
been largely overlooked when considering the formation of soil organic matter. This
has been described as the “eye of the needle” through which all nutrients and organic
matter needs to pass. So although small in size, the turnover rate of the microbial
biomass is seen as key to soil functioning because of its low carbon-to-nutrient ratio.
Release of carbon and nitrogen from dead microbial cells further increases microbial
activity and the outcome of the competitive interaction between plants and microor-
ganisms determine rates of plant productivity. As indicated above, microbial cells
consist of a wide range of compounds, some of which are not easily decomposed.
The incorporation of nitrogen-15 labelled compounds (Knicker et al. 1996;
Knicker and Lüdemann 1995; Clinton et al. 1995) into the soil organic matter, or
carbon-13 labelled glucose (Baldock et al. 1989) into structures resembling
proteins and nucleic acids suggests a dominant biological function provided by the
microbial community. Clinton et al. (1995) separated applied nitrogen-15 that was
subsequently immobilised into humified organic matter and poorly transformed
plant fragments and showed that most of the nitrogen-15 was incorporated into a
pool of humified organic matter. The lack of a nitrogen-15 signal in the spectra
assigned to poorly transformed plant fragments along with evidence that most of
the nitrogen-15 shown to be in protein nitrogen was attributable to microbial activ-
ity, suggests that microbial material can contribute to a pool considered by some to
be mainly derived from plant material. The carbon-13 spectra indicated that
labelled glucose was incorporated into new compounds within microbial biomass.
Longer term studies of nitrogen-15 recovery by plants (e.g., Mead et al. 2008; Mead
and Preston 1994) have shown that the pool of applied nitrogen-15 immobilised in
soils can be very stable suggesting that the contribution of microbial communities
to the formation of soil organic may be significant and persistent.
The growth and incorporation of carbon, nitrogen and other nutrients into micro-
bial biomass has been widely shown, so what happens to this biomass? A common
assumption on the nature of soil organic matter is that it is consists of decaying plant
matter. The soil biomass is commonly excluded from such definitions. However, it
has been suggested that the proportion of decaying plant matter in soil organic mat-
ter that is soil microbial biomass is greater than expected. For example, using the
glucosamine content of decaying wooden blocks colonised by specific wood decay
fungi showed that mass loss of wood was in fact greater than indicated by the loss
of mass alone because of the growth of wood decay fungi within the wood material
which was included in the mass of the block (Jones and Worrall, 1995; Joergensen
and Wichern 2008). Although Jones and Worrall (1995) did not demonstrate trans-
fer of microbially derived molecules to the soil organic matter pool, the transfer of
nutrients out of decaying wood blocks does suggest that ultimately microbially
derived organic matter may be transferred into soils by hyphal growth (Wells et al.
1998). The significance of this observation is clearly overlooked in most organic
matter decay studies although more recent studies support the transfer of microbial
compounds to the soil organic matter pool (Kindler et al. 2009).
Few studies have determined quantitatively the contribution of microbially
derived organic matter to total soil organic matter. As previously stated the microbial
biomass is small and generally accepted as less than 5% of soil carbon. Engelking
et al. (2007) recently showed that the incorporation of carbon into microbial residues
can exceed the amount in the microbial biomass. Based on recent studies, between
30% and 80% of soil carbon may be derived from both live (biomass) and dead
(necromass) soil microorganisms (Simpson et al. 2007a; Kindler et al. 2009; Appuhn
et al. 2006). Clearly, the proportion will depend on a range of factors not yet fully
studied, such as vegetation specific effects on microbial residues (Six et al. 2006;
Liang et al. 2007). Mikutta et al. (2009) studied the ratio of plant-derived pentoses
to microbial amino sugars in mineral organic associations in soils of different ages
under the same dominant vegetation. Their results suggest that microbial growth and
activity and the turnover of cells to form necromass may play an important role in
the formation and control of mineral organic associations in some soils. Clearly,
there is a view now with respect to the stabilisation of soil organic matter that micro-
bially derived organic matter is important in this process. This new knowledge will
challenge the importance of other sources of organic matter, such as roots, with
regards to their contribution to the soil organic pool (Nierop 1998).
Recent studies confirm that we need to focus further on the factors controlling the
soil microbial community (soils, vegetation and management) so that we can head
towards a universally accepted model on the nature of soil organic matter formation.
This in turn will allow us to manage this important pool of soil organic matter
(Jastrow et al. 2007; Carletti et al. 2009; Chabbi and Rumpel 2009). For example,
land use change can result in a shift from microbial-derived soil organic matter to
plant derived organic matter with fungi being more sensitive to land-use change than
bacteria (Jolivet et al. 2006). New methods and approaches will continue to improve
our understanding of microbially derived soil organic matter (Nelson and Baldock
2005; Zhang et al. 2007; Simpson et al. 2007b; Glaser et al. 2006).
The implications of the significant role of soil microbes in soil organic matter
formation are clearly aligned with emerging views on the usefulness of our current
models of soil organic matter turnover. It may be timely to review the existing
models and to consider more carefully other proposed models for understanding
soil organic matter dynamics. In their review, Sutton and Sposito (2005) concluded
that there was evidence to support a new view emerging on the nature of soil humic
substances and their formation. If this view is eventually widely adopted, a strong
case for the involvement of soil microorganisms in the formation of soil organic
matter is verified as this group of organisms possess the physiological diversity to
create the full range of smaller molecules required in the new model of soil organic
matter formation.
We should examine more closely what we know about the inputs and transfor-
mations of organic matter and the processes responsible for this in the soil environ-
ment. Clearly, large amounts of organic material are added to the soil surface or
incorporated into the soil profile by soil fauna or mechanical disturbance and this
is well represented in a variety of models of carbon cycling that still form the con-
ceptual basis explaining origin and fate of photosynthetically derived carbon. These
models also include the important inputs from root turnover and root exudates.
However, the number and complexity of models has lead to a lack of focus on the
role of soil microbes in developing and increasing soil organic matter and for the
time being the diversity of these models has become a research focus. It is clearly
time for a major revamp of the black box approach to studying soil processes and
recognise soil microbes both live and dead and their interactions with soil animals
more routinely in models of soil organic matter dynamics. Increases in soil organic
matter may be driven by the activities of a small microbial biomass over very long
periods of time. If this was the case then a large pool of soil organic matter contain-
ing carbon of a range of ages and in a large diversity of molecules as well as fresh
plant material derived from roots and aboveground plant parts would result. The
challenge is to represent this complexity in the next generation of models of soil
organic matter. It is thus necessary to study the factors controlling turnover of
added and soil carbon in more detail with a particular focus on the role of soil
microbial biomass and specific microbial assemblages and species.
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Chapter 5
Intimate Associations of Beneficial Soil
Microbes with Host Plants
Oksana Y. Shtark, Alexey Y. Borisov, Vladimir A. Zhukov,

Nikolay A. Provorov, and Igor A. Tikhonovich
Introduction
Symbiosis, or “coexistence of diverse organisms”, is the term which was proposed

firstly by Anton de Bary (1879). Roughly, there are three types of symbiosis: com-
mensalism (an interaction which is beneficial for one symbiotic partner but neutral for
the other), antagonism (or parasitism, where one symbiont develops to the detriment
of another – see elsewhere in this Book) and mutualism (where both symbionts benefit
from mutual coexistence). Mutualism is a special ecological and evolutionary strategy
for two (or more) dissimilar organisms to share (allocate) all of their physiological
O.Y. Shtark (*)

Laboratory of Genetics of Plant-Microbe Interactions, Department of Biotechnology,
A.Y. Borisov
Laboratory of Genetics of Plant-Microbe Interactions, Dept of Biotechnology,
V.A. Zhukov
Laboratory of Genetics of Plant-Microbe Interactions, Department of Biotechnology.
N.A. Provorov
I.A. Tikhonovich
120 O.Y. Shtark et al.
functions between them rather than them being separated. This is distinguished prin-
cipally from individual adaptations which are dependent on morpho-physiological
and behavioural reactions of one free-living organism. Here there is an evolutionary
opportunity to develop certain functions and possibly lose others leading to genetic
and metabolic integration according to a dialectical law “negation of negation” which
may be applicable to evolution. As a result some intimate genetic and metabolic coop-
eration between the symbiotic partners of complicated super-organismic systems
develop resulting in new adaptations (functions) with their environments. In mutual-
istic (mutually beneficial) symbioses between plants and microbes, novel types of
cells and tissues (physiologically and/or structurally) and even organs have often
evolved (Douglas 1994; Tikhonovich and Provorov 2007).
In the process of evolution, symbiosis plays a key role in developing highly
organized lifestyles and possibly new types of ecological interactions (Douglas
1994; Margulis 1998; Margulis and Sagan 2002). The eukaryotic cell in particular,
is considered to be an evolutionary product of beneficial symbioses with different
prokaryotes (Taylor 1974). In accordance with this theory, cell organelles such as
mitochondria and photosynthesising plastids originated from free-living bacteria
which turned to a symbiotic life style and lost their capabilities for autonomous
existence (Margulis 1970; Margulis 1988; Dyall and Johnson 2000; Margulis and
Sagan 2002; Embley et al. 2003). Lichens for example are the products of beneficial
symbiotic interactions between algae and/or cyanobacteria with fungi. Moreover, it
has been shown that in the geological time in which plants achieved a terrestrial
existence symbiotic associations with arbuscular mycorrhizal fungi also developed
(Pirozynski and Dalpé 1989; Remy et al. 1994; Redecker et al. 2000).
According to contemporary nomenclature, symbioses may be categorized in accor-
dance with a range of indicators (Lewis 1985; Provorov et al. 2008). According to the
level of dependence of the partners on each other, symbioses can be generally considered
as either obligatory or facultative in terms of their ecology and genetics. The concept of
“ecological obligation” means that organisms are only able to occupy new ecological
niches in the symbiotic state (Smith and Read 1997), while “genetic obligation” indi-
cates a state where autonomic existence of the symbionts is impossible because they
have lost genes encoding for products with essential functions (Provorov 2005). If one
of the partners penetrates into the other, this is endosymbiosis, which can be intra-cav-
ernous, intratissular or intracellular; whereas ectosymbiosis is coexistence without deep
penetration of one partner into the other. Trophic interactions between partners also show
a range of dependencies from: biotrophy (where the nutrient source is a living partner’s
cells) through to necrotrophy (where the nutrient source is a partner’s dead cells).
Symbioses of microbes with plants (as well as animals) are quite widespread in
nature and enable the higher organisms to occupy particular ecological niches (Douglas
1994). In the case of facultative or ecologically obligatory interactions, the microsym-
biont through intimate association with the host achieves ecological advantages com-
pared with free-living forms. In particular the microsymbiont benefits from priority
utilization of host metabolites or protection from predators. There are however, con-
comitant disadvantages since genetically obligatory microsymbionts are not able to
5 Intimate Associations of Beneficial Soil Microbes with Host Plants 121
survive outside the symbiotic system wherein their reproduction is strictly controlled
by the host (Provorov et al. 2008). With regard to the host plant, mutually beneficial
plant–microbe symbioses are generally facultative or ecologically obligatory and usu-
ally subdivided according to nutritional (microbes providing the host with nutrients, in
particular with nitrogen or phosphorus) or defensive (microbes supply the host with
the resistance to pathogens) benefit (Vance 2001; Lum and Hirsch 2003; Provorov
2009). Usually, in natural environments both types of interactions are available simul-
taneously for the same plant due to presence of different types of microsymbionts.
The taxonomy, morphology and function of mutually beneficial plant–microbe
symbioses is diverse; they include intracellular, intratissular (endophytic) ones or
associative ectosymbiosis (epiphytic) forms. Nevertheless, despite superficial differ-
ences between the intracellular and intratissular forms of endophytic symbiosis and
associative ectosymbiosis or epiphytic forms, all beneficial plant–microbe symbio-
ses are based on similar genetic, cellular and molecular mechanisms. Moreover, they
have a series of traits in common with plant–pathogenic systems and represent an
evolutionary plant–microbe continuum (Jones et al. 2007; Provorov 2009).
This chapter focuses on the ecologically and agriculturally most important beneficial
plant–microbe endosymbiotic systems, such as arbuscular mycorrhiza (AM) and root-
nodule (RN) symbiosis, or root nodules. These are characterized by the formation of
special complex symbiotic compartments, as well as by interactions with plant growth-
promoting rhizosphere bacteria (PGPR) and/or beneficial endophytic types, where from
the outset of interaction special symbiotic structures are not formed (Fig. 1). These
groups of beneficial microbes improve host mineral nutrition, acquisition of water,
promote plant development and offer protection from pathogens and pests.
The taxonomy of the partners, the processes of the formation of symbioses and
their functions will be reviewed. Development of symbiotic root nodules will be
described using pea (Pisum sativum L.) as an example of an evolutionary advanced
model of nodulation. Special attention will be given to developmental genetics,
evolution of the symbioses and their inter-relationships, and then, using this per-
spective the prospects for improving effectiveness of beneficial plant–microbe
systems in sustainable agriculture will be discussed.
Mycorrhizas
The Main Types of Mycorrhizas
Mutually beneficial symbioses of plant roots with fungi are referred to as mycor-
rhizas (from the Greek ‘mycos’, meaning fungus and ‘rhiza’, meaning root), this
term was coined by Frank in 1885 (Frank 1885). Several forms of mycorrhiza are
recognized: endomycorrhiza, including arbuscular, orchid and ericoid mycorrhiza,
when part of the fungal hyphae penetrates into the plant cell, and ectomycorrhiza,
when the fungal mycelium remains outside plant cells. As arbuscular mycorrhiza is
Fig. 1 Mutually beneficial plant-microbe symbioses. a–f – Arbuscular mycorrhiza (AM), g–m –
Root-nodule (RN) symbiosis, n – Symbiotic association of root with plant growth-promoting
rhizobacteria (PGPR). a, e, f – Spores of Glomus sp. a – Extraradical spore, e – Intraradical spore,
f – Sporocarp. b–d – Light microscopy of macerated root fragments stained with black ink accord-
ing to Vierheilig et al., 1998. b, d – Roots of P. sativum colonized by Glomus intraradices. c –
Root of chives (Allium schoenoprasum L.) colonized by G. fasciculatum. eh – Extraradical hypha,
ap – Appressorium, ih – Intraradical hypha, ar – Arbuscule, v – Vesicle, s – Spore. g – Root nod-
ules in pea (Pisum sativum). A nodule is indicated by an arrowhead. i–m – Indeterminate symbi-
otic root nodule development in P. sativum inoculated with a derivative of Rhizobium
leguminosarum bv. viciae strain containing a Tn5-gusA insertion constitutively expressing gusA
probably the most widespread terrestrial symbiosis (Fitter 2005) and the most
important form of mycorrhizas in agriculture it will be described detail. Other
mycorrhizas will be treated in less depth.
Arbuscular Mycorrhiza (AM)
Arbuscular Mycorrhiza (AM) is an ancient mutually beneficial plant–fungal symbi-

otic system formed by at least 80% of terrestrial plants, including the majority
of angiosperms and gymnosperms. This symbiotic association has existed for
400–500 millions years, and was considered to play a decisive role in plants achiev-
ing a terrestrial existence (Pirozynski and Dalpé 1989; Remy et al. 1994; Redecker
et al. 2000). The evolutionary age, life-style and genetic structure of AM fungi is
unusual and because they have existed in a morphologically unaltered state they could
therefore possibly qualify as living fossils (Parniske 2008). The taxonomy of fungi
able to form AM has been revised several times (reviewed in Koide and Mosse 2004).
Latterly, after analysis of 18S rRNA genes sequences the AM fungi have been placed
in a new phylum Glomeromycota (Schüßler et al. 2001) which replaced the zygomy-
cete order Glomales. The new phylum contains the genera Glomus, Acaulospora,
Scutellospora, Gigaspora, Paraglomus and Archaeospora together with Geosiphon
(which is non-mycorrhizal but is macrosymbiont in an association with photosynthetic
nitrogen-fixing cyanobacteria Nostoc). The Glomeromycota are unique as the only
monophyletic mycorrhizal fungus lineage that has co-evolved with land plants
throughout their history (Brundrett 2002). This is why AM fungi are considered as
obligate biotrophs that colonize plant roots obtaining photosynthates, such as carbo-
hydrates (hexoses), and niches for both their growth and reproduction (Smith and
Read 1997; Bago and Becard 2002). Although it should be noted that according to
recent studies, some AM fungi can be cultivated in vitro, in the absence of plant, in a
dixenic system with the bacterium Paenibacillus validus (Hildebrandt et al. 2006).
The hyphae of AM fungi are usually aseptate and coenocytic, with hundreds of
nuclei located in the cytoplasm (Smith and Read 1997). Spores of AM fungi are
Fig. 1 (continued) (from Voroshilova et al., 2009, modified). i–l – Longitudinal vibratome sections
of roots and nodules, with GUS staining of bacteria in infection threads (ITs) and colonized nodule
cells. m – Cross-section of nodule with Feulgen/alcian blue staining. i – Transcellular IT growth in
the outer root cortex and resumption of cell proliferation in the inner cortex cells 5 days after inocu-
lation (DAI). j – Nodule primordium at 9 DAI with proliferating cells harbouring ITs. k – An emerg-
ing juvenile nodule at 12 DAI. l – A nodule at 28 DAI showing normal histological organization. (I),
nodule meristem; (II), colonization zone; (III), nitrogen fixation zone. The ‘IT entrance’ to the nod-
ule usually appeared at an epicentric position. m – Mitosis in the nodule meristem cell that forms
transcellular ITs. Neighbouring cells have the preinfection structures. it – infection thread (IT), iz –
infection zone, np – nodule primordium; nm – nodule meristem, mi – dividing plant cells (mitoses).
h – Transmission electronic micrograph of ultrathin section of P. sativum root nodule demonstrating
the symbiosome, or bacteroid (ba). n – Fluorescent microscopy of wheat (Triticum monococcum L.)
root colonized by Pseudomonas chlororaphis strain carrying a constitutive reporter fusion with
Green Fluorescent Protein (GFP) gene. rh – root hair, mc – micro-colony of bacteria. Bars: g – 5
mm; e, k, l – 100 µm; a-c, f, i, j, n – 50 µm; d, m – 10 µm; h – 2 µm
relatively large in size (up to 400 mm) and are the main reserve of genetic material
and carbon sources for subsequent generation of mycelium (Smith and Read 1997).
Individual spores contain numerous nuclei and this raises the question of how the
different polymorphic DNA-sequence variants that are present within a single cell
are distributed between genomes or nuclei. Not surprisingly, this question is subject
to continuing debate (Smith and Read 1997; Pawlowska and Taylor 2004; Hijri and
Sanders 2005; Rosendahl 2008). Although there is no confirmed report of a sexual
stage in the life-cycle of AM fungi, it is possible that genetic material is exchanged
and recombined through mechanisms such as anastomosis between hyphae
(Giovannetti et al. 2004; De la Providencia et al. 2005). Anastomosis allows the
exchange of nuclei (De la Providencia et al. 2005), but has so far only been
observed between hyphae of closely related fungal isolates. It would be interesting
to determine the level of relatedness that is required for these fusions to occur.
Molecular evidence for recombination in AM fungi has been controversial
(Vandenkoornhuyse et al. 2001; Gandolfi et al. 2003; Rosendahl 2008). As an
important step towards the genetic manipulation of these fungi, transient transfor-
mation by particle bombardment has been achieved (Helber and Requena 2008).
Highly differentiated intracellular structures called arbuscules are formed on colo-
nization of roots by AM fungi (Smith and Read 1997; Harrison 2005). In addition to
growth within the root cortex, they develop an extensive extraradical mycelium in the
surrounding soil. Different AM fungi have different hyphal growth patterns, anastomoses
and branching frequencies. Such differences probably reflect varying strategies and the
occupation of several niches within the soil. For example, many Glomus species form
highly branched and anastomosing hyphal networks. These networks are more resistant
to soil disturbance than the mycelia of species of Scutellospora or Gigaspora, which
form longer hyphae and can, probably, explore soil at greater distance from the root (De
la Providencia et al. 2005; Voets et al. 2006). Most of the fungal biomass in members
of the Gigasporaceae family is found in the hyphae that are located outside the plant
root, whereas in members of the Glomeraceae family, most of the hyphal biomass is
inside the root (Maherali and Klironomos 2007).
The intraradical and extraradical growth phases are a single continuum via which
the fungus is able to translocate mineral nutrients, primarily phosphates and water
from the soil to the interior of root systems; thus, by living in association with AM
fungi the host plant becomes capable of existing in nutrient-poor and drought-affected
soils (Ames et al. 1983; Jakobsen 1995; Ruiz-Lozano and Azcon 1996; Harrison 1997;
Smith et al. 2001; Liu et al. 2002; Quilambo 2003). This is why AM can be considered
as nutritional plant–microbe symbioses. In addition, well developed AM-symbiosis
improves host resistance against biotic for example phytopathogenic attack (Cordier
et al. 1998; Dassi et al. 1998; Hause et al. 2002; Fritz et al. 2006; Liu et al. 2007), and
abiotic stresses, in particular heavy metal contamination of soils (Turnau et al. 2007).
An important function of extraradical mycelium in ecosystems is the formation of a
common ‘hyphal web’, linking different plants (Molina et al. 1992). Also extraradical
mycelium has an active role in soil structure formation (Celik et al. 2004; Rillig 2004).
The AM fungi can be involved in bioremediation including phytoremediation, a tech-
nique based on the use of plants for the remediation of contaminated soil (Requena
et al. 2001; Celik et al. 2004; Turnau et al. 2007).
Ectomycorrhiza (ECM)
Ectomycorrhiza (ECM) is predominantly found on trees in temperate forests, such

as: Betula, Fagus, Picea, Pinus, Populus, Quercus and Salix. It has also been found
in a few plant families of the southern hemisphere and the monsoon forests of
south-east Asia (Jackson and Mason 1984; Smith and Read 1997). A wide range
of fungi predominantly belonging to the phyla Basidiomycota and Ascomycota
form ECM (Smith and Read 1997). These fungi are polyphyletic (they have four
or more origins) (LoBuglio et al. 1996; Hibbert et al. 2000). Most ECM fungi are
epigeous (having above-ground fruiting bodies), but up to a quarter of them are
hypogeous (with underground fruiting bodies) (Molina et al. 1992). The ECM
fungi do not show a high degree of host specificity in the colonization of the sec-
ondary or tertiary roots of woody species. The hyphae of ECM fungi cover the root
like a sheath and grow inwards between epidermal and cortical cells forming a
Hartig net. Penetration of the root cortex is achieved through mechanical force and
secretion of pectinases (Werner 1992). The hyphae never penetrate into cell lumens
or into the stele. A complex network of fungal hyphae is the site of nutrient
exchange (metabolic integration) between the fungus and host trees; predomi-
nantly nitrogen is exchanged for photosynthates, but due to their polyphyletic
origins it has been suggested that ECM fungi have considerable functional diver-
sity (Brundrett 2002).
Orchid Mycorrhiza
Orchids have mycorrhizal associations with soil fungi that are considered to be
essential for seed germination and assist with the growth of adult plants (Rasmussen
1995; Currah et al. 1997). Most orchids have fairly specific fungal associates that
vary between host species and habitat (Currah et al. 1997; Sen et al. 1999). Most of
these fungi are assigned to the anamorphic form genus Rhizoctonia (Currah et al.
1997), some species of which are close relatives of pathogenic forms (Pope and
Carter 2001). The benefits provided by orchids to mycorrhizal fungi, if any, are not
clear, as these fungi seem to grow as well without their hosts as they do with them.
Saprophytic (myco-heterotrophic) orchids without chlorophyll have fully exploit-
ative mycorrhizal associations that supply both the energy and nutrient requirements
of the host (Leake 1994). Many of these plants associate with fungi that are not
related to the mycorrhizal fungi of chlorophyllous orchids, including ECM associ-
ates of trees, wood-rotting and parasitic fungi. These associations have a high degree
of host-fungal specificity and species of Corallorhiza, Gastrodia and Galeola may
only associate with a single fungal genus (Leake 1994; Currah et al. 1997).
Ericoid Mycorrhiza
Mycorrhizal fungi that associate with members of the Ericaceae and Epacridaceae
include several groups of ascomycetes which generally do not form mycorrhizas
with other vascular plants (Smith and Read 1997). Studies of DNA sequences of
fungi from these plants in different Continents have revealed that two or more
distantly related groups of fungi are involved in ericoid mycorrhizas (McLean et al.
1999; Monreal et al. 1999; Sharples et al. 2000). Hymenoscyphus-like fungi associ-
ate with the Ericales and bryophytes throughout the world, but other taxa are more
restricted to specific geographic regions (Chambers et al. 1999; Read et al. 2000).
It is not certain whether ericoid mycorrhizal fungi exist primarily as soil sapro-
phytes, or as mycorrhizal associates of plants. Ericoid mycorrhizal associations are
considered to be capable of neutralizing highly acidic soils and acquiring organic
nutrients (Smith and Read 1997). Substantial nutritional benefits accrued by the
host have been shown in some experiments, but not in others (Bell and Pate 1996;
Jansa and Vosatka 2000) which may indicate facultative associations.
Individual members of Ericales form monotropoid or arbutoid mycorrhizas
(ECM-like associations). They generally have much greater host-fungus specificity
than other ECM associations (Smith and Read 1997).
Arbuscular Mycorrhiza (AM): Development and Function
Two principal morphological types of AM are known, the Paris and Arum types,
named after the two plant species (Paris quadrifolia and Arum maculatum) in which
these types were first described more than 100 years ago (Gallaud 1905). In the Arum
type, the intraradical fungal hyphae grow linearly in intercellular spaces forming fine
intracellular arbuscules, whereas, in the Paris type, cortical cells are colonized by
thick, coiled and fine arbusculate, coiled hyphae. When the hypha of the coiled type
are growing through longitudinally continuous air spaces they assume a typical linear
morphology, but in the absence of abundant aerenchyma coils are formed (Brundrett
2002). Both types are widely distributed throughout the plant kingdom (Smith and
Read 1997; Smith and Smith 1997). In spite of some AM fungi forming one specific
morphological pattern of AM association, other fungi have been demonstrated to be
capable of forming both types of colonization depending on the host involved (Smith
and Read 1997; Brundrett 2002). While the Paris type is most common in contempo-
rary bryophytes, ferns and gymnosperms, the Arum type is found predominantly in
cultivated herbaceous plants, and has been more frequently studied than the Paris
type (Smith and Read 1997; Smith and Smith 1997). Therefore, in this chapter atten-
tion is mainly focused on the Arum type of interaction.
Cycle of AM Development
Development of an AM association involves two major stages: pre-symbiotic and

symbiotic (Bago and Becard 2002). The first stage starts when the dormant fungal
spores germinate and unbranched germ tubes appear (Fig. 2). This is a reversible pro-
cess: spore germination can be induced by several abiotic stimuli (Powell 1976; Koske
1981), but where root exudates are absent development of the germ tube stops and it
Fig. 2 Control of development of root nodule (RN) symbiosis and arbuscular mycorrhiza (AM) in
pea (Pisum sativum) by the sequential functioning of regulatory symbiotic genes.The programme of
RN symbiosis formation (according to (Borisov et al. 2007)) involves: (1) sub-programme of nodule
tissue differentiation: Ccd – cortical cell division, Npd – nodule primordium development; Anm –
apical nodule meristem development, Nmp – nodule meristem persistence; (2) sub-programme of
colonization (infection and bacteria differentiation): Hac – root hair curling, Crh – colonization of
curled root hair, Iti – infection thread growth initiation, Ith – infection thread growth inside root hair
cells, Itr – infection thread growth in root tissue, Itn – infection thread formation in nodule primor-
dium, Idd – infection droplet differentiation, Bad – bacteroid differentiation, Nop – nodule persis-
tence. The programme of AM formation (according to (Marsh and Schultze 2001)) involves: Pre-Pen
– pre-penetration; Pen - root penetration; Arb - arbuscule development. Stages of RN symbiosis
formation are located directly above the responsible genes. Sign ‘–‘ means that genes which
are located beneath do not involve in corresponding sub-programme of root nodule development.
The continuous arrows point to the stages of RN symbiosis or AM formation which are blocked
by the gene mutations; broken arrows point to the stages of AM formation which are partially repressed
is autolysed or invaginated back into the spore (Giovannetti et al. 1993; Giovannetti
et al. 1996; Bago et al. 1998). Spores may germinate several times (Koske 1981).
The role of the root exudates in AM fungal mycelial growth from germinated
spores has been well reported. Flavonoids (Fig. 3a) and polyamines have been
found to provide induction, but are not major stimuli for mycelial growth (Nair
et al. 1991; Tsai and Phillips 1991). During the few last years a considerable change
in our understanding of signalling process at the pre-symbiotic stage of AM
development has happened. The compound inducing primarily ramification of
hyphae, the so-called, branching factor, has been isolated recently from the root
exudates of Lotus japonicus (Akiyama et al. 2005) and identified as 5-deoxy-
strigol, a strigolactone with sesquiterpene structure (Fig. 3b). Strigolactones were
shown to stimulate AM fungi by activating their respiration (Besserer et al. 2006).
Plant root strigolactones are also chemical signals which induce seed germination
of parasitic weeds (Akiyama and Hayashi 2006). This fungus also produces diffusible
Fig. 3 Signal molecules essential for the earliest stages of arbuscular mycorrhiza (AM) and root
nodule (RN) symbiosis formation. a – The plant molecule, flavonoid (flavonol). The OH-group may
be removed or replaced with different substituents to produce various flavonoid compounds, for
example:- removed at (1) (flavone); replaced at (1) with 3rd ring (isoflavone); removed at (1) and (4)
and replaced at (3) with glucose (apigenin-7-monoglucoside). The O may by replaced at (2) with H
(anthocyanin). b – The plant molecule, sesquiterpene lactone, strigolactone (5-Deoxy-strigol). c –
Rhizobial lipo-chito-oligosaccharide (LCO) molecule, Nod factor. The possible replacements
include: R2, methyl; R3, R4, carbomoyl; R5, carbomoyl or acetyl; R6, sulfate, acetyl, fucosyl, meth-
ylfucosyl, acetylmethylfucosyl, or sulfomethylfucosyl (this position is crucial for a specific recogni-
tion of “Afghan” pea by Rhizobium legumonosarum bv. viciae; see Sections 3 and 4); R7, arabinosyl.
A – non-reducing terminus of Nod factor; B – reducing terminus of Nod factor
signal molecules called Myc factor (Mycorrhization factor) (Kosuta et al. 2003).
The chemical nature of the Myc factor has not yet been identified although
the molecule is thought to have non-proteinaceous nature because of its thermo-
stability, amphiphilic properties, and small mass (< 3 kD).
The branched primary hyphae reach the root surface and form attachment
structures called appressoria (Fig. 1b, 2). Their formation is evidence of a successful
recognition of a host-plant (Nagahashi and Douds 1997). After contact by the
fungus with the root surface the symbiotic stage of AM development starts: from
the apressorium the colonising hyphae grow through the epidermis and into the
root. It was demonstrated recently that the host plant plays an active role in fungal
growth into both the epidermal layer (Genre et al. 2005; Genre et al. 2008) and
cortex (Genre et al. 2009) forming a new intracellular structure, termed pre-
penetration cytoskeleton apparatus. It represents a ‘tunnel’ resembling the cortical
pre-infection threads formed during nodulation (see later in Section 4.3).
Passing from the epidermis, the intraradical hypha grows through the cortex
where it branches and forms intercellular mycelia. In the inner cortical layer(s)
close to endodermis as was shown for Lotus (Demchenko et al. 2004), the fungus
invades into cells and there differentiates into highly dichotomically ramified
tree-liked structures, the arbuscules (Fig. 1b, 1d, 2). The intercellular hyphae
continuously form new arbuscules which are ephemeral structures: several days
after emergence they are degraded in the plant cell (Gianinazzi-Pearson 1996;
Bonfante et al. 2009).
The arbuscules are subcellular compartments which ensure close metabolic integra-
tion between the partners. This is manifested as an intensive exchange of metabolites,
primarily in transport to the host of phosphates absorbed by the fungus from soil. This
function is fulfilled despite a separation of fungal and plant cytoplasms. The arbuscule
remains separated from the plant cytoplasm by an extension of the plasma membrane,
called the periarbuscular membrane, similar to peribacteroid membrane formed during
nodulation (Provorov et al. 2002) (see later in Section 3.2). This envelopment of the
arbuscule also results in the formation of a new apoplastic space between the periar-
buscular membrane and the arbuscule, called the periarbuscular space which contains
a structurally differentiated matrix containing polysaccharides and enzymes of both
plant and fungal origin (Smith and Read 1997; Harrison et al. 2002).
A pronounced differentiation is characteristic for the plant cells containing
arbuscules. The cytoskeleton of the invaded cells indicates extensive, dynamic rear-
rangements of the cortical microtubules, which presumably enables the trafficking
of membrane and cell wall precursors into the extending membrane and new
apoplastic compartment. The vacuole is reduced or degraded, the amount of
a-tubulin increases, the nucleus is deformed (sometimes it is surrounded by the
branches of arbuscule) and chromatin is decondensed due to a high transcriptional
activity. The number of ribosomes and Golgi vesicles is greatly increased correlat-
ing with the synthesis of periarbuscular membrane (Takemoto and Hardham 2004;
Genre and Bonfante 2005).
Intraradical mycelium formed inside the cortex creates a network of channels in
which carbon is transported from the plant to the fungus (Smith and Read 1997).
When the carbon supply is sufficient, lipid-rich vesicles (Fig. 1c) are formed inter-
cellularly within the cortex, as fungal storage organs. Hexoses, in particular glu-
cose, were found as the major form in which carbon is taken up and metabolized
by AM fungi (Pfeffer et al. 1999; Douds et al. 2000). Glucose can then be directly
incorporated into trehalose and glycogen, the first substantial compound in the
fungal carbon pool (Shachar-Hill et al. 1995). Later on, when the carbon supply is
sufficient storage lipids are synthesized, predominantly triacylglycerols in AM
fungi (Bago et al. 2002), which are also the principal form of carbon translocated
from hyphae present in the root cortex to more distal regions (Smith and Read
1997; Smith and Smith 1997). As a result of biotrophic carbon assimilation, growth
of AM fungi is activated and an abundant net of extraradical mycelium is developed
due to active ramification and anastomosis of hyphae coming out from the root into
the rhizosphere (Smith and Read 1997; Bago et al. 1998; De la Providencia et al.
2005). In the symbiotic stage, carbohydrates can only be taken up within intraradi-
cal structures, no uptake of hexoses by the extraradical mycelium has been detected
(Pfeffer et al. 1999; Douds et al. 2000).
A growing colony of AM fungus includes intraradical and extraradical mycelia

and is in an assimilative condition, when most of nutrients taken up from the soil
are used for growth. Mature AM with dense extraradical mycelium, actively taking
up minerals, is characterized by a transfer into dissimilative stage, when a portion
of these nutrients is transported from the fungus to the host playing an important
role in plant development (Smith and Read 1997; Bago et al. 1998).
Spore formation is a turning-point in AM life cycle. They are formed on the
extraradical mycelium (Smith and Read 1997; Bago et al. 1998) (Fig. 1a), but in
several AM fungi spores are formed on intraradical mycelium as well (Bago et al.
1998) (Fig. 1e). Spores contain reserve nutrients predominantly lipids and smaller
amounts of glycogen (Sancholle et al. 2001). Some fungi can form sporocarps,
which may be as simple as a few spores surrounded with mycelium (Fig. 1f). Extra-
and intraradical spores, sporocarps, root fragments containing mycelium with ves-
icles as well as living extraradical hypae can all operate as propagules, starting new
root colonization (Smith and Read 1997; Daplé et al. 2005; Souza et al. 2005). In
addition to propagating the AM fungus, spores probably contain cells of permanent
bacterial endosymbionts, therefore single-spore cultures do not guarantee truly
monoxenic culture of AM fungi (see below).
Improvement of Plant Phosphorus Uptake by AM Fungi
Phosphorus (P) is one of the mineral nutrients essential for plant growth (constitut-
ing up to 0.2% of the dry weight of the plant cell) and development. It has diverse
regulatory, structural, and energy transfer roles and consequently is required in
significant quantities (Bieleski 1973; Schachtman et al. 1998). Biochemical data
support the suggestion that AM fungi improve phosphorus uptake from soils
(mainly as the water soluble H2PO4− ion, referred to as Pi) since the process involves
several fungal transport systems some of which have an extremely high affinity for
Pi (Ezawa et al. 2002). After transport into the hyphae, a major part of Pi is trans-
formed into linear polyphosphate chains (polyP) through reactions catalyzed by a
polyphosphate kinase. Granules rich in polyP, accompanied by P-containing esters,
are packed into cylindrical vacuoles for translocation within hyphae along tubulin
fibrils (Ashford 2002). After reaching the arbuscules, P-compounds are degraded
by phosphatases allowing the release of Pi across the partners’ interfaces. The cru-
cial role of arbuscules in Pi export was proved by the dependence of their develop-
ment on the soil phosphorus status: arbuscules are formed actively under Pi limiting
conditions to moderate its supply, while an absence or excess of Pi inhibits their
formation.
Molecular mechanisms controlling subsequent phosphate transport from extra-
radical hyphae to intraradical ones and efflux from the arbuscule are largely
unknown, but it is well documented that plants possess many classes of phosphate
transport proteins, including those which are expressed only in AM symbiosis
(Karandashov and Bucher 2005; Javot et al. 2007). On the fungal side, the presence
of a G. mosseae Pi transporter was shown in cells containing arbuscules (Balestrini
et al. 2007). The import of Pi by plant phosphate transporter is suggested to be a

signal which permits continuous development of the arbuscule and consequently
sustains the fungal persistence within the root (Bonfante et al. 2009).
Increase of Plant Drought Tolerance
Drought stress is a major agricultural constraint in the semi-arid tropics. In most cases
symbiosis with AM fungi has been shown to increase host plant growth rates during
drought stress and plant resistance to drought. Several mechanisms explaining this
phenomenon have been proposed: an influence of AM on plant hormone profiles,
increasing intensity of gaseous exchange and photosynthesis in leaves, direct water
transport via fungal hyphae from soil into the host plant, enhanced water uptake through
improved hydraulic conductivity and increasing leaf conductance and photosynthetic
activity, nitrate assimilation by fungal hyphae, enhanced activity of plant enzymes
involved in defence against oxidative stress, plant osmosis regulation, and changes in
cell-wall elasticity (reviewed in: Quilambo 2003; Augé 2001; Augé et al. 2004).
Using bean mutants not forming AM associations, it was determined that about
half of the considerable promotion of stomatal conductance by AM fungi was attrib-
utable to soil colonization by fungal hyphae and about half to plant colonization by
AM fungus (Augé et al. 2004). A path analysis modelling approach revealed that soil
hyphal colonization had larger direct and total effects on the of tolerance of beans to
dehydration compared with plant root AM colonization or several other soil or plant
variables (Augé et al. 2003).
Soil structure refers to pore space as well as to aggregates, while the moisture
characteristic of a soil depends on the size and distribution of its pores. Fungi may
be the most effective soil organisms in stabilizing soil structure, and AM fungi
often comprise the largest portion of the soil microbial biomass (reviewed in: Augé
et al. 2003). The AM fungal hyphae grow into the soil matrix and create conditions
conducive to the formation of microaggregates and then their packing into macro-
aggregates (Celik et al. 2004; Rillig 2004) due to production copious amounts of
the glycoprotein glomalin (Rillig 2004). Through AM fungi-mediated effects on
soil structure, it seems logical to suggest that AM colonization of a soil might affect
its moisture retention properties and, in turn, the behaviour of plants growing in the
soil, particularly when it is relatively dry (Augé et al. 2004).
A comparative analysis of the results from a wide series of studies embracing
interactions between more than 60 genera of plants and five genera of AM fungi
(reviewed in: Augé 2001) showed that different effects of AM on host-plant/water
relations either under normal environmental conditions or during drought stress are
either related or unrelated to improved mineral nutrition of the host by fungi. Usually
the type of response depended on the prevailing environmental conditions and
the particular plant–microbe association studied. It was generally concluded that
mycorrhizal effects on plant/water relations are not as dramatic and consistent as
those relating to phosphorus acquisition and host growth.
Increase of Plant Resistance to Pathogens
Plants colonized by AM fungi have been demonstrated to manifest increasing resis-

tance to some pathogens. In particular, AM fungi have been observed to reduce the
susceptibility of tomato to pathogenic fungi such as Phytophthora parasitica (Cordier
et al. 1998; Dassi et al. 1998) and Alternaria solani (Fritz et al. 2006) as well as leading
to an inhibition of the bacterial leaf pathogen Xanthomonas campestris in Medicago
truncatula (Liu et al. 2007). Whether such increased resistance to pathogens is a
consequence of improved plant fitness or is due to a specific defence response
induced by AM fungi is unknown. At the same time AM formation has been shown
to be accompanied by a complex pattern of local and systemic changes in plant cell
defence reactions (Ruiz-Lozano and Azcon 1996) and gene expression, including the
induction of a functional defence response (Liu et al. 2007). Most probably, systemic
plant defence reactions caused by AM fungi occur as a type of SAR (systemic
acquired resistance) or ISR (induced systemic resistance) (see for review: Pieterse
et al. 2001; Vallad and Goodman 2004) as several authors refer to an induction of the
biosynthesis of signalling molecules in arbuscular mycorrhizal plants typical of the
signal transduction pathways such as pathogenesis related proteins (PR-proteins)
(Dassi et al. 1998) and jasmonate (Hause et al. 2002).
A range of processes occurring in plants as a result of pathogen invasion (plant
defence responses) also take place during interactions with AM fungi. These include
signal perception, signal transduction and defence-related gene activation.
Hypersensitive responses have been observed to take place with compatible and non-
compatible combinations of plants with AM fungi (Salzer et al. 1999). Reactions
similar to an ‘oxidative burst’ are typical for the AM fungus penetration into epider-
mal cells (Salzer et al. 1999). Some elements of signal transduction pathways acti-
vated during pathogenesis are also activated at the early stages of AM development.
During appressorial formation in different plants catalase and peroxidase activities are
increased and are associated with salicylic acid accumulation (Spanu and Bonfante-
Fasolo 1988; Blilou et al. 2000a; Lambais 2000). The process of salicylic acid accu-
mulation also correlates with expression of genes encoding for the lipid transport
protein (LTP) and phenylalanine-ammonium-lyase (PAL) (Blilou et al. 2000b), the
later being of relevance to lignification of the plant cell wall. Localized expression of
defence-related genes or an accumulation of gene products in arbuscule-containing
cells has also been observed (Harrison 1999; Bonanomi et al. 2001).
In AM, as in other compatible biotrophic interactions, the defence response appears
to be weak and occurs transitorily during the early phases of colonization (Harrison
1999). By the time mycorrhizal fungi are well established in the roots, responses are
often similar to or lower than those in non-mycorrhizal control plants, suggesting that
the suppression of plant defence responses may contribute to successful, compatible
AM fungal colonization (Harrison 1999; Garcia-Garrido and Ocampo 2002).
Important evidence supporting the defence-related reactions has been obtained
from AM-defective mutants induced in legumes, such as Pisum, Medicago, and
Lotus spp. While in the wild-type plants the AM development is characterized by
weak or suppressed defence responses (possibly, due to special fungal signals), in
mutants they appear stronger and involve changes at several different levels, including
increased production of the signalling compound salicylic acid (Blilou et al. 1999),
expression of defence-related genes (Harrison 1999), and accumulation of defence-
associated proteins (Dassi et al. 1998) or phytoalexins (Bonanomi et al. 2001;
Blilou et al. 1999; Gollotte et al. 1993). Cytological studies have identified the cell
wall appositions on the inside of root cell walls adjacent to the appressoria of pea
mutants (Gollotte et al. 1993) and death of invaded epidermal cells of Lotus mutants
(Bonfante et al. 2000).
Bacterial Endosymbionts of Mycorrhizal Fungi
A few fungi, including some Glomeromycota species (AM fungi and Geosiphon
pyriforme) (Scannerini and Bonfante 1991; Bianciotto et al. 1996; Bianciotto et al.
2000; Schüßler and Kluge 2001; De Boer et al. 2005) and in the ECM basidiomycete
Laccaria bicolor (Bertaux et al. 2003) are reported to have endocellular bacteria. In
the cytoplasm of several AM fungal species (Glomus versiforme, Acaulospora laevis,
Gigaspora margarita) are Gram-negative rod-shaped bacteria, which have not been
grown on cell-free media (Scannerini and Bonfante 1991; Bianciotto et al. 1996;
Mosse 1959; MacDonald and Chandler 1981; Bianciotto et al. 2004; Jargeat et al.
2004). In the most of fungal species evaluated intracellular bacteria were detected
through all the stages of the fungal life cycle, i.e. spores, germtubes, and extra- and
intraradical hyphae, except arbuscules (Bianciotto et al. 1996). It has been shown that
a G. margarita spore contains an average about 20,000 bacteria (Bianciotto et al.
2004; Jargeat et al. 2004). These bacteria were initially assigned to the genus
Burkholderia on the basis of their 16S ribosomal RNA gene sequence, but were more
recently reassigned to a new taxon named Candidatus Glomeribacter gigasporarum
(Bianciotto et al. 2003); the provisional Candidatus designation is given to uncultured
bacteria (Murray and Schleifer 1994; Murray and Stackebrandt 1995).
The physiological role of the endosymbiotic bacteria in AM fungi is unknown
(Jargeat et al. 2004), but two bacterial genes have been found in the genomic library
developed from G. margarita spores including a putative phosphate transporter
gene (pst) and a vacB-like gene involved in host cell colonization by enteroinvasive,
pathogenic bacteria such as Shigella flexneri and Escherichia coli es components
of the bacterial endosymbiont genome (van Buuren et al. 1999). It is assumed that
these genes originate not from G. margarita, but endosymbiotic bacteria, and might
be of particular interest for future determination of the potential role of the bacte-
rium in mycorrhizal symbiosis (Jargeat et al. 2004; Ruiz-Lozano and Bonfante
1999; Ruiz-Lozano and Bonfante 2000).
The bacterium Candidatus Glomeribacter gigasporarum is supposed to be
vertically transmitted within AM fungi (Artursson et al. 2006). Cells of the bacteria
were demonstrated to be transmitted through five fungal vegetative generations of
asexual G. margarita spores (Bianciotto et al. 2004). The asexual reproduction
typical of AM fungi and the coenocytic nature of their mycelium may facilitate the
migration of the endosymbiotic bacteria from spores into hyphae, and thereby
allowing the vertical transmission to take place (Artursson et al. 2006). Additional
support to the hypotheses regarding the vertical transmission of Candidatus

Glomeribacter gigasporarum bacteria within AM fungi, and their obligate
endocellular nature comes from the fact that Candidatus Glomeribacter gigaspo-
rarum has a surprisingly small genome size for a bacterium of only around 1.4 Mb
in total and consisting of a chromosome of approximately 750-kb and an additional
replicon of approximately 650 kb (Jargeat et al. 2004). Small genomes are however,
often a feature of obligate endocellular bacterial species, for example beneficial
endosymbionts of insects: Buchnera (aphid’s symbiont) (Shigenobu et al. 2000;
Hosokawa et al. 2006) and other gut endocellular bacteria of different incects
(Hosokawa et al. 2006), Sitofhilus zeamais primarily endosymbiont (SZPE) (Dale
et al. 2002), as well as animal endocellular parasitic bacteria: Mycoplasma,
Rickettsia (reviewed in: Provorov et al. 2008). Considering processes like reductive
evolution where only those genes absolutely essential for survival in an intracel-
lular environment are retained (Dale et al. 2002), Candidatus Glomeribacter
gigasporarum represents a typical candidate for a permanent endosymbiont with its
small genome size (Artursson et al. 2006). One of the major future challenges
within this research area is to understand the functional significance of AM fungal
endosymbiotic bacteria. One important step in this direction will be the ability to
remove the bacteria from the fungal cytoplasm, enabling comparisons of fungal
effects on plants, in the presence and absence of the bacterial symbiont.
Legume-Rhizobia Root-Nodule (RN) Symbiosis
Leguminous plants can grow in the complete absence of combined nitrogen in soil/
substrate due to the fixation of atmospheric nitrogen by symbiotic nodule bacteria as
described elsewhere in this Book. Leguminous plants in collaboration with rhizobia
make a large contribution to the global nitrogen balance in natural and agricultural
ecosystems (Vance 2001). Nitrogen fixation occurs within special plant organs, typi-
cally root nodules (Fig. 1g) but in some associations stem nodules are also formed.
The ontogeny of nodulation represents a well-organized process based on the tightly
coordinated expression of specialized plant and bacterial genes. In the nodules bac-
teria differentiate into a specialized symbiotic organelle-like form, termed bacteroids
(Fig. 1h). The main enzyme of nitrogen fixation is a nitrogenase that has a complex
structure (Hirsch 1992; Sprent 2001). The legume nodules provide an ecological
niche for bacteria, as well as structure for metabolic/signal exchange between the
partners and for the control of symbionts by the hosts (Brewin 2004).
Specificity of RN Symbiosis
Leguminous plants have a world-wide distribution and are classified in one of the big-
gest angiosperm families, the Fabaceae, which contains 17,000–19,000 species
divided between three sub-families (Ceasalpinioideae, Mimosoideae, Papilionoideae)
and more than 700 genera (Allen and Allen 1981). With a single exception (Parasponia:
Ulmaceae), the ability for symbioses with rhizobia is restricted to Fabaceae, although
in eight related dicotyledonous families (Rosid I clade) an ability to form nodules with
the nitrogen-fixing actinobacteria Frankia is known (Wall 2000).
By contrast to legumes, their nitrogen-fixing microsymbionts do not constitute
a taxonomically coherent group of organisms. The majority of rhizobia belong to
the a-proteobacteria assigned into the family Rhizobiaceae solely on the basis of
their ability to nodulate legumes. A range of diverse genera (e.g., Azorhizobium,
Bradyrhizobium, Mesorhizobium, Rhizobium, Sinorhizobium) are distinguished
among these bacteria. Moreover, recently some b-proteobacteria (close to
Burkholderia, Cupriavidus, Pseudomonas and Ralstonia) and even some
g-proteobacteria have been discovered that can form nitrogen-fixing nodules with
legumes (Balachandar et al. 2007). All these bacteria (collectively called “rhizobia”)
vary enormously in their overall genome organization, location of “symbiotic”
(Sym) genes and their molecular organization and regulation (Spaink et al. 1998;
MacLean et al. 2007).
According to the early surveys of symbiotic specificity (Fred et al. 1932), legumes
were suggested to comprise a range of taxonomically restricted Cross-Inoculation
Groups (CIGs) within which cross-inoculation can freely occur, while species from
different groups do not cross-inoculate. The best studied examples of this classifica-
tion are represented by four CIGs: “Trifolium – Rhizobium leguminosarum bv. trifolii”,
“Pisum, Vicia, Lathyrus, Lens – R. leguminosarum bv. viciae”, “Galega – R. galegae”,
“Medicago, Melilotus, Trigonella – Sinorhizobium meliloti”.
It was later demonstrated however (Provorov 1994; Broughton and Perret 1999)
that such strictly defined specificity is limited to the herbage papilionoid legumes
living in temperate zones and representing the “galegoid complex”. For many tropical
and subtropical legumes a strict separation into CIGs is not typical, and a variable
degree of symbiotic promiscuity is observed. It is important to note that Lotus sp.
demonstrates promiscuity with respect to different rhizobia. Also, the species of
tribe Phaseoleae (Phaseolus vulgaris, Vigna unguiculata, Glycine max) may be
inoculated with strains representing different rhizobia genera isolated from the
taxonomically distant legume species. Moreover, some legumes may have the
taxonomically distant symbionts (soybean – Sinorhizobium fredii and Bradyrhizobum
japonicum) which share the genes required to nodulate a common host. Many
tropical rhizobia can inoculate very broad spectra of hosts: e.g., strain NGR234
(Sinorhizobium fredii) inoculates plants from 112 legume genera and also the
non-legume Parasponia.
The specificity of legume-rhizobia interactions is expressed only during the pre-
infection interactions when rhizobia recognize the roots of appropriate host plants
and colonize their surfaces. The interaction starts when the root-derived signals, in
particular, flavonoids (Fig. 3a), activate the rhizobial nodulation (nod/nol/noe)
genes (stage Ngi – nodulation gene induction) (Ovtsyna and Staehelin 2005). These
genes control the synthesis of lipo-chito-oligosaccharide (LCO) Nod factors (nodu-
lation factors) (Fig. 3c) which induce the early stages of nodule development
(Spaink 1995; Schultze and Kondorosi 1998a; D’Haeze and Holsters 2002).
With the exception of the genes nodA, B, C and D, the majority of nodulation
genes are host specific: they are responsible for affinities of different bacterial
species and strains to the particular legume hosts. When rhizobia are entering into
the vicinities of host roots, the nod/nol/noe genes are activated by the NodD pro-
tein (a sensor for root-derived signals) interacting with the universal nod-box
sequences located in the promoters of other nodulation genes (Long 1989). The
products of genes nodA, nodB and nodC catalyze the biosynthesis of Nod factor
core structures which are common for all rhizobia species (Spaink et al. 1991). In
turn, the products of other nod genes perform a decoration of this core with differ-
ent substitutes, which could be unique to a specific strain.
Nod factors represent a unique group of bacterial signalling molecules not known
outside legume–rhizobia associations. They are among the most potent developmental
regulators: their effect is expressed at concentrations as low as of 10−8–10−12 M. Nod
factors cause varying changes in plants leading to the formation of a root-nodule sym-
biosis; therefore they can be considered as hormone-like molecules. The core structure
of these molecules, common for all rhizobia species, consists of 3–6 residues of
N-acetylglucosamine and a fatty acid (acyl) chain (Fig. 3b).
The symbiotic specificity is dependent mainly on the chemical modifications
in Nod factor structures (Ovtsyna and Staehelin 2005). Narrow specificity typical
for rhizobia inoculating the galegoid legumes is usually associated with the presence
of unusual highly unsaturated acyl groups (2–4 double C=C bounds per 16–20
carbon atoms in the chain) while broad host specificity is correlated with the
presence of saturated mono-unsaturated acyl groups (none or only one double
C=C bound). Greater fine tuning of specificity towards the particular host species
and even towards some genotypes is correlated with chemical substitutions in the
oligo-chitin part of Nod factor. For example, the sulphation of the R6 position at
the reducing terminus encoded by nodP, nodQ, nodH genes is required for inter-
action between S. meliloti and alfalfa (lucerne) (Medicago) while the acetylation
encoded by nodX is necessary for R. leguminosarum bv. viciae to inoculate the
“Afghan” pea (Pisum sativum) lines (Broughton and Perret 1999). Interestingly,
for interaction with “Afghan” pea, nodX may be substituted functionally by nodZ
gene, controlling a dissimilar modification in R6 (fucosylation instead of acetyla-
tion) (Ovtsyna et al. 1999; Ovtsyna et al. 2000), suggesting a flexibility in the
mechanisms for signal reception (see also Section 4.3). These recognition and
early signalling processes are controlled in a “gene-for-gene” manner indicating
the similarities between early symbiosis development and host–pathogen interac-
tions (Tikhonovich et al. 2000).
It is likely that the control of specificity of interactions between legume plants
and nodule bacteria is much more complex than current evidence suggests.
Recognition of the structural differences in the Nod factor molecule may not turn
out to be the one and only mechanism controlling the specificity of legume–
rhizobial interactions. Further control of host specificity may result from interac-
tions between the bacterial surface molecules (some polysaccharides and proteins)
(Becker and Pühler 1998; Lugtenberg 1998) and the lectins located on root hair
surfaces (Jones et al. 2008). Transfer of the pea lectin gene Psl into the clover roots
via the Agrobacterium rhizogenes mediated transformation resulted in an ability for
clover to be nodulated by pea rhizobia (Diaz et al. 1995). Similar results were
obtained after transfer of the soybean lectin gene into trefoil plants (van Rhijn et al.
1998). Recently, a rhizobia–legume signalling system, alternative to the “flavonoid/
Nod factor” circuit has been reported. Other low-molecular weight compounds hav-
ing non-flavonoid structures play important roles in RN symbiosis development
determining polarization of a bacterial cell and controlling adhesive properties of
rhizobia (J.A. Downie, 2006 personal communication).
Giraud et al. (Giraud et al. 2007) have recently reported results of a complete
genome sequencing of two symbiotic, photosynthetic, Bradyrhizobium strains,
BTAi1 and ORS278. It was shown that canonical nodABC genes and typical
lipochitooligosaccharidic Nod factors are not required for symbiosis in some legumes
(especially, aquatic legumes Aeschynomene sensitiva (from Africa) and A. indica
(from North America)). Mutational analyses indicated that these unique rhizobia
use an alternative pathway to initiate symbioses, here a purine derivative may play
a key role in triggering nodule formation. These results suggest that signal interac-
tions and specificity of symbiosis formation are more complicated and variable than
it was supposed; but in any event, the activity of signalling cascades results in
transcriptional changes in the root, nodule morphogenesis and nitrogen fixation.
Development and Functioning of RN
Unfortunately, only very limited numbers of Rhizobium–legume interactions have

been studied in detail (Hadri et al. 1998) but it is possible to identify at least two
major types of root nodule, namely those with indeterminate and those with deter-
minate nodule meristems (NMs) (reviewed in: Sprent and James 2007). In this
section, the development of symbiotic structures in the best-studied indeterminate
nodules formed by evolutionarily advanced legumes from the “galegoid complex”
(e.g. Pisum, Medicago, Trifolium) is described. These plants use one of the most
complicated and evolutionarily advanced nodulation programmes which may pro-
vide a conduit for a greater variety of simpler nodulation programmes used by
several representatives of the Fabaceae (Sprent 2001).
Early cytological responses to Nod factors in root hair cells have been described
in several legumes (Cardenas et al. 1998; Esseling et al. 2003). The first event,
detectable only a few seconds after Nod factor application, is a Ca2+ ion influx at
the root hair tip, causing a several-fold increase in cytosolic calcium concentration
(Felle et al. 1999). This is associated with a transient depolarization of the plasma
membrane (Lhuissier et al. 2001; Shaw and Long 2003a). Within 20–30 min
following application of Nod factors, there is suppression of peroxide release from
the roots (Shaw and Long 2003b), and a reorganisation of the actin and microtubule
networks in the root hair cells (Timmers et al. 1999; Sieberer and Emons 2000;
Sieberer et al. 2002). There is also a second calcium event, the induction of periodic
oscillations of cytoplasmic calcium concentration (Ca2+ spiking), which is observed

in the perinuclear area (Ben Amor et al. 2003; Oldroyd and Long 2003; Shaw and
Long 2003a). Following application of Nod factors, the earliest observed
morphological event is a cessation of tip-growth of root hairs which occurs within
one hour. The arrest of apical growth is associated with the swelling of root hair tips
(Lhuissier et al. 2001).
Nodule morphogenesis in clover (Nutman 1969), pea (Bond 1948; Newcomb
1976; Brewin 1998; Tsyganov et al. 2002; Voroshilova et al. 2009) and alfalfa
(lucerne) (Vasse et al. 1990) starts on rhizobial colonization of roots with strong
deformation and curling of root hairs (stage Hac – Hair Curling) (Fig. 1i, 2), this
reaction is mediated by Nod factor action. Then the curl is colonized by bacteria at
high density (stage Crh – Colonization of the pocket in the curled root hair)
(Tsyganov et al. 2002) (Fig. 2). Within the curls, the root hair cell wall is “weak-
ened” and the plasmalemma invaginates initiating growth of a special tunnel struc-
ture – Infection Thread (IT, stage Iti – Infection Thread Growth Initiation) – which
starts growing through the root hair cell first (stage Ith – Infection Thread Growth in
root Hair cell) (Fig. 2). These processes mainly involve the plant molecular
machinerychanging cell wall composition and biogenesis of intracellular membranes
(endoplasmic reticulum and Golgi vesicles) ensuring construction of a pathway for
bacteria into the host (Gualtieri and Bisseling 2000; Brewin 2004).
Bacteria move, thereafter into the root cortex through IT growing into the root
perpendicular to the root surface in a “cell-to-cell” or “transcellular” manner
(Brewin 1998; Brewin 2004) and a certain amount of IT branching can be observed
(Fig. 1-i). The transcellular growth is associated with the development in cortical
cells of “Pre-Infection Threads” (PITs) which represent the cytoplasmic zones
with specially reorganized cytoskeletal components which facilitate a rapid pas-
sage of ITs (van Brussel et al. 1992). In IT the walls are built up using plant cell
wall polymers, while the internal space contains a martix synthesised by both
partners. Several proteins are secreted by the plant into the IT matrix controlling
the rhizobial activity, among them extensin-like and proline-rich proteins are most
abundant (Brewin 2004). Although IT develops either between or within the plant
cells, the rhizobia inside IT have a topologically intercellular location (Brewin
1998).
Simultaneously with the root hair curling and IT growth initiation, and on
Nod factor action, cell division in the inner cortex of the root is initiated due to
re-activation of the mitotic cycle in G0/G1-arrested plant root cells that leads
to formation of Nodule Primordium (NP) (Fig. 1j) this later develops into the
nodule meristem (NM) (Fig. 1k) (Vasse et al. 1990; Hadri et al. 1998; Foucher and
Kondorosi 2000; Demchenko and Demchenko 2001; Young et al. 2003; Voroshilova
et al. 2009). Consequently, the mitotic reactivation (Fig. 1m), dedifferentiation and
proliferation of the cortical cells induced by Nod factors (stage Ccd – Cortical Cell
Division) (Fig. 2) leads to formation of a mature persistent nodule meristem
through a series of successive developmental stages. Then the advancing IT enters
the NP where plant cells with endocytosed bacteria can be found.
Further growth of the NP tissues and ITs results in a formation of juvenile nodules
as they emerge from the root (Fig. 1k). At this stage, the nodules do not yet have the
histological differentiation characteristic for mature indeterminate nodules described
by Vasse et al. (1990). Nevertheless, the transient histological patterns in juvenile
nodules are in preparation for the future developmental pathways of mature nodule
structures. First, the IT is visible which in combination with the surrounding cells
provides growth and forms the “entrance” for bacteria to the root. Second, peripheral
layers of NP cells which are not penetrated by ITs can be distinguished. Furthermore,
dividing cells on the distal side of the NP form a hemispherical structure located
above the plant cells already colonized by bacteria and surrounding the original
“entrance” pathway for the IT into the root. At the distal part of this hemispherical
structure actively dividing plant cells are observed while in the proximal cell layers
of this hemispherical structure simultaneous plant cell divisions and growth of
branching ITs occur. At the same time the orientation of IT growth is completely
reversed and turned backwards toward the nodule apex (Libbenga and Harkes 1973;
Timmers et al. 1999). It is proposed that the presence of proliferating cells harbour-
ing infection threads is a prerequisite for normal formation of the Apical Nodule
Meristem (Stage Anm) (Voroshilova et al. 2009) (Fig. 2). Therefore, only after com-
pleting the colonization of the plant cells with ITs, the plant cells leave the mitotic
cycle under the influence of at least one plant cell cycle regulator (Cebolla et al.
1999) and undergo several rounds of endoreduplication in preparation for hosting
intracellular bacteria and the control of their differentiation passing to the symbiotic
(bacteroid) state (Mergaert et al. 2006). In parallel to establishment of the final nod-
ule anatomy, the histological zones (I, II, III, IV) of the inner tissue of indeterminate
nodules can be observed as described by Vasse et al. (1990) (Fig. 1l).
The key stage of endosymbiosis development is represented by the bacterial “release”
from infection droplets into the plant cytoplasm via an endocytosis-like process. Infection
droplets are “unwalled” regions of the ITs (they usually arise at the sides or at the tips of
growing of ITs) which invaginate into the plant cells and in which the bacteria come into
“direct” contact with the plant cell plasma membrane. At these contacts, the membrane
vesicles which contain single bacterial cells are segregated into the plant cell cytoplasm.
Therefore, from the start of the “release”, bacteria inside plant cytoplasm are surrounded
by specialised (PeriBacteroid) Membranes (PBMs) which originate from the plasma-
lemma of infection droplets and are completed by products from endoplasmic reticulum
and Golgi vesicles. The bacterial cell(s) surrounded by PBM represent a major intracel-
lular symbiotic compartment – the symbiosome (Roth and Stacey 1989). Within symbio-
somes the bacteria differentiate into bacteroids (stage Bad – Bacteroid Differentiation)
(Fig. 1h, 2). They are considerably larger than free-living bacteria and have an altered
shape (in pea nodules the bacteroids are of X- or Y-shape) (Fig. 1h).
Bacteroid differentiation is correlated with a repression of many bacterial genes, with
the exception of oxygen-regulated symbiotic genes, the products of which are directly
involved in the processes of symbiotic nitrogen-fixation (e.g. fixNc) (Tsyganov et al.
2003). Irrespective of their related to symbiosis, the expression of bacterial genes gradu-
ally declines as the programme of bacteroid differentiation advances (Jimenez and
Casadesus 1989; Quispel 1998; Perret et al. 1999; Margolin 2000; Tsyganov et al. 2003).
Decline occurs in parallel with the gradual morphological changes observed in the
bacterial cells during such developmental process. Even actively expressed genes at
the beginning and/or intermediate stages of symbiotic interactions, such as the
flavonoid-induced nodulation genes and dctA appear to be subject to this decline in
expression during bacteroid development (Tsyganov et al. 2003).
These observations (Tsyganov et al. 2003) agree with previous reports (Vasse
et al. 1990) suggesting that development of symbiosomes after release (“endocy-
tosis”) of bacteria into plant nodule cells is not a simple result of endocytosis,
but rather a gradual process involving several additional stages, each controlled
by different plant genes. Within this perspective, the process of Bacteroid
Differentiation (a separate stage in symbiosis development, Bad) could resemble
an adjustable process of adaptation to the symbiosome environment, believed to
be of a stressful nature (Brewin 1998; Santos et al. 2000; Nogales et al. 2002). The
final product of this adaptive process could be the mature, actively nitrogen-fixing
bacteroid, which is transformed into an organelle-like state and has lost the
capacity to resume a free-living growth (Jimenez and Casadesus 1989; Quispel
1998; Tsyganov et al. 2003).
Synthesis of nitrogenase (the enzyme catalysing reduction of N2 into NH4+) and
other proteins involved in nitrogen fixation is induced in bacteroids. Therefore,
symbiosomes are organelle-like units of plant cell responsible for nitrogen fixation
(Brewin 1998; Tsyganov et al. 2003). A pronounced differentiation is typical for
rhizobia-infected plant cells, such as an increase in internal membrane structures
participating in the PBM formation and biosynthetic processes. Polyploidization
and chromatin decondensation are typical for these cells correlating with an
elevated transcription activity (Pawlowski and Bisseling 1996). Biochemically
plant cell differentiation is expressed as a de novo synthesis of many proteins
including leghaemoglobin and nodule-specific isozymes of carbon and nitrogen
metabolism (Vance and Heichel 1991). Leghaemoglobin binds oxygen actively
ensuring its transport towards symbiosomes (which are characterised by the
intensive respiration necessary to support energy consuming nitrogen fixation)
and microaerobic conditions inside the nodules (required for the nitrogenase
activity). The carbon and nitrogen metabolic enzymes responsible for the energy
supply to nitrogenase and for the assimilation of fixed nitrogen are nodule specific
(Fedorova et al. 1999).
The processes described above result in formation of a complex nodule of
indeterminate type (Fig. 1g). Its major structures are: (1) central rhizobia-infected
tissue where nitrogen fixation occurs (histological zone III); (2) peripheral vas-
cular bundles which transport photosynthates into and nitrogen-compounds out
of the nodule; (3) apical meristem which ensures nodule growth (histological
zone I) (Fig. 1l). The latter is responsible for a continuous renovation of
“infected” (colonised) tissue and its differentiation into zones corresponding to
the different stages of endosymbiosis development (Brewin 1991, 1998, 2004;
Mergaert et al. 2006).
The final stage of symbiosis development is represented by the senescence of
nodule tissue in the basal histological zone IV of the nodule where the symbiosomes
are transformed into lytic compartments, bacteroids and other symbiotic sub-cellular
compartments are digested by the host cells. Moreover, in alfalfa (lucerne) the
release of bacteria into the decaying nodular tissue was observed followed by
intensive propagation of bacteria without any signs of bacteroid differentiation
(Timmers et al. 2000). This process, localized in a special histological zone V
(occasionally, even in the actively nitrogen-fixing nodules), may be responsible for
an increase in the number of nodule bacteria cells in the environment after the
decay of the nodule and for the movement of bacteria towards an improved nitrogen-
fixing activity in symbiosis with the host plant.
A fine balancing of partners’ interactions is also highly dependent on exopoly-
saccharides (EPS), lipopolysaccharides and cyclic glucans located on bacterial
surfaces (Breedveld and Miller 1994; Leigh and Walker 1994; Brewin 1998; Cheng
and Walker 1998; Niehaus et al. 1998; Broughton and Perret 1999). The surface
rhizobia components are suggested either to suppress the plant defence systems or
to make bacteria tolerant of them but in either case they play an important role in
all stages of the colonisation process: during infection thread formation and growth,
endocytosis of bacteria and symbiosome differentiation.
In addition to the local mechanisms of endosymbiont control, legumes possess
a systemic autoregulation of nodulation by signals circulating between roots and
shoots. Mutants impaired in autoregulation were selected as supernodulating
(phenotype Nod++). Usually these mutants retain nodulation in the presence of high
nitrate doses inhibiting symbiosis in wild-growing plants (phenotype Nts – Nitrate
tolerant symbiosis). Therefore, autoregulation represents a strategy through which
the host plant can balance the symbiotrophic nitrogen nutrition with the energeti-
cally cheaper combined nitrogen nutrition. Involvement of the shoots in control of
Nod++ phenotypes was demonstrated by means of shoot/root reciprocal grafting
(Caetano-Anolles and Gresshoff 1991; Sagan and Duc 1996). The autoregulatory
response may be mediated by an abortion of excessive IT in the cortical cells via a
mechanism similar to hypersensitive reaction between plants and some pathogens
(Vasse et al. 1993).
The ontogeny of nodulation exists along with other developmental and regula-
tory plant processes (van Buuren et al. 1999; Beveridge et al. 2007) in which
phytohormones, play important roles. Physiological and developmental effects of
phytohormones on nodulation have long been recognized and are extensively
described (Guinel and LaRue 1992; Penmetsa and Cook 1997; Guinel and Geil
2002), however, the mechanisms of phytohormone regulation of nodule initiation
remain relatively obscure. The gaseous plant hormone ethylene and its precursor
1-aminocyclopropane-1-carboxylic acid (ACC) are some of the earliest phytohor-
monal signals associated with the ‘negative’ regulation of nodule number. For
example, treatment with ethylene or ACC inhibited nodulation in a wide range of
legumes. In contrast, exposure to inhibitors of ethylene activity, for example
aminoethoxyvinylglycine (AVG), and silver ions increased nodulation in many of
the legumes tested. Such inhibitors also partially restored nodulation in selected
low-nodulating pea mutants (Guinel and LaRue 1992). Ethylene is supposed to
regulate the number of developing nodules by controlling the position of nodule
initiation and the ability of plant cells to initiate nodule foci (Nukui et al. 2000;
Lohar et al. 2009). It has been reported that transgenic ethylene and ACC in
sensitive L. japonicus lines increased nodule number per plant by comparison with
wild type (Lohar et al. 2009). The Lotus transgenic lines described were not, however,
super- or hypernodulated over a large portion of the root, and did not exhibit nitrate
tolerance in nodulation, demonstrating that ethylene regulates nodulation at other
sites and is not directly functional in autoregulation of nodulation and nitrate
inhibition of nodulation.
Immediately after induction of nodulation the processes aimed at regulating the
performance of the endosymbiont start inside the host. The importance of these
processes is evident from a much greater potential for propagation of bacteria with
respect to plant cells. The perisymbiotic interfaces (IT walls, symbiosome membranes)
ensure separation of the partners and play an important role in regulation of the internal
bacterial population. In addition to these “physical” barriers, a range of processes
occur inside nodules which are similar to the defence reactions induced by pathogens.
They include synthesis of flavonoids, phenolics, chitinases, callose, peroxidases,
extensins and some pathogen-regulated (PR) proteins (reviewed in: Tikhonovich and
Provorov 2007). In nodules however, these reactions are not as intensive as those
occurring during pathogenesis because the level of defence components in plants
undergoing nodulation is much lower than in plants responding to pathogen attack. It
is likely therefore, that these reactions are probably regulated in a different way, by
regulatory symbiotic genes that control the number of symbiotic microorganisms and
the development of symbiotic compartments rather than determining their activity.
Expectedly, early stages of both determinate and indeterminate nodules are
encoded by orthologous genes in different legumes. Moreover, recent achievements
in molecular cloning and sequencing the legume genes responsible for nodule
development have shown that some of these genes are involved not only in nodula-
tion, but also in mycorrhization. The developmental genetics of early stages of RN
and AM symbioses is described in the next section.
Developmental Genetics of RN and AM Symbioses
In RN and AM symbioses the genetic systems of the partners are highly integrated
because the complex developmental processes which lead to the formation of inter-
cellular and sub-cellular symbiotic compartments are controlled by both organisms.
The development and function of the symbioses is reliant to greatest extent on the
plant. Developmental genetics in RN is now well described because both plants and
nodule bacteria can be subjected to genetic analysis during nitrogen-fixing nodule
formation and functioning. There has been less investigation of AM systems. Mainly
this is due to the difficulties encountered in culturing AM-fungi, caused by their
obligate symbiotic lifestyle and impossibility of using selective media. Additionally,
genetic analysis of AM fungi is more complex because of their heterokaryotic nature
and lack of sexual process (Smith and Read 1997; Pawlowska and Taylor 2004).
The plant genes involved in development of RN and AM symbioses may be

divided into two groups, depending on how they are identified. The genes of one
group, Sym genes (from symbiosis, the term was suggested by Lie (1971), have
been identified with the use of formal genetic analysis (commencing with the selection
of plant mutants defective in nodule development). The other group of genes
comprises nodulins (from nodule) (van Kammen 1984), mycorrhizins (from mycor-
rhiza) (Gianinazzi-Pearson 1996; Niehaus et al. 1998), and symbiosins (from
symbiosis) (Kistner et al. 2005; Küster et al. 2007). These genes were identified by
molecular genetic methods, through identification of proteins and mRNAs synthe-
sized de novo in root nodules (nodulins) or roots colonized by AM fungi
(mycorrhizins). The genes whose expression is induced during the development of
both RN and AM are called symbiosins (Küster et al. 2007). Genes of these groups
are suspected to be playing different roles in the processes which may be referred
to as the “management of microsymbionts” within plant roots.
Most of the known nodulins/mycorrhizins/symbiosins are cloned and sequenced;
however, functions for many of them have been identified only preliminarily
using the sequence data of the encoding genes and location of gene products in
the symbiotic compartments.
The products of several nodulin genes represent the structural elements of newly
constructed temporary compartments (ITs, symbiosomes) developed during sym-
biosis (Albrecht et al. 1998). Also, some of nodulins may play several roles in
modulating the hormonal status of developing nodules (Kumagai et al. 2006; Wan
et al. 2007). Recently in silico and microarray-based transcriptome profiling has
allowed the identification of nodulins and of other classes of these genes, mycor-
rhizins and symbiosins. All of these are activated in response to an AM fungal
signal, or by either rhizobial or AM fungal stimuli, respectively (Kistner et al.
2005). Several hundred genes were found to be activated in different stages of
either symbiosis, with almost 100 genes being co-induced during nodulation and in
AM formation. These co-induced genes can be associated with different cellular
functions required for symbiotic efficiency, such as facilitating transport processes
across the perisymbiotic membranes that surround the endosymbiotic bacteroids in
root nodules and the arbuscules in AM roots (Küster et al. 2007).
Most of the nodulins, mycorrhizins and symbiosins seem to play subordinate
roles in symbioses, having an influence on the functionality and stability. In turn,
the main, regulatory role controlling symbiotic programmes is attributed to the Sym
genes. These genes are usually not expressed outside of the symbiotic structures and
there are many examples of high levels of functional and sequence homologies
between them in different legumes. The first genes of this group were identified in
spontaneous mutants obtained from natural populations of legumes (Nutman 1946)
and afterwards in experimentally induced mutants deficient in nodulation and
nitrogen fixation (Nod– phenotype) (Jacobsen 1984). Subsequently, it was demonstrated
that mutations in some of these genes also affect the ability of plant to form AM
(Gianinazzi-Pearson 1996). The existence of common genes necessary for both
AM and RN symbioses development suggests that both symbioses were more
closely linked than was previously suspected. Cloning and sequencing of the common
symbiotic genes helped to show that AM and root-nodule symbioses share a common
signalling pathway, which probably was established during evolution of the plant–AM
symbiosis and was recruited afterwards into RN development (Parniske 2008).
Developmental Genetics of RN Symbiosis
Mutant plants which cannot form nodules (Nod–, no nodules) or form nodules
devoid of nitrogenase activity (Fix–, no nitrogen fixation) may be induced either by
physical or by chemical mutagens or be present in natural populations or in culti-
vars. Usually the mutants are selected on the basis of impaired growth in N-free
substrates. More than 80 genes have been identified which are responsible for nod-
ule development (Hadri et al. 1998; Borisov et al. 2007) by using these mutants in
different legumes (Pisum sativum L., Glycine max (L.) Merr., Clover spp.,
Medicago sativa L., Phaseouls vulgaris L., Cicer arietinum L., Vicia faba L.).
More than 40 genes were identified in pea (Pisum sativum L.) which is still one
of the most convenient models for the genetic analysis of nodule development.
As a rule, plant alleles determining Nod- phenotype are recessive, while Fix– alleles
may be either recessive (in pea, clover, alfalfa) or dominant (in soybean). Some
“symbiotic” mutations alter the root or shoot morphology, while the majority of
these mutations do not affect development of “non-symbiotic” organs.
Analysis of genetic variability of the symbiotic properties of pea plants was
pioneered by Govorov (1928) and Razumovskaya (1937). They found that some
genotypes of pea originating from Afghanistan (which is part of the Central-Asian
centre of origin for these plants) fail to form nodules with root nodule bacteria taken
from European soils. Although some plants from the region with different genotypes,
did form the nodules when inoculated with bacteria from Europe. Genetic analysis of
local Afghan host strains by Lie (1971), identified the first symbiotic (Sym) pea genes.
The later genetic analysis of experimentally induced mutants in pea allowed the
dissection of the major stages of symbiosis development into simple (elementary)
steps, each step being controlled by a few genes or even single ones (Borisov et al.
1997, 2007; Tsyganov et al. 1998, 2002). To identify the order of expression of
genes controlling the sequence of developmental stages an analysis of single and
double mutant lines has been made. The developmental epistasis detected in some
cases permitted the genes to be arranged in their order of activity.
The results of phenotypic characterization of single and double mutants suggested
that the integrated programme of nodule development is composed not from a chain of
successive stages (each begining only when the preceeding stages had been completed)
rather by two sub-programmes which: (a) are implemented to some extent indepen-
dently from each other; and (b) may be dissected into succeeding steps (Fig. 2).
The stages in the sub-programme of plant tissue colonization and differentiation
of bacteria into symbiotic forms (the so-called bacteroids) are as follows: Hac –
root hair curling; Crh – colonization of the pocket formed by curled root hair cells;
Iti – infection thread growth initiation; Ith – growth and development of the infec-
tion thread in the root hair cell; Itr – growth and development of the infection thread
in the root tissue; Itn – growth and development of the infection thread in the young
root nodule tissue; Idd – infection droplet differentiation; Bad – bacteroid differen-
tiation, i.e., transformation of bacteria into organelle-like symbiotic forms; and Nop –
structural and functional nodule persistence. The sequence of stages in nodule
tissue differentiation is defined as follows: Ccd – cortical cell divisions, division of
the cells of internal root cortex; Npd – nodule primordium development; Anm –
apical nodule meristem formation; and Nmp – nodule meristem persistence.
These sub-programmes are controlled by the same sets of plant Sym genes
(Fig. 2), but, their functional orders in the colonisation (infection) and symbiosome
differentiation and nodule tissue development subprograms may be not identical
(Tsyganov et al. 2002; Borisov et al. 2007). Selected stages of both sub-programmes
are described in previously in detail, here the order of pea (P. sativum) regulatory
gene functioning identified after experimental mutagenesis followed by classical
genetic analysis and phenotypic characterisation is given (Fig. 2).
Plant Genes Implicated in AM Development
The most productive approach allowing the identification of plant mycorrhization

regulatory Sym genes is to search among those of legumes implicated in nodulation.
Duc et al. (1989) were first to demonstrate that some legume mutants that were
impaired in nodule development also blocked AM formation. As a result of analysis
of diverse collections of legume mutants with impaired nodulation several AM
developmental stages controlled by common symbiotic genes were identified. In
particular, for pea eight common Sym genes (Fig. 2) and two developmental stages
were identified: Pen – root penetration, and Arb – arbuscule development (accord-
ing to Marsh and Schultze 2001). Concurrently two additional stages were found
for Lotus japonicus: Coi (Marsh and Schultze 2001) – cortex invasion; Ici (Senoo
et al. 2000a) – inner cortex invasion.
To date the vast majority of mutants isolated in different legumes are defective in
the early stage of AM formation and block fungal penetration (Pen−) into the root
epidermis (previously, the phenotype has been referred as Myc−1 (Gianinazzi-Pearson
1996)). These include the first AM mutants isolated from populations of P. sativum
and Vicia faba (Duc et al. 1989), as well as mutants identified in Medicago truncatula
(Sagan et al. 1995), Medicago sativa (Bradbury et al. 1991), and more recently in L.
japonicus (Senoo et al. 2000b). All these mutants are Nod– and carry mutations in
early symbiotic genes, for example PsSym8,1 PsSym9 and PsSym9 for pea (Fig. 2).
Commonly associated with this type of mutantion is the formation of complex
and somewhat abnormal appressoria as compared with the wild-type (WT). This
can be seen clearly in the mcbep (mycorrhizal colonization blocked at epidermis)
mutants of L. japonicus (Senoo et al. 2000b), the alfalfa (M. sativa) line MN-NN1008
(Bradbury et al. 1991), and the P2 (Pssym9) mutant of pea (Duc et al. 1989).
1
Ps, Lj, Mt are abbreviations to the Latin plant names Pisum sativum, Lotus japonicus, Medicago
truncatula, respectively.
Irrespective to fungal genotype, each of these mutations prevents penetration of the

root epidermis and results in the formation of complex hyphal branching, multiple
and unusually swollen appressoria. This effect has been suggested to be a conse-
quence of the continuing but unsuccessful attempts by the fungus to penetrate
through the root surface (Bradbury et al. 1991; Senoo et al. 2000a). This is possibly
why it was found that the number of appressoria in the roots of Pen– mutants is
often increased as compared with the wild-type (Bradbury et al. 1991; Senoo et al.
2000b) although sometimes there may be decreases (Duc et al. 1989) or no difference
(Bradbury et al. 1991; Bonfante et al. 2000; Senoo et al. 2000b). Also the number
of appressoria varies between Pen– mutants depending on the species of the AM
fungi (Bradbury et al. 1991).
It has been shown that plant defence responses and signalling events are altered
in Pen– mutants, for instance: cell wall thickening and increases in the deposition
of callose and phenolics by epidermal and hypodermal cells at the points of contact
with appressoria (Gollotte et al. 1993); increased levels of endogenous salicylic
acid and transcripts of a number of defence related genes in the mutant roots com-
pared with the wild-type plants (Blilou et al. 1999; Ruiz-Lozano and Bonfante
1999). By using the M. truncatula mutant TRV25 (Sagan et al. 1995) possessing an
epidermal block, a common MtDMI3 gene (orthologue for PsSym9 pea gene)
(see later in Section 4.3.1) has been found which is essential for the assembly of
the pre-penetration apparatus in epidermal cells (Genre et al. 2005).
By contrast to the mutants in which an infection process aborts after appressorial
formation, a large collection of mutants were described in L. japonicus which are
blocked prior to cortex invasion (Coi–) although they form normal appressoria and
are capable of penetrating the rhizodermis (Wegel et al. 1998; Bonfante et al. 2000;
Parniske 2000). The mutants having phenotypes (Ici–) that are affected to varying
degrees in the rate of intercellular progression by hyphae. These include pheno-
types in which inner cortical invasion does not occur as in the case of the mcbee
(mycorrhizal colonization blocked between epidermis and exodermis] and the
mcbex (mycorrhizal colonization blocked in exodermis] mutants of L. japonicus
(Senoo et al. 2000a). It should be noted, that in the case of the mcbex mutants,
although they predominantly express an Ici– phenotype, the overproduction of
deformed appressoria and occasional formation of abnormal arbuscules is found. In
pea and alfalfa, the stages Coi or Ici was not identified, and in all mutants blocked
for cortex colonization then epidermis colonization is similarly affected (Duc et al.
1989; Bradbury et al. 1991; Sagan et al. 1995). Very likely, these stages are absent
in such plants because they lack an exodermal layer which distinguishes them from
L. japonicus. An exodermis is known to be an additional barrier for fungal penetra-
tion (Brundrett 2002).
The cloned legume genes controlling Pen and Coi stages are shown to be
responsible for the signal transduction cascade inside the root which is common for
both RN and AM (see later in Section 4.3).
Some mutants permit the colonization of the inner cortex but do not form arbus-
cules, as exemplified by R69 of P. vulgaris (Shirtliffe and Vessey 1996) and the
Nod−/Fix–, MN-IN3811 mutant of alfalfa (Bradbury et al. 1991), or form truncated
(stumpy) arbuscules (RisNod24, RisNod26 (Pssym36) isolated in P. sativum

(Gianinazzi-Pearson 1996)), whereas appressoria formation, root penetration and
hyphal proliferation within the cortex proceed normally. The phenotype was
designated as Arb– (Marsh and Schultze 2001), while previously it was denoted as
Myc–2 (Gianinazzi-Pearson 1996). This is the latest stage of AM development which
can be revealed at present using the legume mutants defected in nodulation.
The two P. sativum mutants SGEFix–-1 (Pssym40), SGEFix–-2 (Pssym33), form-
ing root nodules which do not fix atmospheric nitrogen, were characterized for the
dynamics of AM development. Mutation in the Pssym33 gene caused decreased
mycorrhizal colonization and delayed arbuscule development (Rmd– phenotype),
whereas mutation in Pssym40 accelerated AM colonization and arbuscule turnover
(Rmd++ phenotype) (Jacobi et al. 2003). Since the both mutants produce leaky phe-
notypes dependent on temperature, it seems likely that these genes are involved in
some kind of hormonal regulation of the host plant and in symbiosis development.
Interestingly, several hypernodulating mutants, defective in systemic autoregula-
tion of nodulation, are also characterized by increased mycorrhizal colonization
and arbuscule development. The examples of such mutants are P88 (Pssym29)
induced in P. sativum, those of M. truncatula impaired in the MtSUNN gene –
MycA++ phenotype (Morandi et al. 2000; Schnabel et al. 2005a), as well as those of
L. japonicus mutated in the LjHAR1 – Arb++ phenotype (Solaiman et al. 2000;
Nishimura et al. 2002a), similar to those mentioned above. Hence both local
signal transduction and systemic autoregulation may be common in nodular
and AM symbioses.
The AM mutants obtained by using the approach described above allows the
identification of genes common in both AM and RN symbioses, but not AM-specific
genes. The direct screening of mutants with impaired AM development was until
recently, only possible for non-legumes including tomato (Barker et al. 1998; Gao
et al. 2001; David-Schwartz et al. 2001, 2003) and maize (Zea mays L.) (Paszkowski
et al. 2006). One tomato rmc mutant demonstrated Pen–, Coi– and Rmc (reduced
mycorrhizal colonization) phenotypes which were fungus-specific (Gao et al.
2004). The phenotypes with fungal penetration into epidermal or hypodermal cells
were shown to be associated with an enhanced and more prolonged defence gene
expression (Gao et al. 2004). So, it has been supposed that defence-like responses
may be the basis of the mechanisms underlying the specificity of interactions
between AM fungi and the rmc mutant. The phenotypes were similar to those of
Pen–, Coi– and Rmd-, respectively, observed in legume mutants. The same is true for
the maize mutant phenotypes taci1 (decreased mycorrhization level) and Pram1
(hypermycorrhization) (Paszkowski et al. 2006). A rice (Oryza sativa) mutant
affected in OsIPD3 gene manifested rmc phenotype (Chen et al. 2008). The
OsIPD3 gene is determined to be an orthologue of M. truncatula MtIPD3 gene
common in both AM and RN symbioses (Chen et al. 2008).
Although phenotypically similar to other Pen– mutants selected from pre-
existing Nod– mutants, the rmc mutant was identified by direct screening for abnor-
mal AM. It may therefore harbour a mutated gene specific to the mycorrhizal
symbiosis. At the same time, nearly all cloned legume genes required for nodulation
and AM development have their putative homologues in the non-leguminous plants

(Zhu et al. 2005; Chen et al. 2008) including Arabidopsis thaliana, which forms
neither RN nor AM sybmioses (Zhu et al. 2005). These data suggest strongly that
the pre-existing genes have been recruited from the diverse cellular processes to
function as the symbiotic (AM) genes.
The highly interesting group of the mutants induced in non-legume plants rep-
resented by pmi and pmi2 of tomato (David-Schwartz et al. 2001, 2003) and nope1
of maize (Paszkowski et al. 2006) mutant lines is those impaired in the pre-symbiotic
stage Pre-Pen (pre-penetration) (according to (Marsh and Schultze 2001)). These
mutants are principally distinguished from those described in legumes as their muta-
tions are unique to the AM symbiosis.
Only recently were the first AM-specific legume mutants (ram1, ram2) char-
acterized in M. truncatula by using direct screening of a mutated plant popula-
tion (Marsh et al. 2006, 2008). These mutants have been shown to represent at
least two complementation groups, which are not allelic to any of the known
symbiotic mutants. These mutants had normal nodulation phenotypes. By con-
trast to the non-nodulating and non-mycorrhizal M. truncatula dmi mutants,
which form extensive and complex appressoria on the root epidermis (Sagan
et al. 1995), these new mutants exhibited no sustained contact with the root
surface. It is supposed therefore, that these mutants may be altered in their
reception of fungal signals required in order to establish normal symbiosis
(Marsh et al. 2008). Also the direct screening of a mutagen-treated pea (P. sativum)
population has been initiated and a series of putative mutants presumably defec-
tive in AM development were isolated (Shtark et al. 2007). A “nurse plant”
inoculation system (Rosewarne et al. 1997) has several advantages by compari-
son with conventional methods of inoculation with AM-fungi, and is supposed
to be a potential tool for the isolation of mycorrhizal mutants from different
legumes allowing the identification of new AM-specific plant genes. The genetic
analysis of the mutants which is now in progress will identify new pea genes
controlling the development of AM symbiosis.
Therefore, based on the analysis of all the diverse mutant phenotypes, the fol-
lowing stages of AM development can be distinguished : (1) Pre-Pen, (2) Pen, (3)
Coi, and (4) Arb. The present classification is preliminary and will be modified
along with accumulation of novel data on genetic and molecular control of sym-
biosis. In the next section, the comparative analysis of present knowledge of the
molecular mechanisms of AM and RN symbioses in the leguminous (crop and
model) plants is presented.
The Use of Model Legumes for Studying Molecular

Genetics of Symbioses
The large sizes of genomes in crop legumes (e.g., in soybean and pea) as well as
their low capabilities for transformation, greatly complicates the cloning of symbiotic
genes, analysis of primary structures and their subsequent genetic manipulation.

In the early 1990s other legume species, such as: Lotus japonicus (Regel.) Larsen
(Handberg and Stougaard 1992) and Medicago truncatula Gaertn. (Barker et al.
1990; Cook 1999) were used as a consequence as model plants for such studies.
These species are characterized by small genomes (~ 470–500 Mb, according to
(Young et al. 2003)) and can be genetically transformed with relative ease (Cook
et al. 1997; Cook 1999; Stougaard 2001; Udvardi 2001). In addition, their short
lifecycles and high volumes of seed production make them attractive and convenient
models for studying the molecular basis of RN and AM symbioses.
Genetic analysis in model and crop legumes was started using experimental
mutagenesis. Large-scale programmes of insertion, chemical and X-rays mutagen-
esis, performed by several research groups, resulted in the identification of numer-
ous symbiotic mutations in L. japonicus and M. truncatula (Penmetsa and Cook
1997; Schauser et al. 1998). The genes involved in early stages of RN symbiosis
(so-called early Sym genes) were of primary interest in these studies, particularly
because dissecting the mechanisms by which the Nod factor signal is perceived and
transduced in host plants were considered to be the main targets for symbiosis
research (Albrecht et al. 1999). The results of this research showed that after the
perception of Nod factor RN symbiosis uses the same signalling pathway as does
AM (so-called “common symbiosis pathway” (CSP)), though with slight differ-
ences. The general scheme of CSP work is described below.
General Scheme of Functioning of the Common Symbiosis Pathway
The data obtained during the last 10 years allows the reconstruction of the symbi-
otic signalling pathway which starts in RN and AM symbioses with recognition of
the Nod and Myc factor, respectively, and goes on as signal transduction inside the
root (Fig. 4). In legumes Nod and Myc factors are most likely perceived by specific
receptor complexes, though the structure of such complexes remains unknown for
the possession of the Myc factor (Parniske 2008). In turn, the receptor for the Nod
factor is thought to be a heterodimer composed of at least two LysM containing
receptor kinases (LjNFR5/MtNFP/PsSYM10 and LjNFR1/MtHCL/PsSYM37) (see
below). This receptor complex (at least in pea and possibly also in M. truncatula) is
thought to be needed at the start of interactions and for penetration by the bacteria
into the cortex through root hairs (Zhukov et al. 2008). At this later stage, the pres-
ence of some alternative homologous proteins able to substitute for these kinases in
the receptor complex is suggested (Smit et al. 2007; Zhukov et al. 2008). Probably,
the diversity of receptor kinases could compensate for the variability of Rhizobium
strains and increase the specificity of their interactions with rhizobia.
Reception of the Myc factor is less specific, but it is absent in the non-
mycotrophic plants (Navazio et al. 2007). The Myc factor was demonstrated to
induce expression of symbiosis-specific nodulin (symbiosin) MtENOD11 in the
roots of Medicago truncatula (Kosuta et al. 2003). At the same time plant defence
responses are not induced by this factor (Navazio et al. 2007). It is supposed that
Fig. 4 The schematic representation of symbiotic signal transmission by the products of legume
Sym genes.Products of genes involved in nodule formation only are given in white boxes. Products
of common genes which are involved in both arbuscular mycorrhiza (AM) and root nodule (RN)
formation are in grey boxes. Products of genes which expression is induced de novo by signal
from microsymbionts are below the horizontal line separating the picture, whereas the products of
genes which are already present in root cells are above this line. Products of common genes which
are in the same box are homologues in different plant species (Lj – Lotus japonicus, Mt –
Medicago truncatula, Ps – Pisum sativum). The continuous arrows represent a direct transmission
of signal; dotted arrows mean its indirect transmission (e.g. in the case of Myc factor reception the
signal goes indirectly to SYMRK)
the Myc factor could be produced constitutively by the fungus, without any physical
contact with the plant partner (Navazio et al. 2007).
After the first step in reception of Nod or Myc factor, the signal is transmitted to
the common signalling pathway. The first element in this pathway is an LRR-
receptor kinase, or SYMRK (symbiotic receptor kinase) (LjSYMRK/MtDMI2/
PsSYM19), which is required for both RN and AM development (Endre et al. 2002;
Stracke et al. 2002). Earlier it was suspected that LRR-receptor kinase could be a
Myc factor receptor, but now it is thought more likely that this kinase indirectly
perceives the signal from the fungus. Interestingly, the activity of this kinase is also
required for a complete development of late stages of the symbioses, at least for
rhizobial infection, since silencing of MtDMI2 by inducible anti-sense mRNA at a later
stage, during nodule formation, results in Fix- phenotype (Limpens et al. 2005).
The symbiosis receptor kinase SYMRK acts upstream of the Nod and Myc
factor-induced Ca2+ spiking in the perinuclear region of root hairs within a few
minutes after the application of the Nod factor (Wais et al. 2000). Perinuclear cal-
cium spiking involves the release of calcium from a storage compartment (probably
the nuclear envelope) through as-yet-unidentified calcium channels. Currently, it is
known that the potassium-permeable channels (LjCASTOR and LjPOLLUX/
MtDMI1/PsSYM8) (Ane et al. 2004; Imaizumi-Anraku et al. 2005; Edwards et al.
2007) may compensate for the resulting charge imbalance and could regulate cal-
cium channels in plants (Peiter et al. 2007; Riely et al. 2007). Also, nucleoporins
LjNUP85 and LjNUP133 (to date described only in Lotus) are required for calcium
spiking, although their mode of involvement is unknown. Probably, LjNUP85 and
LjNUP133 may be a part of specific nuclear pore subcomplex that plays a crucial
role in the signalling process requiring interaction at the cell plasma membrane and
at nuclear and plastid organelle-membranes to induce Ca2+ spiking (Kanamori et al.
2006; Saito et al. 2007).
Calcium spiking is characteristic for both RN and AM formation, but the fre-
quency and amplitude of oscillations differ in these processes (Oldroyd and Downie
2004). These Ca2+ spikes are supposed to activate a calcium- and calmodulin-
dependent protein kinase (LjCCAMK/MtDMI3/PsSYM9) that is also necessary for
Nod factor signalling and AM development (Catoira et al. 2001). This kinase con-
tains an autoinhibition domain, which if removed leads to a spontaneous activation
of downstream transcription events and the induction of nodule formation in
absence of rhizobia (Gleason et al. 2006). Thus, CCaMK appears to be a general
regulator in both symbioses activating different cascades of signals for RN and AM
in response to different Ca2+ spiking, because the next steps of nodulation signalling
are independent from those of AM: the mutations in downstream Sym genes do not
change the mycorrhizal phenotype of legume plant. Interestingly, the mutations in
any Sym genes do not influence the defence reactions, suggesting that signalling
pathways of mutualistic symbioses and pathogenesis are substantially different.
The calcium–calmodulin-dependent protein kinase of Lotus japonicus (LjCCaMK)
is known to form a complex with LjCYCLOPS, a phosphorylation substrate, within the
nucleus (Parniske 2008). Ljcyclops mutants severely impair the infection processes of
bacterial or fungal symbionts (Albrecht et al. 1998). During RN symbiosis, Ljcyclops
mutants exhibit specific defects in their infection-thread initiation, but not in nodule
organogenesis (Yano et al. 2008), indicating that LjCYCLOPS acts in an infection-
specific branch of the symbiotic signalling network (Parniske 2008). LjCYCLOPS
encodes for a protein with no overall sequence similarity to proteins of known function,
but contains a functional nuclear localization signal and a carboxy-terminal coiled-coil
domain. Further work is needed to shed light upon the role(s) of LjCYCLOPS (as well
as its homologue MtIPD3 in Medicago (Messinese et al. 2007) and possible a homo-
logue in pea) during development of symbioses. No other Sym genes cloned to date
have been found to be involved in the mycorrhization process in legumes.
It is supposed that CCaMK probably phosphorylates specific transcription
factors already present in cell: LjNSP1/MtNSP1 and LjNSP2/MtNSP2/PsSYM7,
which influences changes of expression in several genes related to symbiosis
development (Kalo et al. 2005; Smit et al. 2005). The activity of these proteins
leads to transcriptional changes in root tissues, for instance, increasing the level of
early nodulins ENOD40, ENOD11, ENOD12, ENOD5, which are known to be
potential regulators of infection thread growth and nodule primordial formation
(Albrecht et al. 1999; Heckmann et al. 2006; Murakami et al. 2006). Also, the
changes in cytokinin status of host plants are detected, followed by up-regulation
of genes encoding for nitrogen-fixing symbiosis-specific cytokinin receptors LjLHK1
and MtCRE1 (Gonzalez-Rizzo et al. 2006; Murray et al. 2007; Tirichine et al. 2007).
Moreover, transcription regulators LjNIN/MtNIN/PsSYM35 and MtERN, are induced
specifically downstream of the early Nod factor signalling pathway in order to
coordinate and regulate the correct temporal and spatial formation of root nodules
(Schauser et al. 1999; Borisov et al. 2003; Marsh et al. 2007; Middleton et al. 2007).
The presently identified genes are responsible for a signal cascade which aims
to induce the nodulin, mycorrhizin and symbiosin genes responsible for building
the symbiotic structures and initiating their biochemical functions. It is supposed
that the signalling pathway did not appear de novo in legumes when they become
able to form nodules, but developed from a pre-existing system of AM formation
into which the novel, nodule specific genes were recruited. New genes had been
involved in RN development, especially those encoding receptors recognizing
hormones (e.g. cytokinins) and hormone-like molecules (Nod factors).
Plant Receptors Involved in Nodule Formation
The spectrum of secreted signal molecules – Nod factors – appears to be a unique

feature of strains of nodule forming bacteria (Geurts et al. 1997). In turn, the speci-
ficity of interactions is determined by plant receptors recognizing the structure of
the Nod factor molecule (Limpens et al. 2003; Radutoiu et al. 2003, 2007; Oldroyd
and Downie 2008). Intensive work in the past 10 years has led to the identification
and cloning of legume genes encoding for receptor kinases perceiving a Nod factor
during signal interactions with nodule bacteria.
In Lotus japonicus two genes LjNFR1 and LjNFR5 (Nod factor receptor) were
identified mutations of which elicited no response to Mesorhizobium loti inoculation
or to Nod factor application, while remaining able to develop AM (Schauser et al.
1998). Positional cloning of these genes performed by fine genetic mapping
followed by analysis of genomic libraries showed the respective nucleotide and
putative protein structures of these genes (Radutoiu et al. 2003; Madsen et al.
2003). Both LjNFR1 and LjNFR5 genes appeared to encode receptor kinases with
3 LysM domains presented in the extracellular C-terminal part of the putative

protein. LysM domains are found among enzyme proteins of some prokaryotes and
eukaryotes, while receptor kinases with the LysM motif have been found only in
plants. At a moment, LysM motifs have been shown to bind oligosaccharide
molecules, therefore LysM-containing receptor kinases are supposed to represent
the molecules capable of recognizing a bacterial Nod factor, although predicting the
structure for a putative Nod factor-binding site needs further research (Radutoiu
et al. 2003, 2007).
The transmembrane domain in both LjNFR1 and LjNFR5 putative proteins sug-
gests that receptor kinases are located in the plasma membrane of the root cell, and
when LysM domains perceive the Nod factor, Serine/Threonine kinase domain
gives a start to a signal transduction cascade. It was found that kinase domain in
LjNFR5 lacks an “activation loop” the absence of which makes kinase incapable of
auto-phosphorylation. It is supposed that LjNFR5 needs the other protein, LjNFR1,
for proper functioning, and therefore, they might work in tandem.
In Medicago and pea, the system recognizing Nod factor appears to be more com-
plex. For LjNFR5, orthologues in Pisum and Medicago were found among known Sym
genes (PsSym10 and MtNFP, respectively) (Amor et al. 2003; Madsen et al. 2003;
Arrighi et al. 2006). Mutations in both Pssym10 and Mtnfp leads to an absence of any
reactions to Nod factors but does not affect mycorrhization, indicating that receptors
for Nod factors are placed upstream of the common signal transduction pathway.
Obviously, if LjNFR5/PsSYM10/MtNFP needs the second component of the Nod fac-
tor recognition complex in order to work properly then it might be the protein product
of an orthologue of LjNFR1. The phenotype of pea and Medicago mutants in homolo-
gous genes however, turned out to differ from that of Lotus nfr1 mutants.
Historically, the first example of legume symbiotic genes involved in the recog-
nition of rhizobia and perception of a Nod factor was pea gene PsSym2 identified
more than 30 years ago as a gene causing resistance in pea lines from Afghanistan
(Pssym2A allele) to infection by European strains of Rhizobium leguminosarum bv.
viciae (Lie 1984). This resistance could be overcome by R. l. bv. viciae strains
found in soils from the Middle East and North-West Russia that produce a Nod
factor with an acetyl moiety substituted on a reducing terminus of a lipo-chito-
oligosaccharide chain (Götz et al. 1985). The strains of R. l. bv. viciae from Europe
producing a Nod factor without this substitution failed to nodulate “Afghan” peas
(Fig. 3c). This situation illustrates the “gene-for-gene” interactions, when one gene
of microorganism (nodX, encoding acetyltransferase, performing acetylation of
Nod factor (Davis et al. 1988) interacts with one gene of plant (PsSym2, probably
encoding a Nod factor receptor).
For years, PsSym2 has been a model for researchers working with unique phe-
notypes of “Afghan” peas, but the DNA sequence of this gene remains unknown.
The attempts to analyze the region of the M. truncatula genome orthologous to the
region of localization for PsSym2 in pea by means of genetic mapping of the phe-
notypic manifestations of PsSym2 lead to the identification a series of genes encod-
ing for receptor-like kinases containing LysM-domains. These studies were
switched from pea to M. truncatula and as a result one of these kinases, MtLYK3,
has been shown to be a Nod factor receptor, highly homologous to LjNFR1 of
L. japonicus (Limpens et al. 2003).
Medicago hcl mutants carrying mutations in MtLYK3, as well as LYK3-silenced
transformants, have phenotypes characterized either by defects in root hair curling
and an absence of the bacterial penetration into the root cortex or by successful root
hair curling and the infection thread growth initiation (Wais et al. 2000; Catoira
et al. 2001; Limpens et al. 2003; Smit et al. 2007). A similar phenotype was also
described for “Afghan” pea carrying Pssym2A allele and pea Pssym37 mutants
(Kozik et al. 1995; Zhukov et al. 2008). Both Medicago and pea plants were mycor-
rhized normally suggesting that MtLYK3/HCL, PsSym2 and PsSym37 are important
only for RN symbiosis.
The map position of PsSym37 appeared to be similar to that of PsSym2 and syn-
tenic (i.e. it is found on the same chromosome) to the location of MtLYK3/HCL in
M. truncatula and LjNFR1 of L. japonicus. Sequencing a pea homologue of LjNFR1
in Pssym37 mutants allowed the finding of nucleotide substitutions, suggesting an
homology of PsSym37 with MtLYK3/HCL and LjNFR1. Intriguingly one pea
Pssym37 mutant with an amino acid change in its receptor portion may be comple-
mented with the Nod+ phenotype by the double acetylated Nod factor structure, like
“Afghan” pea lines (Zhukov et al. 2008). The putative protein PsSYM37 in “Afghan”
lines does not contain any amino acid changes compared with one in “European”
lines with normal nodulation, suggesting that PsSym37 could only be a functional
analog of PsSym2 gene, but not of PsSym2 itself.
The region of the Medicago genome containing MtLYK3/HCL includes about
10 homologous genes encoding for receptor kinases with similar structures (Limpens
et al. 2003). In such a region, new variants of receptor molecules could appear, for
instance, as a result of non-reciprocal recombination events (Michelmore and
Meyers 1998). Accordingly, the gene MtLYK4 is highly homologous to the neigh-
bouring genes MtLYK3 and MtLYK5, whereas its receptor part is homologous to that
of MtLYK3 and the kinase part is homologous to that of MtLYK5. Among these
genes, probably only MtLYK3 participates in interactions with rhizobia, as its
expression is specific to roots and nodules (Limpens et al. 2003). Presumably, the
role of LysM-containing kinases encoded by other genes of this region provides
recognition of chitin elicitors similar to Nod factor in their structure. The ortholo-
gous region of the pea genome must also contain a series of homologous kinase
genes, one of which could be the elusive PsSym2 gene. Sequencing one such gene,
namely PsK1, showing the differences between “Afghan” and “European” pea lines
in receptor part of the putative PsK1 protein allows supposition that PsK1 or another
gene of this region could actually be PsSym2 encoding the changeable component
of receptor complex recognizing the Nod factor structure (Zhukov et al. 2008).
Several years ago, a “two-receptor model” was proposed based on studies of
bacterial mutants (Ardourel et al. 1994). This model suggests a signalling receptor
inducing early responses with low specificity towards Nod factor structure, and an
entry receptor that controls infection with more stringent specificity and, in the case
of successful recognition the Nod factor, allows rhizobia to enter the root hair and
root tissues. Indeed, blocks in infection occurring at this stage in pea Pssym37
mutants and “Afghan” pea lines interacting with “European” rhizobia, as well as
Medicago hcl mutants, tend to corroborate this model.
It could be concluded that the system of Nod factor recognition works in pea
and M. truncatula in a similar fashion, and PsSYM10/MtNFP plays the role of a
low-specific signal receptor, whereas PsSYM37/MtLYK3 performs more stringent
recognition of Nod factor as an entry receptor. In fact, other LysM-containing
receptor kinases potentially capable of binding lipochito-oligosaccharide molecules
(and therefore for determining the structure of a Nod factor) could function as
alternative components of Nod factor receptor complex, substituting PsSym37/MtLYK3
(Smit et al. 2007; Zhukov et al. 2008). The adaptive role in the functioning of such
LysM-containing kinases as exchangeable subunits of receptor complex could be
explained by a necessity for interactions with several strains of rhizobia, which
appear to be highly variable based on the structure of their secreted Nod factors.
In Lotus, which is evolutionarily remote from pea and Medicago, mutations in both
LjNFR1 and LjNFR5 genes lead to an absence of any responses to a Nod factor present
in the rhizosphere. This fact shows that mechanisms of Nod factor reception in Lotus
differ from those of pea and Medicago, despite genes encoding for receptor kinases
which recognize Nod factors being highly homologous in Lotus, Medicago and pea.
Cytokinin Signalling and Reception in Legumes
Nodule primordium formation and development is regulated in legume plants by

changes in local concentrations of plant hormones – auxins, cytokinins and ethylene
(Schultze and Kondorosi 1998b). Cytokinin appears to play a key regulatory role in
this process, for the following reasons. Several legumes (e.g. Medicago sativa L. and
Trifolium pratense L.) are reported to form spontaneous nodules in the absence of
rhizobia which are structurally similar to nitrogen-fixing nodules (Truchet et al. 1989;
Blauenfeldt et al. 1994). Additionally, experimental mutagenesis in Lotus japonicus
revealed mutants which spontaneously form nodules (Tirichine et al. 2006). It was
shown that spontaneously nodulating mutant lines carry mutations in a known gene
for the calcium/calmodulin-dependent kinase LjCCaMK (Tirichine et al. 2006) and in
a novel symbiotic gene for the cytokinin receptor LjLHK1 (Tirichine et al. 2007).
LjCCaMK is known to detect the “calcium spikes” and activate the programmes
of symbiosis development; such mutations which are associated with gains in func-
tions leading to a loss of auto-inhibition of the activity of this kinase and spontane-
ous nodule formation (Gleason et al. 2006; Tirichine et al. 2006). Similarly,
LjLHK1 (Lotus histidine kinase) perceiving the changes in cytokinin concentrations
could normally activate cell divisions in the root cortex leading to nodule primor-
dium formation, while the mutant with an altered histidine kinase receptor has
cytokinin-independent activity (Tirichine et al. 2007). It was concluded that cyto-
kinin signalling is required for cell divisions that initiate nodule development and
defines an autoregulated process where cytokinin induction of nodule stem cells is
controlled by shoots.
In Medicago, homologous gene MtCRE1 (cytokinin receptor 1) has also been

shown to be involved in nodulation signalling. RNA interference of this gene leads
to significant increases in lateral root development and a reduction in nodulation,
associated with perturbations in both infection and nodule primordia formation
(Gonzalez-Rizzo et al. 2006). Similarly, “loss-of-function” mutants in Ljlhk1 of
Lotus, exhibit abundant infection-thread formation but fail to initiate timely cortical
cell divisions in response to rhizobial signalling (Murray et al. 2007). Conclusively,
cytokinin signalling plays an important role in plant meristem formation and is
directly involved in initiating root nodule organogenesis.
Intriguingly, cytokinin signalling plays a key role in Nod factor-independent
nodulation. Recently, it was shown that nodulation of some legumes by rhizobia
could occur in the absence of the nodABC genes and lipochito-oligosaccharidic
Nod factors, indicating that other signalling strategies could trigger nodule
organogenesis in some legumes (Giraud et al. 2007). Indeed, two symbiotic and
photosynthetic Bradyrhizobium sp. strains specifically induce nodules on both the
root and stem of the aquatic legume Aeschynomene sp., although the nodulation
ability of these strains is restricted to a few species, including A. sensitiva and A.
indica (Giraud and Fleischman 2004). These Bradyrhizobium strains produce a
purine derivative, which is a cytokinin-like molecule (or a cytokinin precursor)
and may act as a signalling molecule (Giraud et al. 2007). It is supposed that this
molecule activates the cytokinin receptor and starts the Nod factor-independent
nodulation. During “conventional” infection, Nod factors induce root hair defor-
mation, leading to bacterial entrapment. In turn, root hair curling and infection
thread formation are not initiated in Nod factor-independent nodulation and bac-
teria enter the plant tissue through cracks in the epidermis, often at the sites of
lateral root emergence (Downie 2007).
Autoregulation of RN and AM Symbioses Development
Beside mutual recognition and signalling, another important feature of the develop-
ment of symbioses is autoregulation by the host plant. The plant is known to govern
the growth and differentiation of the microsymbionts in roots. For RN symbiosis, it
is considered that host legumes control root nodule numbers by sensing external
and internal cues. A major external cue is soil nitrate content, there is a feedback
regulatory system by which earlier formed nodules suppress further nodulation
through shoot–root communication using an important internal cue. The latter is
known as autoregulation of nodulation (AUT), and is believed to consist of two
long-distance signals: a root-derived signal that is generated in infected roots and
transmitted to the shoot; and a shoot-derived signal that systemically inhibits
nodulation.
It has been shown that in Lotus japonicus, the leucine-rich repeat receptor-like
kinase, HYPERNODULATION ABERRANT ROOT FORMATION 1 (LjHAR1),
mediates AUT and nitrate inhibition of nodulation, and is thought to recognize the
root-derived signal. Some cloned orthologous genes in M. truncatula and pea
(MtSUNN and PsSym29) are supposed to serve a similar autoregulation process as

mutations in all these genes lead to supernodulating phenotype (Nod++) (Krusell
et al. 2002; Nishimura et al. 2002a; Schnabel et al. 2005). The root signal is suspected
to be a small peptide related to the CLAVATA3/ESR-related (CLE) family of peptides,
and it could be speculated that LjHAR1 might recognize a Lotus CLE peptide induced
by rhizobial infection as the root-derived AUT signal (Okamoto et al. 2009). Two
L. japonicus genes CLERoot Signal 1 (LjCLE-RS1) and LjCLE-RS2 are strong candi-
dates to encode for this root-derived signal. Another gene, LjASTRAY, encoding for
transcription activator, is also supposed to play some role in autoregulation of the
nodulation process (Nishimura et al. 2002b).
Very little is known about plant regulation of the mycorrhization process. It is
demonstrated that in the case of LjHAR1, mutation also affects mycorrhizal forma-
tion (Myc++) suggesting the shared role of LjHAR1/MtSUNN/PsSYM29 in control-
ling the rate of root colonization by microsymbionts. Therefore, not only the local
signal transduction but the systemic autoregulation may be common for the RN and
AM symbioses. The systemic regulation of symbioses formation and maintenance
also need further investigations.
The next step in unravelling the developmental genetics of symbioses is the study
of gene networks at an intergenomic level i.e., coordinated expression by plant and
microbe genes. For AM, the use of new molecular approaches, in particular tran-
scriptomics, have identified a series of AM fungal genes showing altered expression
levels during the symbiosis formation (Grunwald et al. 2004; Gianinazzi-Pearson
and Brechenmacher 2004; Seddas et al., 2009 personal communication). The stages
of fungal–plant interaction at which induction or repression commence are not well
understood, and so the use of plant mutants impaired at different stages of AM
development might be a useful approach to uncover patterns in the plant and fungal
genetic relationship (Kuznetsova et al. 2010). Work aimed at identifying plant–
rhizobial gene interactions using plant and microbe mutants is in progress.
Associations of Roots with Plant Growth-Promoting

Rhizobacteria (PGPR)
Plant Growth Promoting Rhizobacteria (PGPR) are the most widespread plant
co-habitants. They are taxonomically diverse including assorted groups of Gram-
negative bacteria (e.g., Azospirillum, Burkholderia, Enterobacter, Pseudomonas),
Gram-positive bacteria (Bacillus (and new genus Paenibacillus), Streptomyces) and
even some archaea (Lugtenberg et al. 2001). The PGPR are inhabitants of soil in
the vicinity of plant roots, many are able to attach to root surfaces and to AM and
other fungal hyphae.
The PGPR provide several benefits (affecting the host plant either directly or
indirectly) including: nitrogen fixation, stimulation of root development (due to
phytohormone production), solubilization of soil phosphates, defence of plants
from soil borne pathogens and improving host tolerance to abiotic stresses.
Nutritional Associations
The nutritional root-PGPR interactions were discovered and initially characterized

by J. Dobereiner who identified a broad distribution of Azospirillum – cereal asso-
ciations and described their basic physiology and ecology descriptions, as well as
demonstrating a substantial agronomic potential (reviewed in: Döbereiner 1988).
In her early papers, plant growth promoting activity was attributed mainly to nitrogen
fixation which was estimated to provide 40–60 kg ha−1 and in some circumstances
up to 100 kg ha−1 per season in cereal–Azospirillum associations. It was demon-
strated later, however that a partial role in these plant–PGPR associations was due
to phytohormone (auxin) synthesis (Costacurta and Vanderleyden 1995) which
improves the root growth and assimilatory capabilities and hence aids nitrogen
uptake by plants.
Despite the absence of visible anatomic differentiation in root-Azospirillum asso-
ciations, their development involves a range of molecular interactions some of which
may have commonalities with endosymbiotic associations. For example, in the
genomes of A. brasilense and A. lipoferum DNA sequences were found which are
homologous with genes nodABC and nodQPGEFH of Sinorhizobium meliloti
(Fogher et al. 1985; Vieille and Elmerich 1990, 1992). By contrast to rhizobia, the
expression of nod genes in Azospirillum does not require the presence of root exudates
or their flavonoid components. This is consistent with the absence of promoters of
Azospirillum nod genes in the nod-box sequences which are the targets of the NodD
regulator activating these genes in rhizobia in the presence of root-exuded flavonoids
(see Section 3). The presence of some elements from the system for Nod factor syn-
thesis suggests however, that Azospirillum can elicit some signalling processes in the
roots which are common to the components of nodulation/AM formation cascades.
Furthermore, genes homologous with chromosomal virulence genes of agrobac-
teria (chvA and chvB) were found in Azospirillum (Waelkens et al. 1987). Close
homologues of these genes (ndvA and ndvB) were identified in rhizobia and found
to participate in the synthesis of cyclic b-glucans involved in the formation of sur-
face structures which are important for nodule development (Breedveld and Miller
1994). Genes fixABC were found in azospirilla which have homologues in rhizobia
(Fogher et al. 1985) suggesting similarities in the regulation of associative and
endo-symbiotic nitrogen fixation.
An important prerequisite of high nitrogen-fixing activity in azospirilla may be
the secretion of auxins which can be synthesized via different biochemical path-
ways (Costacurta and Vanderleyden 1995). The direct involvement of bacterial
auxins in the stimulation of root growth was demonstrated using IAA-deficient
mutants of A. brasilense. The production of IAA by azospirilla may be based on
root-exuded tryptophane and be responsible for promoting root growth and an
increase in root exudation which supports rhizospheric nitrogen-fixation. The effi-
ciency of the conversion of tryptophane into IAA by azospirilla however, does not
exceed 1% while the amount of synthesized phytohormone is only about 10−11 M
per seedling per 24 h (Kravchenko et al. 1994). The role of these synthetic pro-
cesses in the development of associative symbiosis needs to be clarified further.
It has been reported that PGPR which produce ACC deaminase (which is
involved in the catabolism of ACC (1-aminocyclopropane-1-carboxylate) – a pre-
cursor of plant hormone, ethylene), can lower ethylene concentration in a develop-
ing or stressed plant, protecting it against the deleterious effects of ethylene induced
stress and facilitating the formation of longer roots (Penrose and Glick 2003).
Also it has been demonstrated using transformants of Azospirillum brasilense Cd
in which ACC deaminase gene (acdS) from Enterobacter cloacae UW4 was
expressed under the control of different promoters that transformants having lower
ACC deaminase activity showed significantly increased IAA synthesis as well. In
addition, the result was increased ability to promote the growth of tomato seedlings
(Holguin and Glick 2003).
The amount and composition of plant exudates which support the development
of Azospirillum-root associations are dependent greatly on host genotype and envi-
ronmental growth conditions. It has been demonstrated that components of wheat
root exudates which support growth and nitrogen-fixing activity of Azospirillum
include organic acids (initially citrate, succinate and malate) but not necessarily
sugars (glucose, sucrose, xylanose) (Kravchenko and Leonova 1993). Interestingly,
in wild wheat genotypes organic acids are dominant components of exudates, while
in agronomically advanced cultivars sugars dominate demonstrating that during the
domestication and breeding of this crop there has been a decrease in the potential
for the development of beneficial PGPR communities in the rhizosphere.
Another mechanism ensuring the effective interaction between the host plants
and their rhizospheric associates is the signalling process which attracts azospirilla
into the rhizosphere and stimulates their attachment to the roots (plant function).
This is dependent on the activities of bacterial flagella and on the surface mole-
cules, initially, exo- and lipopolysaccharides. The effective attachment of azospi-
rilla to the root surfaces is dependent on plant lectins which may be responsible also
for the expression of properties which are beneficial to the bacteria. For example,
purified wheat lectins were demonstrated to enhance the nitrogen-fixing activity of
azospirilla and the accumulation of ammonium in gnotobiotic systems (Antonyuk
et al. 1993). It is interesting to note that some azospirilla produce their own lectins
which may mediate the adsorption of bacteria to root surfaces and also improve
root susceptibility to inoculation (Nikitina et al. 1996) suggesting an operation of
effective positive feedbacks between the partners in associative symbiosis.
Defensive Associations
A very efficient defence from the phytopathogens may be provided by microbes

creating protective barriers solely on root surfaces. The best studied examples are:
Pseudomonas (P. fluorescens, P. chlororaphis, P. putida), some Serratia (S. marc-
escens) and Bacillus (B. cereus, B subtilis) species. Many of these bacteria are
capable of preventing attacks by pathogenic fungi (such as: Fusarium, Rhizoctonia,
and Verticillium). Diverse mechanisms may be involved in host protection offered
by PGPR (Lugtenberg et al. 2001).
The best studied mechanisms are the competitive exclusion of pathogens often
related to their direct suppression by the bacterial antibiotic substances. For exam-
ple, many Pseudomonas strains produce phenazines, e.g. phenazine-1-carboxamide
(PCN), which are active against Fusarium oxysporum (Chin-A-Woeng et al. 2000)
and F. culmorum (Shtark et al. 2003). The bacterial mutations leading to the PCN-
phenotype usually results in the loss of biocontrol activity. Genes for PCN synthesis
are transcriptionally activated in the host rhizosphere as a result of root exudates
(host plant function). The bacterial synthesis of antibiotics may be accompanied by
production of volatile dyes, for example of cyanides, their involvement in antifungal
activities was demonstrated during the cultivation of PGPR-Fusarium combinations
(Thomashow and Weller 1996). Cyclic lipopeptides, for example: tensine, syringo-
mycin, syringopeptide, produced by Pseudomonas species also exhibit potent sur-
factant and broad-spectrum antibiotic properties (Raaijmakers et al. 2006).
An important mechanism for the suppression of pathogens by biocontrol
microbes may result from competition for nutrients. Among the processes involved
in this competition are bacterial siderophores which may possess much greater
affinities for ferric ions than those for fungal siderophores (Miyazaki et al. 1995).
The direct evidence for the importance of bacterial synthesis of siderophores in the
suppression of pathogens was obtained by using genetically modified PGPR strains
with increased or arrested siderophore production (Buysens et al. 1994; Raaijmakers
et al. 1995). The value of siderophores in biocontrol effects under natural conditions
is currently predominantly associated with their ability to induce forms of systemic
resistance in plant (Preston 2004). See below and other chapters in this Book.
Competitive exclusion of pathogens by PGPR is best achieved when the bacteria
exhibit high root-colonizing activity. Application of the technique of genetic labelling
with Green Fluorescent Protein (GFP) suggested that these bacteria do not regularly
result in the colonization of root interiors, and only sometimes may be observed
inside the outer root tissues (Bloemberg et al. 1997, 2000). Root exudates may
account for up to 30% of the net plant photosynthetic output and the optimal
ecologicalniches for PGPR are zones of greatest rhizo-deposition. Most PGPR cells
are concentrated on the root surface where the micro-colonies or bio-films form
(O’Toole and Kolter 1998). Since the interactions of plants with root-associated bac-
teria are not specialised, the bacteria will colonize the roots of a broad spectrum of
hosts. Specificity of the defensive association may be expressed however, at the point
when antimicrobial compounds are being synthesized and this does not always cor-
relate with bacteria taxonomy; many strains of Bacillus and Pseudomonas which
have plant-protective properties have close relatives amongst phytopathogenic types
(Stephens and Murray 2001; Catara 2007).
The importance of root colonization in the expression of beneficial defensive
properties in symbionts is evident from research demonstrating that genes encoding
for bacteria surface components that are required for the root adhesion (lipopoly-
saccharides, flagella) are also very important in this type of defensive process
(Whipps 2001). Inactivation of these genes usually results in the loss of plant-pro-
tecting properties in PGPR strains while increasing the gene activities (via gene
amplifications or fusions to constitutive “strong” promoters) improves host protec-

tion markedly (Lugtenberg et al. 2001).
Microscopic observations demonstrated that suppression of Fusarium may be
correlated with P. chloloraphis attachment to the root surfaces and to pathogen
hyphae (Bolwerk et al. 2004). As a result of this attachment, some PGPR strains
commence their biocontrol functions by behaving as hyper-parasites of pathogenic fungi.
This suppression may be correlated with the production of bacterial enzymes which
destroy the pathogen cell walls. Some Serratia and Bacillus strains produce extra-
cellular chitinases which inhibit the pathogen during developmental stages such as
conidial germination and the growth of hyphae (Popova and Khatskevich 2004).
Several mechanisms which increase bacterial variability in the genes controlling
surface components may be important in the enhancement of root colonizing activ-
ity in defensive PGPR. For example, one of the Tn5 insertions leading to enhance-
ment of root-colonizing activity in Pseudomonas was located in the gene mutY
encoding for repair of A:G base pairs in DNA after mutagen treatments (Lugtenberg
et al. 2004). It was found that mutY:Tn5 mutation does not provide an immediate
increase in root colonization. This is due to the secondary mutations which are
generated in the background of mutY:Tn5 insertion since mutants with improved
root adsorption may be selected positively by plants during the interaction of
bacterial inoculum with root surface.
Another example of the involvement of enzymes from DNA metabolism in the
control of plant-protective activities of PGPR was found in gene sss coding for site-
specific recombinase in P. fluorescens. Inactivation of this gene resulted in an impair-
ment of bacterial traits related to biocontrol of Fusarium (root colonization, synthesis
of phenazines, cyanide and chitinases). The Sss recombinase appeared to be respon-
sible for the “phase variation” in Pseudomonas resulting from the rearrangements of
genes involved in biocontrol (Lugtenberg et al. 2001). Introduction of the multiple sss
copies into Pseudomonas strains possessing a low biocontol activity may improve
them significantly (Dekkers et al. 2000).
Sometimes the biocontrol activities in PGPR do not correlate with intensive colo-
nization of host roots and plant protection results from only a small number of bacte-
ria cells. This occurs when PGPR inoculation results in the induction of systemic
resistance mechanisms that make the root non-accessible by pathogens. Initially this
effect of PGPR was called ISR (Induced Systemic Resistance) and was attributed
exclusively to nonpathogenic systems (Van Loon et al. 1998); SAR reactions, by
contrast, were considered to be typical of the interactions with plant pathogens.
Nevertheless, it was later found that the reactions of both types occur in either patho-
gen or nonpathogen systems and are distinguished by the nature of their endogenous
elicitors (reviewed in: Preston 2004; Vallad and Goodman 2004). The conventional
SAR reaction is characterized by an accumulation of salicylic acid signalling mole-
cules and pathogenesis-related proteins (PR-proteins), whereas ISR reaction is based
on signal transduction pathways regulated by jasmonates and ethylene. The systemic
defence responses of both types may be elicited exogenously by PGPR cells attached
to the roots or penetrating their outer tissues. Some molecules produced by PGPR
(cell wall and cyclic lipopolysaccharides, flagella components, exoenzymes, phyto-

hormones, type III secretion system (TTSS) effectors, siderophores, salicylic acid,
and toxins) may be perceived by the plant and elicit a defensive response (Preston
2004). Pronounced plant species-specificity has been observed in the manifestation
of ISR reactions, caused by PGPR (Liu et al. 1995; Van Wees et al. 1997).
The activities of plant-protecting PGPR are under direct control of the host
which provides nutrients and energy for the bacteria existing in the rhizosphere.
The greatest expression of plant protective activities by PGPR is typically seen in
the zone of root elongation, where these bacteria are also the most numerous
(Kamilova et al. 2006a). The importance of nutritional interactions by the symbi-
otic partners in providing biocontrol of pathogens is evident from the prevalence of
genes, whose products are responsible for producing different catabolic enzymes,
among the rhi genes of P. fluorescens which are activated specifically in the host
rhizosphere (Rainey 1999).
Active growth, expression of the biocontrol-related genes, and production of antifun-
gal factors such as antibiotics and siderophores are induced in plant-protecting PGPR
by root-secreted organic acids (malate, citrate, succinate, fumarate) while the majority
of mono- and di-saccharides are less important for the host-beneficial activities
(Lugtenberg and Dekkers 1999; Lugtenberg et al. 2001; Shtark et al. 2002, 2003), in a
comparable manner to Azospirillum-root associations. The difference between organic
acid and sugar formation was confirmed via mutational analysis: the root colonization
activity of P. fluorescens mutants with impaired utilization efficiency of organic acids
(e.g., defective malate dehydrogenase) is sharply decreased, while the mutants for sugar
utilization (e.g., devoid of glucose-6-phosphate dehydrogenase) are unaffected
(Lugtenberg and Dekkers 1999; Lugtenberg et al. 2001).
Highly effective plant defence may be due to an ability of the host to regulate
PGPR biocontrol functions by modulating the composition of root exudates. When
plants invaded by a pathogen are inoculated with PGPR, the amount of organic acids
in exudates may increase and stimulate the growth of bacteria and antibiotic produc-
tion (Kamilova et al. 2006b). Additionally, some plants (including the legumes, pea
and alfalfa [lucerne]) regulate their PGPR functions by exudating specialised signals
from the roots which mimic the bacterial “quorum sensing” regulators required for
root colonization and antifungal activities (Teplitski et al. 2000). These observations
suggest that improvement of biocontrol functions in root-PGPR associations may be
achieved via manipulations of the bacterial genotypes and host genotypes.
Bacterially-mediated synthesis of phytohormones, mainly auxins (IAA) may
produce improvements in plant nutrition and in plant protection. For example, com-
binations of enhanced IAA synthesis and biocontrol activities were detected in
Pseudomonas strains grown in rhizosphere of radish plants that exuded 30–100
times more tryptophane (the precursor of auxins) compared with wheat or tomato.
An important role in the expression of bacterial defensive functions may be
provided by ACC-deaminase (Glick 2004). Investigations in the ability of PGPR to
use ACC as a sole source of nitrogen demonstrated that only some strains possess
ACC-deaminase. Transfer of the gene coding for ACC-deaminase from Erwinia
cloacae to P. fluorescens resulted in an increase in ACC utilization efficiency
s ufficient to enhance the recombinants’ ability to suppress phytopathogenic fungi.

Therefore, in defensive associations ethylene has a role as a negative regulator
similar to its effects in plant associations with azospirilla (Penrose and Glick 2003)
and legume nodulation (Guinel and Geil 2002), although mechanisms of the regula-
tion may be different.
Summarizing what is known about nitrogen-fixing and defensive root-PGPR
associations it may be concluded that a range of common molecular mechanisms
are available: (i) root-exuded organic acids, but not sugars, supporting different
types of symbiotic bacteria; (ii) ethylene acting as a negative regulator for
these symbioses; (iii) surface components, namely, EPS, LPS, lectins and
cyclic b-glucans involved in the associations. A range of genes common to
rhizobia are induced in root associated azospirilla. Moreover, inoculation of the
model legume Medicago truncatula by Pseudomonas fluorescens results in induc-
tion of some plant genes common to nodulation/AM formation signalling cascades,
e.g., gene MtDMI3 (Sanchez et al. 2005) coding for calcium calmoduline-dependent
kinase (CCaMK) (Catoira et al. 2000) (Section.4.3.1). There is a possibility of
finding some sequence homologous in Pseudomonas similar to rhizobial nod
genes (as were found in Azospirillum genomes).
Despite relatively low specificity of plant associations with PGPR, plant
genotype has been shown to influence their effectiveness (i.e. genetic integra-
tion exists between the partners), and a series of genome loci (QTL) was identi-
fied controlling its quantitative variation (Wu et al. 1995; Smith and Goodman
1999; Preston 2004).
Mutually Beneficial Associations of Plants

with Endophytic Bacteria
Healthy naturally propagated plants grown in the field or in pot cultures are colonized
by populations of endophytic bacteria, averaging 103–106 cfu g−1 fresh plant weight
and sometimes reaching 1010 cfu g−1 (reviewed in: Hallmann and Berg 2006).
The spectrum of endophytic bacteria isolated from the roots of various plants covers a
wide range of species from about 70 genera; representatives of the genera
Pseudomonas, Bacillus or Streptomyces are most frequently encountered as endo-
phytes (reviewed in: Hallmann and Berg 2006). Newly developed molecular methods
enable complete analyses of the diversity of culturable and non-culturable bacteria
(Van Overbeek et al. 2006). Although most of the known genera include some phy-
topathogenic endophytes, many of the individual species are known to be beneficial
to higher plants (Sturz et al. 2000; Rosenblueth and Martínez-Romero 2006).
Both individually and collectively, the associations of endophytic bacteria
represent a continuum from mutualism, commensalisms and latent pathogenesis
through to active pathogenesis (Schulz et al. 2006). According to a working hypoth-
esis proposed by the current authors, asymptomatic colonization (without causing
visible plant disease symptoms) is a balance of antagonisms between host and
endophytes. Endophytes and pathogens both possess many similar virulence factors.
Indeed the loss of a virulence factor in model experiments resulted in transforming
a pathogen into a harmless endophyte (reviewed in: Hallmann and Berg 2006). It is
vexed question, however, as to which form found in Nature was transformed into
the other: pathogen into endophyte or vice versa?
Some endophytes are seed-borne, but others have mechanisms for colonizing
plants that have yet to be elucidated (Schulz et al. 2006). Although there are
occasional poorly substantiated reports of intracellular colonization of bacteria
providing a consistent and effective increase in the productivity of crops, it is
still considered that the intercellular apoplastic space is the most suitable niche
for endophytes (Sturz et al. 2000). It is suggested that many bacterial ‘endophytes’
may not colonize living tissues per se, but occupy protective niches in dead surface
tissues or closely adhering soil of rhizosheaths. Opportunities for passive ‘crack
entry’ into healthy, undisturbed roots under field conditions have been presumed
but after studies with laboratory-grown plants these may not be demonstrated to
be feasible. Consistent entry of endophytes into living root tissues in the field is
supposed to require a bacterial capability to hydrolyse the hydrophobic incrusta-
tions of the walls of epidermal, hypodermal, endodermal, and other cortical cells.
The lumen apoplast of xylem is considered as an unsuitable niche for endophytes,
especially in grasses, because of the reduced fitness that such colonization imposes
on plants when subjected to stresses in the field (McCully 2001). Although this
niche, is of course, frequently colonized by plant pathogens, notably wilt patho-
gens such as fungal Verticillium and Fusarium spp., as well as bacterial Ralstonia
spp. (Denny 2006) and Erwinia spp.
Plant associations with endophytic bacteria can increase plant growth and pro-
mote general development or improve plant resistance to pathogens and other
environmental stresses enhancing the host’s ability to acquire nutrients, or by pro-
duction of plant growth-regulating, allelopathic or antibiotic compounds (Sturz
et al. 2000; Berg and Hallmann 2006). Sometimes improved plant resistance can be
linked to induced systemic resistance caused by bacterial elicitors coming from the
endophyte (Kloepper and Ryu 2006). Certain cropping sequences have been shown
to favour the accumulation of particular plant growth-promoting bacterial endophyte
populations. These may lead to the development of beneficial host-endophyte allel-
opathies, with implications for the formation and maintenance of fertile, disease-
suppressive soils. Manipulating bacterial populations in soils and within crops will
be crucial if endophytes are to be utilized in crop production systems, and special-
ised techniques will be required to do so. It is necessary to study the natural associa-
tions between bacterial endophytes and their hosts for the purposes of employing
such systems most productively in sustainable agriculture (Sturz et al. 2000). Some
human pathogens, such as Salmonella spp., have been found as endophytes, and
these bacteria are not removed by disinfection procedures that eliminate superfi-
cially occurring bacteria. Delivery of endophytes to the environment or agricultural
fields should be carefully evaluated to avoid introducing plant, animal and human
pathogens (Rosenblueth and Martínez-Romero 2006).
Synergism of the Beneficial Soil Microbes in the Rhizosphere
The rhizosphere is a dynamic environment where the interactions between plants

and microbes are profoundly influenced by carbon fluxes (Toal et al. 2000). In addi-
tion to the interactions between plants and microbes there are also interactions
within the rhizosphere microbial community (Barea et al. 2005). The AM fungi and
bacteria can interact synergistically with each other to stimulate plant growth
through a range of mechanisms that include improved nutrient acquisition and the
inhibition of fungal plant pathogens.
With regard to AM symbiosis, these interactions result in modification of the envi-
ronment in the vicinity of AM mycelia to such an extent that the term ‘mycorrhizo-
sphere’ has been adopted (Linderman 1988). Both ECM (Garbaye 1994) and AM
(reviewed in: Artursson et al. 2006; Barea et al. 2005) fungi can interact with differ-
ent bacterial species which frequently attach to fungal mycelium. For those bacteria
known to stimulate mycelial growth of mycorrhizal fungi and/or enhance root
mycorrhization the term ‘mycorrhiza-helper-bacteria’ has been proposed (Garbaye
1994). This effect on mycorrhiza establishment is associated with the secretion of
bacterial hormones and increasing the rates of root exudation.
Rhizospheric bacteria considered to be examples of PGPR are also known to
affect the pre-symbiotic stage of AM development, namely germination rate and
mycelium growth (Barea et al. 2005). Furthermore, according to new information
some AM fungi are capable of forming fertile spores even in the absence of plant
roots when they are cultivated dixenically together with bacteria P. validus isolated
as AM fungal spore contaminants (Hildebrandt et al. 2006). In turn, AM formation
is accompanied with alterations to plant physiology which can affect the rhizo-
spheric microbial populations both quantitatively and qualitatively (Barea et al.
2005; Artursson et al. 2006). It was shown that co-inoculation with AM fungi
improves the establishment of both introduced and indigenous PGPR (Barea et al.
2005). Simultaneous inoculation with AM fungi and phosphate-solubilising PGPR
has been shown to be a most effective plant treatment by comparison with
mono-inoculation variants (Barea et al. 2005). Multi-microbial interactions, includ-
ing those between locally isolated AM fungi, phosphate-solubilising bacteria and
Azospirillum, have also be reported, which indicate clearly that microbes act syner-
gistically when inoculated simultaneously (Muthukumar et al. 2001).
Several studies have demonstrated that bacterial antagonists of fungal pathogens do
not exert any anti-microbial effect against AM fungi (reviewed in: Barea et al. 2005).
The results from in vitro or in situ laboratory experiments demonstrated no negative
effects from Pseudomonas strains including those over-producing the antifungal com-
pound 2,4-diacetylphloroglucinol on G. mossea spores germination. Moreover, the
Pseudomonas strains improved plant growth and nutrient (N and P) acquisition by
mycorrization (Barea et al. 1998) under field conditions. Nevertheless, in several cases
of double inoculation with AM fungi and PGPR being used as biocontrol agents great
variability in plant growth-promoting effects were observed between experiments
indicating that more studies are needed for elucidation and effective use of whole
plant–growth substrate-AM fungus-RGPR systems (Barea et al. 2005).
The establishment of AM has been shown to improve nodulation and nitrogen-
fixation (reviewed in: Barea et al. 2005). In addition to the existence of common
genetic system controlling the development of RN symbiosis and AM (genetic
interference) (Section 4), there is trophic interference in symbioses. The AM can
enhance the activity of rhizobia through a generalized stimulation of host nutrition.
Nodulation processes have a significant requirement for phosphorus (Barea and
Azcon-Aguilar 1983), so it would seem logical to need phosphorus-acquiring symbioses
such as AM (Sprent and James 2007). But some localized effects may also occur at the
root or nodule level (Barea et al. 2005). Also AM inoculation improves RN symbiosis
development and functions at low levels of water potential and compensates for the
negative effects of salinity on them (Quilambo 2003; Barea et al. 2005).
The common mechanisms available for RN symbioses and PGPR associations
may result in their synergistic effects on plant growth. There is a general experience
of enhanced nodulation in legumes by simultaneous inoculation with Azospirillum
and PGPR (Okon et al. 1995; Sessitsch et al. 2002). Some mechanisms of syner-
gism between rhizobia and PGPR were studied in pea where inoculation with the
root growth promoting actinobacteria Streptomyces lydicus resulted in increased
nodule mass, size and biochemical activities of the bacteroids as well as an accu-
mulation of ferric ions in the nodules (Tokala et al. 2002).
Thus, there have been many studies concerning interactions between AM fungi
and bacteria, but the underlying mechanisms of the functional properties behind
these associations still require much further experimental confirmation (Artursson
et al. 2006). These interactions are crucially important for sustainable, low-input
agricultural cropping systems which rely on biological processes rather than agro-
chemicals in order to maintain soil fertility and plant health. The potential of micro-
bial synergisms suggest that the prospects for establishing multi-partite symbioses
which improve different agronomic traits in leguminous crops (Barea et al. 2005;
Artursson et al. 2006) are high. The results of experiments with multi-microbial
systems, including AM fungi, rhizobia and PGPR, support the importance of
encouraging the physiological and genetic adaptation of microbes to the environment
(Requena et al. 1997). Thus, locally sourced isolates are recommended for use in
biotechnological applications.
Adaptive Evolution of the Mutually Beneficial Plant–Microbe

Symbioses
It is generally accepted that the capacities of terrestrial plants for regulating the
functions of microorganisms colonizing root systems developed during the earliest
stages of evolution. Comparative data obtained by various methods may be used to
probe the origins of plant–microbe symbiotic interactions and lead to the conclu-
sion that AM is “the mother of plant root endosymbioses” (Parniske 2008) and,
probably, some other symbioses as well. Therefore, most probably, mutualism was
an early form of plant–microbe symbiotic interaction, and not antagonism as is
sometimes postulated (Dyakov et al. 2007).
Fossilised plants have been found (Kidston and Lang 1921; Remy et al. 1994)
which contain structures similar to those of present-day AM and are related to
Silurian and Devonian flora. This was the time (400–500 millions years ago) when
terrestrial plants appeared (Schüßler et al. 2001; Redecker 2002). The importance
of AM associations for these plants is evident since they lacked conventional root
systems, but were equipped with rhizomes and rhizoids which could anchor them
to substrates but not take up nutrients effectively (see for review: Brundrett 2002).
Possibly true roots co-evolved with AM-fungal partners, and anatomy of mycotrophic
plants tends to confirm this (Brundrett 2002). Most of contemporaneous plants still
have a dependence on AM; where this is lacking then such individuals are thought
to result from later evolutionary events (Brundrett 2002).
Discussion continues as to which morphological form of AM development (linear,
so-called Arum type, or coiled, Paris type) is the original ancestor. Indeed, it is difficult
to detect an evolutionary trend in AM types, since they are found throughout the plant
kingdom (Smith and Smith 1997; Dickson et al. 2007). Based on the analysis of fossils
the Arum type could be the oldest mycorrhizal form (Kidston and Lang 1921; Remy
et al. 1994). It has been suggested that coiling AM is more advanced than linear types,
since the former appears to allow the host greater control of the fungus. Additionally,
linear colonization is more rapid and efficient, but this may result in greater energy
costs for the plant (see for review: Brundrett 2002). On the other hand, coiling AM is
the most common form in contemporary bryophytes, ferns and gymnosperms and
thus might be considered as an ancestral condition (Smith and Smith 1997).
Studies of molecular phylogeny based on the conserved genes (16S or 18S rDNA
nucleotide sequences) using polymorphism analyses provides new views on the
origins and evolution of bacterial and fungal symbiotic organisms and their interac-
tions with hosts. Apparently AM fungi are a monophyletic group Glomeromycota
(Schüßler et al. 2001), indicating that they have originated from a common ancestor.
Interestingly this phylum includes along with obligatory biotrophic AM fungi, the
genus Geosiphon represented by the single species G. pyriformis which is both free-living
and able to form symbioses with the photosynthetic (and also nitrogen-fixing)
cyanobacteria Nostoc. Possibly this symbiosis might in evolutionary time have
preceded AM (Schüßler 2002; Parniske 2008), but not those marine ancestors of
terrestrial plants and unicellular or possibly parasitic fungi as was suggested earlier
(Parniske 2000; Schüßler et al. 2001). Thus, it is possible that Glomeromycota
evolved before the land plants. The low specificity of AM fungi in relation to puta-
tive host plants demonstrates that their gene systems, which control AM develop-
ment, are rather conservative and might have been only slightly reorganized for
different symbioses in various host groups. Curiously, a recent study demonstrates
that some AM fungi are capable of completing their life cycles in the absence of
plant roots, but rely instead on the simultaneous growth of bacteria Paenibacillus
validus which is supposed to be the natural partner of these fungi (Hildebrandt
et al. 2006).
In contrast to AM fungi, many other microsymbionts including nodule bacteria

(Sprent 2001) and ectomycorrhizal fungi (LoBuglio et al. 1996; Hibbert et al. 2000;
Moncalvo et al. 2000) are polyphyletic in their origin. Consequently, these plant–
microbe symbioses evolved in parallel and most probably after AM. It is known
that legumes evolved much later than AM (Lavin et al. 2005); hence, their symbio-
sis with nitrogen-fixing nodule bacteria are considerably younger (60–70 million
years) (Sprent 2001; Sprent and James 2007).
Parallel evolution and convergence plays an important role in the evolution of
plant–microbe interactions, as plants possess universal gene systems for the control
of basic functions required in symbiosis, including the maintenance of interior
homeostasis, novel tissue and cellular structures (symbiotic compartments) and the
utilization of nutrients provided by the partner organisms (Gualtieri and Bisseling
2000). Based on the universal organization and regulatory mechanisms of the plant
genetic material (Cronk et al. 2002), the gene systems could have been rearranged
in similar manners during co-evolution with different microbes. Various microor-
ganisms when adapting to an existence within host plants, obviously, followed
convergent evolutionary pathways developing similar mechanisms for dialogue
with their defensive (regulatory) systems (Provorov 2009).
According to the phylogeny of flowering plants developed on the basis of plastid
gene rbcL structure (Doyle 1998), which differs from earlier systems based on plant
morphology, all plants that are able to form nitrogen-fixing nodules with rhizobia
and actinobacteria in the genus Frankia, belong to a monophyletic Rosid I clade.
A common ancestor of Rosid I, obviously, could not form nodules with rhizobia but
had some pre-adaptations to form nitrogen-fixing symbioses which developed in a
parallel manner in several plant families which evolved later. Those pre-adaptations
can be subdivided into two groups: the first includes concerns the capacity for being
colonized by AM fungi and of controlling the mycobiont once inside host tissues;
the second includes capacities which determine the formation of special cellular,
tissue and organ structures characteristic of nitrogen-fixing nodules (symbiotic
organs and compartments).
Developmental genetics using structural analyses, cell biology, genetics and
functional comparative genomics gives many clues for reconstructing the origins
and evolutionary pathways of plant–microbe symbioses since they reveal broad
involvement of common plant genes and stages in the developmental programmes
of root symbiotic nodules and ancestral AM symbiosis. These commonalities sug-
gest that AM was a source of pre-adaptations for other forms of interactions (Sprent
2001; Kistner and Parniske 2002; Provorov et al. 2002; Sprent and James 2007;
Parniske 2008) responsible for host regulatory reactions. These in turn may be
controlled by plant genes recruited from AM programmes into those for nodule
developmental and possibly, into those of plant resistance to pathogens.
Regarding the second type of these pre-adaptations contributing to nodulation,
it is possible that the ancestor of Rosid I was able to form outgrowths (nodule-like
structures) which carried features of depository and systems of metabolic exchange
with aerial plant organs. These were used for the integration of mechanisms of
nitrogen-fixation with consequential implications for the derivation of energy and

nitrogen-carbon exchange. Evidence for this may be deduced from the occurrence
of nodule formation induced by some rhizobia non-secreting host-specific Nod fac-
tors (through cracks in the root epidermis or on the stem from rudimental air-roots)
(Downie 2007; Giraud et al. 2007). Furthermore, spontaneous formation of pseudo-
nodules has been found in some current native legumes (Bonnier 1961) and some
alfalfa (lucerne) and clover lines (Truchet et al. 1989). Several genes were found in
those leguminous lines controlling the formation of pseudo-nodules either under
sterile conditions or in the presence of avirulent, Nod factor defective rhizobial
mutants (Caetano-Anollés et al. 1992).
Summarizing the knowledge available so far permits the development of a hypoth-
esis for consequential placement during evolution of different types of microorgan-
isms which can occupy intercellular and sub-cellular symbiotic compartments within
the root cortex, as a result of which dicotyledonous plants have acquired an ability to
form nitrogen-fixing symbioses with nodule bacteria (Provorov 2009). Hence, nitrogen-
fixing prokaryotes exploited an ancestral programme in the plant for microsymbiont
“hosting” which appeared during co-evolution of most ancient plants with AM fungi.
The ability to form both symbioses was incorporated in parallel into different families
of Rosid I under natural selection pressures acting in favour of the ability for
symbiotrophic nitrogen nutrition. The first step could be a replacement of AM fungi
by Frankia spp. which have a similarly mycelial-like growth habit and in turn was
replaced later by another nitrogen-fixing prokaryote, rhizobia.
These analyses also enable us to suggest a new insight into the origins of legume
nitrogen fixing symbionts (rhizobia). Conventionally, their origins were considered
mainly to have come from plant pathogens or from beneficial plants symbionts
(PGPR, endophytes) in which the systems for Nod factor synthesis arose before
later distribution into a broad spectrum of soil or plant-associated microbes via
horizontal gene transfer (Provorov 1998; Terefework et al. 2000; Suominen et al.
2001). For example, slow-growing rhizobia might have arisen from Azospirillum-
like strains since there is a visible taxonomic relatedness between Azospirillum and
Bradyrhizobium genera, and in the genomes of some Azospirillum strains there are
close homologues to some rhizobial nod and fix genes (Fogher et al. 1985; Vieille
and Elmerich 1990, 1992; Rivas et al. 2002). This suggestion is consistent with
numerous findings demonstrating that rhizobia can effectively operate as the ben-
eficial PGPRs or as endophytic symbionts in “non-host” (legume and non-legume)
plants (Sessitsch et al. 2002). Although the mechanisms of beneficial effects on
plants may be atypical for rhizobia (involving not nitrogen fixation but rather the
synthesis of hormones, defence of plants from pathogens and solubilization of soil
phosphates), these endo- and epiphytic associations of rhizobia with plants may be
considered as a vestige from the early stages of evolution in morphologically dif-
ferent nodular symbioses.
Here we would like to introduce an alternative hypothesis concerning the origins of
rhizobia and postulate that these bacteria came first from micro-symbionts associated
with AM fungi. Some arguments in favour of this hypothesis are summarized below:
1. The root systems of current land plants evolved in the presence of AM fungi
(Brundrett 2002) suggesting that the genetic systems of plants for controlling
root development and mineral nutrition can overlap with the genetic system con-
trolling AM development at the large scale.
2. AM (as well as the other mycorrhizal fungi) are associated with different types of
endo- and ecto-symbiotic bacteria (including nitrogen-fixers). Moreover these
fungi may operate as effective vectors for introducing bacteria into the plants
(Artursson et al. 2006). Therefore, the multipartite symbiotic communities
(plants + fungi + bacteria) may be considered as being ancestral with respect to the
two-component systems (e.g., to root symbiotic nodules). It is important to note
that colonization of roots by mycorrhizal fungi is not associated normally with
induction of bacterial diseases, and therefore, a certain degree of selectivity occurs
with respect to the bacterial satellites of fungi which invade roots. This selectivity
may be an important factor for the maintenance of multipartite symbiotic
communities otherwise they would have been eliminated by natural selection.
3. There are common regulatory plant genes controlling the development of AM
and nitrogen-fixing nodules (reviewed in: Borisov et al. 2008 and this Chapter];
numerous common plant genes are expressed during both symbioses (Küster
et al. 2007); furthermore some common plant genes have been found which are
expressed in the process of establishing AM and mutually beneficial associations
with rhizospheric bacteria (Sanchez et al. 2005, Vivienne Gianinazzi-Pearson,
2008 personal communication).
4. Stimulation of AM formation by Nod factors produced by rhizobia has been
observed (Xie et al. 1997). Therefore, a primary function of Nod factors in the
evolution of rhizobia could be the stimulation of penetration by fungal vector
and only later were Nod factors acquired with a function for the independent
induction of bacterial symbiotic developmental programmes.
5. Taxonomic relatedness was found between some fungal symbionts and some
rhizobia, for example, Burkholderia-like strains are found among endo-symbionts
of AM fungi (Minerdi et al. 2002) and of ectomycorrhizal fungi (Izumi et al.
2006) as well as amongst legume nodular symbionts (Balachandar et al. 2007).
Of course, not all of the numerous and diverse rhizobia species might have arisen
from the bacterial satellites of the mycorrhizal fungi. Nonetheless, the bacterial
co-habitants of mycorrhizal fungi may be seen as possible precursors of the first
rhizobia in which the genes for Nod factor synthesis developed primarily since Nod
factors are chitin-like compounds which can mimic fungal cell wall intermediates and
therefore they can be very useful tools for the colonization of fungal hosts by associ-
ated bacteria. Moreover, intimate interactions between bacteria and fungi might allow
the acquisition by ancient rhizobia of some genes involved in the synthesis of chitin-
like compounds, (e.g., accepting of nodA by horizontal gene transfer from fungi was
suggested as a mechanism by Hirsch et al. 2001). An extended comparative study of
the rhizobia species and of bacterial satellites of mycorrhizal fungi is required to
identify their inputs into the evolution of nodular symbiosis in legumes.
Since fossil land plants were, possibly, obligate biotrophs, maintenance of sym-
biotic fungi in roots was a more ancient function compared with the independent
assimilation of nutrients from soil (Provorov 2009). Two opposite strategies in the
evolution of plant symbiotrophy can be suggested: a change to autotrophy (for
example, loss of mycotrophy in Brassicaceae (previously Cruciferae) or symbiotro-
phy in commercial legume cultivars grown by the elevated use of mineral fertiliz-
ers) or phototrophic denial and formation of myco-heterotrophic associations, as
for instance, in orchids as well as in some dicotyledonous plants, ferns, bryophytes
and liverworts (Bruns and Bidartondo 2002).
From the perspective of an entire plant–microbe system, mutually beneficial
symbioses are represented as the products of interspecies (reciprocal) altruism which
resemble to some extent antagonistic (pathogenic) interactions at the molecular and
cytological levels (Provorov and Vorobyov 2009a, b). Plant hosts, possessing puta-
tive gene networks for regulating beneficial microbes and for defence against patho-
gens, with direct parallels between mutualism and antagonism are evident logical
suggestions. But viewed from the microbial perspective this is unlikely, since benefi-
cial and pathogenic microbes often demonstrate independent origins (Provorov and
Vorobyov 2010). The evolutionary divergence of beneficial and deleterious micro-
symbionts is suggested to result from specific selective pressures (different forms of
group selection) maintaining genes for beneficial traits in microbial populations
(Provorov and Vorobyov 2009a, b). Using the legume–rhizobia symbiosis as a
model, it was suggested that this selection might have been induced by two factors.
The first one is the positive feedbacks operating between partners at the level of
symbiotic metabolism (carbon and nitrogen compounds) and of partners’ co-evolu-
tion (synergistic selective pressures supporting mutualism in plants and in microbes).
A second one is clonal structures of microbial populations in plants which result in
selection favouring symbiotically effective strains.
New Approaches of Application of Mutually Beneficial

Plant–Microbe Systems in Sustainable Agriculture
Firstly, an existence of plant genes (Duc et al. 1989; Gianinazzi-Pearson 1996;

Küster et al. 2007) and their molecular products (Frühling et al. 1997) common for
both AM and RN led to a conclusion that legumes possess common genetic systems
controlling the development of a tripartite symbiosis (legume plant + AM fungi +
rhizobia). This fact along with the demonstration of synergistic activity in beneficial
soil microbes (reviewed in: Barea et al. 2005) and a suggestion that plant genetic
systems controling the development of RN and, probably, of some other beneficial
plant–microbe associations evolved on the basis of that present in AM (Parniske
2008; Provorov 2009) has great importance for the application of tripartite or even
multi-partite symbiotic systems in low-input sustainable environmentally-friendly
agrotechnologies.
Development of New Types of Microbial Inocula
The use in sustainable agriculture of inocula based on beneficial soil microbes as

described above allows the improvement crop productivity with decreased doses of
mineral fertilizers and pesticides (reviewed in: Xavier et al. 2004; Rai 2006).
Currently the majority of commercial inocula contain pure cultures of single
microorganisms and only occasionally multiple combinations. There are several
objections to the use of mono-inoculation. Firstly, endemic microbial communities
are stable and although plastic the introduced microbe may be allowed to occupy a
very small niche in the whole community as a whole. Secondly, genetic material in
microbes is very plastic, and consequently strains introduced into natural ecosys-
tems can rapidly lose their beneficial traits. Thirdly, the existence of microbial
cooperation in the rhizosphere (Barea et al. 2005) as well as in natural synergistic
associations of different microbes including those between AM fungi and their
endocellular or superficial symbionts (Artursson et al. 2006; Barea et al. 2005)
question the possibility and expediency of applying mono-inoculants and even use
of the term ‘mono-inoculation’ itself. Finally, plants possess relatively stable
genomes and this fact contributes significantly to the effectiveness of symbiosis
(Tikhonovich and Provorov 2007). Therefore, for industrial plant production in
sustainable systems we should use plants having highly effective interactions with
all forms of beneficial soil microbes, which can encourage the development of
multiple niches hosting microbes and regulating their activity. For this it is neces-
sary to develop new multi-component microbial inocula which increase the content
and biodiversity of beneficial soil microbes in agricultural land.
There is experimental evidence of the effectiveness of simultaneous inoculation
of legumes with AM fungi and nodule bacteria leading to increased productivity
and quality of the yield, e.g. groundnut (Ibrahim et al. 1995), pea (Jacobi et al.
1999; Borisov et al. 2002, 2004; Shtark et al. 2006), albaida (Anthyllis cytisoides)
(Requena et al. 2001), and soybean (Labutova et al. 2004). The effect achieved
equalled or exceeded that achieved with mineral fertilizers (Borisov et al. 2002,
2004; Shtark et al. 2006). The effect also exceeded that of mono-inoculation with
AM fungi or with rhizobia either in model experiments or under field conditions
(Jacobi et al. 1999; Borisov et al. 2002; Labutova et al. 2004). In long-term experi-
ments in a desertified Mediterranean ecosystem, it was found that simultaneous
inoculation with AM fungi and rhizobia enhanced the establishment of key plant
species and increased soil fertility and quality; increased soil nitrogen content,
organic matter content, and soil aggregate hydrostability and enhanced nitrogen
transfer from nitrogen-fixing to non-fixing species associated with the natural suc-
cession of the plants (Requena et al. 2001).
There is an example of application of triple inoculum (AM fungi, rhizobia and
PGPR) to the legume A. cytisoides which was successful only when the microorgan-
isms used were isolated from local environment (Requena et al. 1997). In collabora-
tion with an innovation company “Bisolbi-Inter” (Russian Federation) the All-Russia
Research Institute for Agricultural Microbiology (ARRIAM), Saint-Petersburg,
Russian Federation, has developed technology for the production and application of
a new multifunctional biopreparation “BisolbiMix” (Chebotar et al. 2008) contain-
ing a complex of the most effective isolates of endosymbiotic microbes (AM fungi
and rhizobia) and associative bacteria (PGPR) from the collection held at ARRIAM.
A non-sterile substrate-carrier which is derived from washing-filtration by-products
of a sugar-beet factory contains its own microbial community including all the
above groups of beneficial microbes. The preparation can be formulated into a seed
dressing (not effective for all the crop plants tested) or granules. The efficacy of
“BisolbiMix” was demonstrated in field trials with legumes, e.g. pea (Shtark et al.
2006) or non-legumes such as wheat, pumpkin and potato (unpublished results). The
use of microbial formulations containing rhizobia for non-legumes seems to be sen-
sible because it is known that nodule bacteria which do not form nodules on a non-
host legume as well as non-legume roots can operate as PGPR (Prévost and Antoun
2005; Hossain and Mårtensson 2008). Thus, the selection of rhizobia with both
PGPR activity and efficient symbiotic nitrogen fixation should be advantageous in
crop rotations or intercropping systems using legumes and non-legumes.
It is possible, therefore, to develop effective multi-microbial inoculants, but it is
necessary to use local communities of beneficial microbes because this exploits the
natural biological and genetical adaptations of the partners to their environment
(Requena et al. 1997; Gentili and Jumpponen 2006).
Legumes in Sustainable Agriculture
During development of plant–microbe systems for low-input sustainable ecologi-

cally friendly plant cultivation it is necessary to be guided by conclusions of
European Community (EC) experts about global productivity of legumes (http://
www.grainlegumes.com/aep/; http://ec.europa.eu/research/biosociety/food_
quality/projects/002_en.html) for sustainable agriculture. The use of legumes in
agriculture is leading to: improved soil fertility and increased diversity of crops
and soil microbial communities; reductions in the use of non-renewable natural
resources; decreased negative effects from intensive agrotechnologies on the natu-
ral environment due to decreased requirement for mineral fertilizers and pesticides
and decreased production of animal protein and associated wastes; local production
of pollution-free food and forage; and a more stable income for the agricultural
producers. This is why it is necessary to breed legumes which have highly effec-
tive interactions with beneficial soil microbes.
Legume Breeding to Improve Their Symbiotic Effectiveness
For more than 25 years the authors’ laboratory has specialized in the genetics of
plant–microbe interactions using pea (P. sativum L.) as a model plant. Our experience
for improving the effectiveness of beneficial plant–microbe systems with pea is

consequently given as an example. At the same time, the authors’ team knows only
one record of other activity of a similar nature viz. genetic variability in onion
(Allium spp.) has been shown with respect to its responsiveness to AM fungal
inoculation which indicates that onion breeding for improving efficacy of associa-
tion with AM fungi is possible (Galvan et al. 2007). The necessity for this sort of
plant breeding is also considered in isolation and mainly with respect to the effec-
tiveness of RN symbiosis (Herridge and Rose 2000; Rengel 2002; Graham et al.
2004; Howieson et al. 2008).
Analysis of Genetic Variability of Pea with Respect to its Effectiveness

of Interactions with Beneficial Soil Microbes
A high level of genetic variability was demonstrated in analyses of the symbiotic

effectiveness under double inoculation with AM fungi and nodule bacteria of 99
land-races and outclassed heritage cultivars of P. sativum from the collection N.I.
Vavilov’s All-Russia Research Institute of Plant Industry, Saint-Petersburg, Russian
Federation, with different geographical origins (Jacobi et al. 1999; Borisov et al.
2004). In a few genotypes considerable increases in plant dry weight (about 300%),
seed productivity (more than 650%), phosphorus and nitrogen content (more than
900 and more than 300%, respectively) were observed. The most promising highly
symbiotically effective genotypes and those with low symbiotic potential were
included in the Pea Genetic Collection (ARRIAM) to be used for experiments
studying the functioning of tripartite/multipartite symbiosis. Types identified as
highly symbiotically effective genotypes were involved in breeding programmes to
create commercial pea cultivars with great potential for interactions with beneficial
soil microbes were used in collaboration with All-Russia Institute of Leguminous
and Groat Crops (ARILGC), Orel, Russian Federation.
The most promising highly symbiotically effective pea genotypes previously
selected and different commercial pea cultivars created without consideration of
symbiotic effectiveness were involved in 3-year field trials (Orel district) (Shtark
et al. 2006). Seed productivity and plant dry weight were chosen as the main crite-
ria for the evaluation of symbiosis effectiveness in legume crops. The double (actu-
ally multiple, see above comments on the nature of AM fungi) inoculation was
shown to increase seed productivity and plant dry weight in most of the pea geno-
types studied and sometimes this could exceed the effect of mineral fertilizers. The
effectiveness of legume breeding to improve symbiotic potential of legume culti-
vars was proven therefore, under field conditions and the genotypes to be used in
such breeding programmes were identified. The genotype K-7284 (non-commercial)
was selected as a standard of symbiotic effectiveness. Additionally, it was demon-
strated that highly effective genotypes can be also found among commercial pea
cultivars created without consideration for effectiveness of interactions with benefi-
cial soil microbes. Taking into account that most commercial legume cultivars have
accidentally lost their abilities for symbiotrophic nutrition without selective pressure
during breeding of intensive crops, the latter constitutes a very important finding
for plant breeders and gives them the possibility for concurrent generation of culti-
vars with required pea plant architecture, other agriculturally important traits and
high effectiveness of interactions with all types of beneficial soil microbes in a
single breeding programme.
Breeding to Improve Pea Symbiotic Effectiveness
In order to cultivate plants with improved symbiotic potential a special breeding

nursery was created in the experimental trials ground of ARILGC on land
where for the preceeding 5 years before nursery establishment mineral fertilizers
had not been applied. To reduce the incidence and severity of root pathogens a
six-field crop rotation was used where cultivation of winter wheat was followed
by peas. The multi-component preparation “BisolbiMix” was used for the
inoculation of test plants.
Using the breeding nursery as well as a breeding protocol developed from
long-term collaboration of ARRIAM with ARILGC the first (in the whole history
of legume breeding) pea cultivar “Triumph” having increased potential of interac-
tions with beneficial soil microbes was intentionally bred (Borisov et al. 2008). It
arose as a result of crossing a commercial cultivar ‘Classic’ (donor of agriculturally
important traits) and the genotype K-8274 (donor of symbiotic effectiveness trait)
and subsequent individual selection of genotypes with high productivity and capac-
ity for supporting various beneficial microbes.
The cultivar “Triumph” is of middle stem height, semi-leafless and has stable
productivity under different climate conditions, it is comparatively resistant to root
rotting pathogens and pests. Its productivity is not lower than those of the produc-
tivity standards for Orel district using conventional production technologies and
10% greater in comparison with standard cultivars when inoculated with
“BisolbiMix”. As a result of 2-year state trials (2007–2008) the productivity of
“Triumph” was shown to be comparable with that of standard regional cultivars
enabling recommendation for commercial cultivation in the Central region of
Russian Federation (unpublished results). Thus, the innovative concept of the
authors’ research team for plant breeding (applicable not only for legumes, but also
for non-legumes) is bearing its first fruits.
Conclusions
Intimate associations of beneficial soil microbes with the host plants described
above in detail are applicable in sustainable crop production if taken either sepa-
rately or in combination. Many authors are now recognizing the need for using
multi-microbial plant inoculants and the advantages of using indigenous plants (or
cultivars of local breeding) and microbes.
The authors’ team proposes its own concept which offers fundamentally new
approaches to plant production. Firstly, it is necessary to consider plant genetic sys-
tems controlling interactions with different beneficial soil microbes in unison.
Secondly, plants used as a component of this complex plant–microbe system control-
ling its effectiveness should be bred to improve the effectiveness of interactions with
all types of beneficial soil microbes. Increases of plant biomass production due to
plant–microbe symbiosis should be used as the main parameter for an evaluation of
plant effectiveness in interactions with beneficial soil microbes. The plant production
should be done with inoculation composed of multi-component microbial inocula
consisting of AM fungi, rhizobia, PGPR and/or beneficial endophytic bacteria.
Finally, taking into consideration the importance of legumes to global agriculture,
greater emphasis should be placed on plant–microbial systems in the development of
low-input agro-biotechnologies enabling wider cultivation of leguminous crops.
Acknowledgements We are grateful to Timofey A. Nemankin and Alexandr I. Zhernakov for

their help in preparing the figures. We thank Dr. Margarita A. Vishnyakova (N.I. Vavilov All-Russia
Research Institute of Plant Industry, RF), Dr. Tatiana S. Naumkina (All-Russia Institute of Legumes
and Groat Crops, RF) and Dr. Vladimir K. Chebotar (“Bisolbi-Inter” Ltd., RF) for the long-term
collaboration in the fields of legume breeding and microbial inocula development. This work was
supported by the grants of RFBR (09-04-00907, 09-04-13895, 09-04-91054, 09-04-91293, 10-04-
00961, 10-04-01146), NWO 047.018.001, Grant to support leading Russian science school
3440.2010.4, Governmental contracts for research with Russian Federal Agency of Science and
Innovations (02.512.11.2280, 02.740.11.0276).
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Chapter 6
Soil-Borne Pathogens and Their Interactions
with the Soil Environment
Geoffrey R. Dixon and Emma L. Tilston
Introduction
Wheat yields in thirteenth century Europe have been estimated at 385 kg ha−1
(Pretty 1990; Houghton 1996), more than half a millennium later, by 1939 they had
been increased to little more than 2 t ha−1. Subsequently, in the period 1952–1986
scientific and technologically based innovation applied to farming increased yields
by an average value of 2.6% pa. It is predicted that wheat yields will rise to 10.48–
13.69 t ha−1 by 2015, with a current theoretical biological ceiling of 19.2 t ha−1
(Britton 1990). These rising yields have been accompanied by a tenfold increase in
the amount of nitrogen applied to wheat in 1943/1945–1994 (Houghton 1996).
They represent one example of what has been achieved by a combination of geno-
type (plant breeding) and environmental modification increasing the nutrients avail-
able to the plant in step with its physiological demands and fending-off consequential
invasions by pests and pathogens attendant on rapid growth and high yields.
Burgeoning human populations, reaching an estimated 10 billion by 2050, demand
that similar yield increases are continued and accelerated. The environmental and
climatic consequences arising from a policy of raising yield solely by increasing
inputs are becoming apparent as dangerously unsustainable. Dramatic changes in
the manner by which crops are husbanded are required with the aim of achieving
increased yield but with minimal damage to the world’s ecosystems (Dixon and
Margerison 2009). Concomitant with the need to continue and accelerate yield
G.R. Dixon (*)

Centre for Horticulture and Landscape, School of Biological Sciences,
Whiteknights, University of Reading, Berkshire RG6 6AS, UK
E.L. Tilston
Scottish Universities Environmental Research Centre, Scottish Enterprise Technology Park,
East Kilbride G75 0QF, UK
198 G.R. Dixon and E.L. Tilston
increases in order to fill empty stomachs and enhance lifestyles far greater control
of pests and pathogens using intelligent, integrated and environmentally benign
methods based on sound scientific knowledge of the intimate relationships of ben-
eficial microbes, roots, soil and pest and pathogen biology is required for the health
of our planet’s environment and biodiversity. So far, our knowledge of soil
microbiology is at best scant and fragmentary and at worst non-existent. Yet as
shown in this book the agronomic opportunities for enhanced productivity offered
by the soil and its inhabitants are huge.
Soils contain very substantial numbers of phytopathogenic microbes capable of
devastating crops worldwide. These include representatives of the bacteria, fungi,
mycoplasma, protista and viruses. Some pathogens have very limited and highly
specific host ranges while others are generalists, to a lesser or greater degree,
causing diseases across many host taxa. Similarly, their geographical ranges may
be restricted or alternatively spread around the entire globe. The intensity of
pathogenesis varies also from those which devastate crops by ultimately killing
their hosts in the processes of colonisation and reproduction to those which are
only mildly aggressive and possibly almost commensalists. The forms of disease
syndrome generated in crop hosts vary from simple root invasion and rotting
through to altered root growth, e.g. root galling and clubbing, to collar rotting and
damping-off into vascular wilting and colonisation throughout all the aerial organs
of the plant. Some pathogens invade early in the host life-cycle and may then enter
a quiescent or dormant phase from which they re-emerge devastating maturing
flowers and fruit or destroying products stored after harvesting from the crop.
Indeed some pathogens are only apparent from the toxins which they elicit in
stored products.
The extensive range of pathogenic lifestyles available to soil-borne microbes
increases the problems facing plant pathologists striving to unravel their biology.
These difficulties are compounded by the sheer physical obscurity resulting from
dwelling either wholly or at least partially in soil. Attempting to ascribe taxonomic
identity and to understand the life-cycle biology of organisms that conduct all or
part of their life-cycles in soil and subsequently are enclosed in the bodies of their
host plants is a complex and at times confusing task. Adding to these problems are
barriers in understanding the physiology and metabolism of host roots which are
the primary targets for invasion by soil-borne pathogens. Roots possess a modular
structure as described by Hodge (Hodge 2009) permitting responses to their soil
environment and adaptation to changes, some of which are a result of the presence
of pathogenic microbes. In achieving adaptation the roots differentiate between
adopting partnership modes with benign microbes which enhance the potential
efficiency for resource capture (see other chapters in this volume) and adopting
defensive modes as a reaction to the presence of pathogens. The root-cap region
appears to be the main environmental sensing and response control centre. Recent
research indicates apparent root-to-root interactions and the capacity for recognising
‘self’ and non-self’ roots (Falik et al. 2003). Root exudates possibly form one
means for positive communication with benign microbes and negative responses to
pathogens (Badri and Vivanco 2009).
6 Soil-Borne Pathogens and Their Interactions with the Soil Environment 199
Developing husbandries which are both economically and environmentally

s ustainable demands a thorough understanding of the impact and interactions
between host roots, benign microbes and pathogenic organisms. Pathogens are capable
of wreaking either rapid crop destruction or causing the long-term degeneration and
decline of hosts possibly without their presence being easily obvious. This chapter
discusses the abilities of pathogenic microbes to cause diseases as moderated by the
soil environment and influenced by husbandry practices. Soil-borne microbial
pathogens are well evolved and efficiently fitted for their ecological niches, for
example as described by Dixon (Dixon 2009a) for Plasmodiophora brassicae, the
causal agent of Clubroot Disease in the Brassicaceae. Ecologically soil-borne
microbes exploit their edaphic and in planta environments with high degrees of
effectiveness. Their traits of pathogenic success are indicative of organisms with
long established and well tested lifestyles. Herein is one of the major mysteries
associated with these crop pathogens. The pathogenic microbe is frequently found
only sporadically within natural ecosystems or may be apparently entirely absent.
They appear to have evolved their current lifestyles in parallel with their hosts as
these were domesticated by man.
Devastating disease epidemics appear to result from or be associated with
extensive and intensive mono-cropping of vulnerable host genotypes. In this con-
text the pathogens cause “iatrogenic diseases of cultivation”. The consequence of
this status for sustainable crop production demands environmentally balanced
disease control achieved by using husbandry systems which are compatible with
host growth and productivity and the maintenance of benign microbe populations
which benefit the host. This means integrating husbandry, biological, chemical,
genetical and legislative techniques into logical and coherent strategies. Acceptance
of this approach reflects changing agronomic attitudes driven at least partially by
the withdrawal of toxic chemical agents such as the sterilant methyl bromide
(Martin 2003). Considerable re-appraisal of the importance of cultural controls
and the needs for crop protection strategies using integrated systems aiming to
stimulate host growth and reproduction, encourage soil health and repress patho-
gens result from this approach.
The Impact of Soil-Borne Pathogens on Crops
The term pathogen indicates the ability of a microbe to cause disease in a particular
host (Anon 1973). Pathogenesis, the process of disease development, results from
an intimate relationship between host plants and microbes. Through this process the
pathogen derives energy for growth and reproduction without any benefits
devolvingto the host. Disease is, in consequence, a metabolic shift away from the
normal functioning of the host resulting in weaker growth, reduced yields and
ultimately host death. Primary problems for those who study soil-borne organisms
are establishing the mechanisms of pathogenesis, relationships with crop losses and
defining soil environments which enhance the pathogenic load around roots and in
the soil more widely. These issues were succinctly summarised by Garrett in 1956
(Garrett 1956) in his researches into pathogenic ‘Inoculum Potential’ in the vicinity
of infection courts and defined as “the energy of growth of a fungal parasite
available infection of the host at the surface of the host organ to be affected”.
Later, in 1960 a broader definition was favoured by Dimond and Horsfall (1960):
“the number of independent infections that are likely to occur in a given situation
in a population of susceptible healthy tissues”. This perspective embraces the
actions of environment, pathogen virulence, host susceptibility and amount of
inoculum (Baker and Snyder 1965). Garrett’s (1956) view is still very valuable as
a philosophical concept for the studies of plant infection especially by soil-borne
microbes despite technical difficulties which still exist in its quantification.
Quantifying Losses
Measuring and evaluating crop losses caused by aerial and soil-borne microbes
have gained precision progressively over the last half century. This is driven by the
environmental and financial costs of chemical and genetic crop protection.
Resultant reductions in pesticide-use have progressed hand-in-hand with increased
accuracy in their application and efficacy in their modes of action. In 1971 crop loss
models were defined by Chiarappa (Chiarappa 1971) as “mathematical methods
used to describe the relationship between yield reduction and the intensity of harm-
ful organisms”. Disease intensity is defined as “the total amount of disease pres-
ent”. Techniques used to assess disease presence and intensity are reviewed in the
sequence of their historical development by Chiarappa (1971), James (1974),
Walker (1983) and Smith et al. (1984). The methods they described produced quan-
titative relationships between yield reduction and the intensity of harmful microbes
as disease progress curves.
Pathogenic microbes are distributed sporadically throughout soil profiles.
Understanding the spatial distribution of soil-borne pathogenic microbes and their
effects on crop losses evolved slowly compared with knowledge of aerial patho-
gens. Soil-borne microbes are distributed in three dimensions. Their impacts are
distributed unevenly across fields or crops and consequently crop stand and yield
reductions vary widely. While James (1974) postulated “the higher the aggregation
of missing plants, the higher the loss”, Bardner and Fletcher (1974) indicated that
yield compensation complicates that picture. There is “an ability of plants or plant
parts to make up for the yield producing functions of other (damaged) plants or
plant parts”; but “compensation is less effective if killed or injured plants are aggre-
gated”. The spatial distribution of pathogenic microbes, resultant patterns of dis-
eased plants and the expression of damage as crop losses require careful and close
examination (Campbell and Noe 1985).
Over the last 20 years substantial progress has been achieved integrating forms
of crop management and developing precision methods for crop protection. These
are based on the use of modelling, diagnosis, prediction and decision-making
systems and summarised by Madden et al. (Madden et al. 2008). Frequently, estab-
lishing mean population densities does not offer sufficient understanding of the
relationship between patterns of crop loss and inoculum distribution, hence more
detailed localised sampling is needed (Hughes 1996). The concept of a pathogenic
‘threshold’ represents a dividing line between the alternative courses of action
needed for slight, moderate or severe infestations (Hughes 1999). Sampling crops
for pathogen incidence is one means by which judgements can be guided. For preci-
sion integrated pathogen management the objective of sampling is establishing the
actual presence of a pathogen and measuring its spatial variation.
Measuring the content of pathogens in soil in situ is technically difficult as iden-
tified by Nicot et al. (1984). There are problems in relating samples with their origi-
nal locations in fields and then acquiring subsamples for analysis. Regardless of the
statistical methods used analysing the occurrence of pathogenic inoculum as
revealed by laboratory-based testing of soil samples and relating results to the dis-
persion of the pathogen and/or its propagules is far from simple. Pathogens may
be concentrated near the soil surface or randomly distributed throughout the soil
horizons. Determining these patterns in a coherent manner is hugely costly in terms
of analytical and technical time and not suited to routine crop or cultivar evaluation
programmes. At even the most empirical levels where a series of soil cores are
bulked together there is then no means of knowing how the pathogen propagules
were distributed amongst the cores and even less in relating results to positioning
in a field or fields.
Crop pathogens are frequently major causes of gross yield loss with prominent
examples such as potato late blight (Phytophthora infestans) in Western Europe and
rice blast (Magnaporthe grisea) in Asia. But huge losses of yield (biomass) in the
field and in store are only part of the picture. Pathogens reduce crop quality and
value in many direct and indirect ways. The scale of losses caused by soil-borne
pathogens is still subject to approximation despite many years of research (e.g.
Dixon 2009b). Improving the accuracy of estimates is beset with problems such as
sampling errors and spatial variation in the distribution of diseased populations
although geographic information systems (GIS) could help improve the latter’s
accuracy. Early studies tended to focus on the physiological basis for yield loss,
macro- and micro-economics of losses due to single diseases, effects of multiple
infections by varying pathogens and on the incorporation of crop loss data into
management programmes. Zadoks (1987) identified three phases in the study of
disease induced loses: exploratory (for example early studies of late blight epidem-
ics, P. infestans (e.g. Beaumont and Staniland 1933), emergency (during and post-
World War II studies estimating crop losses in staple foods such as cereals and
potato, (e.g. Large 1945; Van der Plank 1963) and implementary (shifting to focus-
ing on host growth and development which led to linking effects of pathogens such
as reduced leaf area or reduced photosynthesis using several equations with disease
incidence, plant stand reductions and yield losses as factors (Madden et al. 2008).
Unfortunately, very little of this research has been applied to soil-borne pathogens.
Savary and others (2006) list the types of decisions flowing from a knowledge of
crop losses as: tactical short-term, during the season decisions (T); strategic short-
term between seasons decisions (S) for example pre-planting for annual crops;
strategic long-term decisions (D) for example the design of breeding programmes
or development of a Integrated Pest Management (IPM) strategies; and very–long

term decisions relating to research prioritization (P). Here threshold theory has
been used (Zadoks 1987) and a damage threshold became defined as the level of
injury (yield or quality loss) where the benefit of control just exceeds its cost. This
approach also defines warning and action thresholds as injury levels where control
is planned and implemented respectively, in a timely manner preventing the damage
threshold from being reached. An example is the EPIPRE project (EPIdemiology,
PREdiction and PREvention) developed in the Netherlands from the late-1970s
onwards. By 1985 (Zadoks 1990) 70% of Dutch winter wheat farmers were
involved and its use had spread to: Belgium, Switzerland, Sweden, France and the
United Kingdom.
Short-term strategic decisions (S & T) related to soil-borne pathogens can
involve: choice of crop and cultivar, tillage and genotype plus the timing of crop
establishment, types of seed treatment, forms and rates of fertiliser, sowing density
and spatial geometry at drilling or transplanting and subsequently the form and rate
of the application of irrigation. Knowledge of the density of soil-borne propagules
(inoculum potential) is used to determine whether a field is suited for a particular
crop and whether use should be made of resistant or tolerant cultivars as with patho-
gens such as pea root rot (Fusarium solani) (Oyarzun 1993), Verticillium wilt of
cotton (V. albo-atrum and V. dahliae) (Gutierrez et al. 1983), potato early dying
disease (V. albo-atrum and V. dahliae) (Francl et al. 1987) or clubroot (P. brassicae)
(Faggian and Strelkov 2009).
Long-term decision making (type 3 knowledge “D”) permits disease risk zoning
as with clubroot (P. brassicae) in Canadian canola (oil seed rape, Brassica napus)
which may permit assessments of the importance of a pathogen, efficiency of its
management and research prioritisation. The final category “P” may be applied for
example, in prioritising pathogen importance in relation to climate change (Coakley
et al. 1999; Garrett et al. 2006).
Concern is increasing at the development of toxins in crops pre- and post-harvest
which have grave consequences for human and animal health. Currently, their
impact is difficult to assess but of vital significance with, for example Fusarium
spp. in temperate cereals (Paul et al. 2005a, b) and tropical and subtropical crops,
aflatoxins from Aspergillus flavus in peanuts (Porter et al. 1984). The disease
intensity–toxin relationship depends on fungal species, fungal strain or physiolog-
ical race, host plant resistance, disease dynamics and environmental factors (Porter
et al. 1984; Bai et al. 2001; Champeil et al. 2004; Sinha and Savard 1997).
Spatial aspects of yield loss are especially significant with soil-borne pathogens.
Physiological compensation can occur between neighbouring organs (leaves, roots,
tillers) and may mitigate disease damage (James 1974; Zadoks and Schein 1979).
There are social and economic needs for knowledge of the losses to crop biomass
and quality. This information permits the solving of more immediate practical prob-
lems which reduces: management costs, pesticide usage, other husbandry resource
inputs (such as: irrigation, fertiliser, energy, labour) and improved maintenance of
natural ecosystem services such as: soil biodiversity, topsoil and water conservation
and with directly marketed crops (the fresh produce industry) the stabilisation of
market and system performance. Conventionally, the dimensions of damage are

described in terms of: biomass (M) or biomass per unit of production area (M.L−2),
and basic factors including labour time (T), energy (E) and variable inputs such as
fertilisers, traction, and monetary and environmental issues (Ordish 1952). By this
process financial costs are aligned with husbandry, environmental and biodiversity
costs. Developing knowledge of an injury-damage-loss paradigm integrates the
study of pathogens and their effects into the broader framework of ecological sys-
tems analysis. This concept has been recognised by Cheatham and others (2009)
who encourage the recognition that defining successful disease management should
reach beyond short-term crop yield evaluations. Disease has direct impacts on eco-
system services through the loss of individual plants or whole crops and indirectly
through management activities such as the use of pesticides or selection of tillage
methods. As Cheetham et al. (2009) observe, enhanced biodiversity could reduce
disease risk by diluting the amount of susceptible tissue in the ecosystem alterna-
tively it might heighten it by adding further plant species which aid the pathogen in
the completion of its life cycle.
Evaluating Losses
An evaluation of the losses caused by soil pathogens permits balanced judgements

of their costs. This type of analysis has been done with increasing precision for
some temperate crops. Analyses with annual crops are reasonably straightforward.
With perennial crops however, it is necessary to consider how long soil-borne
pathogens may remain within the host roots, the impact that this will have on
resistance mechanisms and the effects of subsequent waves of invasion. For
example, Dixon et al. (1989) established with considerable precision the yield
losses resulting from the invasion of lucerne (alfalfa, Medicago sativa) rootstocks
by the soil-borne wilt inducing pathogen V. albo-atrum, over several cropping
cycles each of which lasted up to 5 years. Lucerne is a perennial crop which may
be retained for 5 years and beyond if yield is financially viable. Field studies
assessed disease incidence and yield in two cultivars: cv. Europe which is high
yielding and has a rapid rate of growth especially in years 2 and 3 after sowing but
no perceptible resistance genes and cv. Vertus which is less high yielding in the
early cropping years but possesses polygenic quantitative resistance. Clear positive
correlation (r = + 0.975, P < 0.001) was obtained between disease symptoms in
year 4 and those in year 5. This strongly indicated that this soil-borne pathogen is
capable of perennating in the rootstocks of lucerne thereby boosting the in planta
inoculum potential and adding substantially to infection in each subsequent sea-
son. Conversely, there was strong negative correlation (r = −0.877, P < 0.001)
between symptoms in year 4 and yield produced in year 5. There is, therefore, a
continuation of symptom effects between the seasons with increasing perennation
of inoculum potential. This contention was strongly supported by highly signifi-
cant negative correlation (r = −0.881, P < 0.01) between symptom severity and
yield in year 5.
Estimates of yield loss in annual crops such as potato caused to Verticillium

spp. have also been related to soil inoculum content. These suggested that 10–15
colony forming units (cfus) of V. dahliae per cubic centimetre of soil resulted in
10% yield loss (Francl et al. 1987; Nnodu and Harrison 1979). Similarly, with V.
dahliae inducing wilt in cotton grown in California, USA, correlations were devel-
oped for the lint yields of the wilt tolerant cv. Acala GC-510 compared with the
susceptible cv. Acala SJ-2 and expressed as the equation:
Y = 97.08* X 0.038
Correlation coefficients calculated for the 3 years separately and for all 3 years
cumulatively were 0.88, 0.95, 0.86 and 0.87, respectively. The fitted line predicts
that with about three propagules per gram of soil both cultivars (wilt-tolerant and
wilt-susceptible) should produce equivalent yields. At higher inoculum densities
wilt-tolerant cultivars should have higher lint yields than susceptible ones
(Paplomatas et al. 1992). This extends earlier work (Gutierrez et al. 1983) which
identified a relationship between the concentration of soil-borne propagules of
V. dahliae and yield reduction in cotton. Key host factors in this relationship were
the earliness of symptom development and percentage of plants with foliar
symptoms.
The availability of high-speed and -capacity computing provides technology that
now allows sophisticated forecasting of disease risks using mathematical models of
actual and potential pathogen infestation integrated with meteorological and finan-
cial data. This has been developed recently for Sclerotinia sclerotiorum the soil-
borne cause of stem rot in oilseed rape and white rot diseases in an extensive range
of other farm and horticultural crops. In northern Europe this pathogen is an
increasing threat to overwintered oil seed rape crops. The forecasting model pro-
vides decision support for farmers when considering applying fungicidal sprays at
flowering stage in late April and May (Koch et al. 2007). The crop microclimate is
measured by four weather variables: air temperature, relative humidity, rainfall and
sunshine duration. Minimum environmental conditions for infection from
ascospores, following their release from soil level ascocarps growing from sclerotia
resting on or in the soil surface is calculated from laboratory studies to be 7–11°C
and 80–86% relative h umidity and expressed as an index of ‘infection hours’ (inh).
Disease incidence (DI) correlated significantly with ‘inh’ at post-growth stage 58
(late bud stage) (r2 = 0.42, P £ 0.001). Summating ‘inh’ from continuous infection
periods over 23 h significantly improved the correlation (r2 = 0.82, P £ 0.001). A
parallel GS (growth stage) model calculates the developmental stages of oil seed
rape based on temperature within the canopy and commences model calculations at
GS = 58. The forecasting system named ‘SkleroPro’ has a two tiered approach,
firstly providing a regional assessment of risks assumed when 23 ‘inh’ have accu-
mulated after GS = 58. The next tier offers financially based recommendations
tailored to specific fields. This uses the costs of spray chemicals, expected yield and
price of rapeseed calculating the number of ‘inh’ corresponding with DI at the
economic damage threshold (Inhi). There is a decision to spray when Inh £ Inhi.
Using historical field data (1994–2004) the effect of agronomic practices on
Sclerotinia stem rot may be evaluated. Using a 2-year rotation enhanced disease
risk and lowered the infection threshold by 0.8, conversely, a 4-year rotational
break raised it by a factor of 1.3. The number of plants per square metre, level of
nitrogen fertilisation and soil management had no effect on DI. Evaluation of
‘SklerPro’ in practice using 76 historical (1994–2004) and 32 current field
experiments in 2005 showed that the financially correct decisions were made in
70% and 81% of cases, respectively. Comparing this technique with the conven-
tional use of routinely scheduled spraying programmes using predetermined appli-
cation intervals and growth stages showed that it saved 39% and 81% of fungicides,
thereby increasing the net returns to the grower by €23 and 45 ha−1, respectively and
substantially reducing the environmental impact of crop protection in the oil seed
rape crops and more widely in areas surrounding them.
Horticultural or fresh produce crops present much more complex patterns of
damage and loss compared with broadacre ones because of the intricacies and
inter-relationships of their components of quality responsible for attracting the
retail customer which are affected by pathogenic microbes (Dixon 1981, 1989).
The value of these crops frequently rests solely on the quality of their visual
appeal with flowers, fruit, pot plants, trees, shrubs and vegetables. Alternatively,
value may lie in an estimation of future productivity as with transplanting mate-
rial sold for growing-on or forcing. Some estimation of these effects is obtained
when the pathogen is removed. Supplies of virus-tested Narcissus (N. pseudonar-
cissus) bulbs were developed using heat treatment which removed virus patho-
gens some of which have soil-borne nematode vectors from a range of popular
commercial cultivars. This provided accurate assessments of the damage to yield
and the quality losses which these pathogens caused. A large scale multiplication
scheme for virus-tested Narcissus operated successfully in Kincardineshire,
Scotland in the 1980s which integrated virus removal, rapid twin-scale repropa-
gation of bulbs and subsequent protected cultivation to tonnage quantities, fol-
lowed by farm field scale cropping, inspection and certification. In the cv. Carlton
mean bulb yield was 9–20% greater in virus-tested stocks than that achieved in
comparable visually healthy commercial stocks (Sutton et al. 1988). Virus-tested
stocks flowered 3–4 days in advance of commercial stocks with in some years
increased flower stalk length. These improvements substantially increased the
financial value of the crop.
The land areas devoted to individual horticultural crops are relatively small and
often widely dispersed around the countryside but of very high financial and
frequently social values. This contrasts markedly with the broadacre agricultural
crops where cropped land area is the yardstick of over-riding importance. The
cash value of individual horticultural plants can be very high, for example with
trees and shrubs grown for several seasons possibly propagated from seed for sale
as flowering or bare root specimens. Such plants are very vulnerable to damage
from soil-borne pathogens (such as Phytophthora spp.). In fresh produce grown
for consumption slight blemishes will substantially diminish or totally destroy

market-value. For example field lettuce carrying foliar lesions of downy mildew
(Bremia lactucae) can be unsaleable. Resting oospores of B. lactucae perennate in
soil as part of crop debris germinating when a susceptible host is replanted.
Similarly, grey mould (Botrytis cinerea), may destroy the visual value of a huge
range of vegetable, fruit and flower crops and perennates in crop debris from pre-
ceding crops and as resting sclerotia.
The intensity and continuity of cultivation for fresh produce crops is severely
limited by soil-borne pathogens. These affect the intensities of time scales over
which crops are grown particularly where there is an intention to use sequential
plantings on an area of land over a single season. A build-up of white mould (S.
sclerotiorum) in soil as resting sclerotia can prevent land from being rotationally
cropped with carrots, dwarf beans, lettuce and parsnips for instance. There may
be market demands dictating that crops mature at specific dates. Crop scheduling
can be wrecked by pathogens. For example, downy mildew of pea (Peronospora
viciae) may invade from soil-borne resting oospores into seedlings and eventu-
ally cause uneven ripening in the maturing crop. This disrupts the industrial-scale
machine vining of the processing pea crop causing substantial losses since entire
lorry loads which are sampled and found to have unacceptable tenderometer
readings are rejected. In extreme cases entire crops may be rejected for factory
freezing and downgraded to use as dried peas or as sources of animal feed protein
with very much lower financial value. Outbreaks of pea wilt caused by the soil-
borne Fusarium oxysporum f. sp. pisi, were at least partially responsible for
accelerating the demise of ‘pulling pea’ (hand harvested) crop production in
Essex during the 1960s and the crop was relocated to Lincolnshire and Norfolk
as vining peas grown using industrial principles. Similarly red core disease, col-
loquially known as the ‘Lanarkshire Disease’ (Phytophthora fragariae) was
largely responsible for the demise of strawberry cropping in the Scottish Clyde
Valley and West Coast in the 1950s. The appearance of the variant P. fragariae
var. rubi which infests raspberry limited the cropping of cultivars in Perthshire
from the 1970s onwards.
Losses to the asset value of land caused by soil-borne pathogens are insidious
and frequently un-appreciated. Land which becomes infested with a particularly
intractable disease causing pathogen that is difficult and expensive to eradicate such
as clubroot (P. brassicae), white mould (S. sclerotiorum), onion white rot
(Sclerotium cepivorum) or violet root rot (Helicobasidium purpureum) loses market
value. Using infested land for high value vegetable crops becomes impossible
because yield, continuity and quality are impaired. Similar but as yet only crudely
unquantified effects arise where rural and urban landscapes are damaged by the
presence of soil-borne pathogens. The effects of pathogens such as Phytophthora
ramorum (sudden oak death) attacking temperate trees and shrubs, Armillaria mel-
lea, the cause of honey fungus disease or Phytophthora cinnamomi destroying a
range of economically important crop trees and shrubs and wild jarra forests in
Australia diminish the social value of landscapes which in turn adversely affects
human health and welfare.
Mitigation by Manipulating Husbandry
Rotation
Crop rotation is one of the oldest cultural techniques for the mitigation of crop damage
caused by soil-borne pathogens. Estimates suggest it has been used for at least 2,000
years with for example, triennial rotations practised during Medieval times (Maloy
1993). The English four-course rotation was developed in Norfolk during the early
eighteenth century initiating the Agricultural Revolution and permitting increases in
yield which fed the urban factory workers needed by the subsequent nineteenth
century Industrial Revolution. Scientific explanations for the effectiveness of crop
rotation began appearing in the late nineteenth and early twentieth centuries as
research centres such as Rothamsted Experiment Station (now Rothamsted Research)
commenced their studies. Until the 1930s, rotations of 4 years’ duration were wide-
spread throughout Great Britain (Weston 1944). Consequently, soil-borne pathogens
such as: Gaeumannomyces graminis var. tritici (take-all), Bipolaris sorokinia
(formerly Cochliobolus sativus, root rot) and Tapezia yallundae (formerly
Pseudocercosporella herpotrichoides, eyespot) were of little significance. After World
War II (1939–1945) traditional farm rotations were abandoned in Great Britain and
Western Europe as industrial principles were applied to crop production with the aim
of avoiding the food shortages which plagued populations in the first half of the
twentieth century (Dixon and Margerison 2009). Comparisons between monocrop-
ping and attendant disease decline and rotation whereby the amount of soil-borne
inoculum is steadily eroded by soil microbes (Shipton et al. 1973) have classically
been made with clubroot disease (P. brassicae). This disease is a prime cause of loss
in highly valuable field vegetable brassica crops where huge financial penalties result
from monocropping and waiting for disease decline to emerge, consequently this
strategy is virtually impossible to test in practice. Potential rotational alternatives
(such as leek and onion) have lower market values and hence are not realistic solu-
tions in practice. Now that this disease is making substantial inroads into the
Canadian canola crop (Strelkov et al. 2008) and infested farms are banned by statute
from cropping for 5 years it may at last be possible to evaluate the usefulness of rota-
tion as a tool for the control of P. brassicae.
Resting spores of P. brassicae (clubroot) are long lived, reputedly surviving for
20 years or more and they are highly resistant to degradation, being able to survive
passage through the highly acidic guts of grazing animals (Gibbs 1931); and their
cell wall has a structure that is impervious to predation by soil microbes.
Consequently, the resting spores retain viability in soil despite exposure to repeated
seasons of adverse weather. Although rotations of 6 or more years are recom-
mended (Dixon 1981), these are of limited use in the management of land infested
with P. brassicae because of the characteristics of the consumer market for fresh
Brassica vegetables. Swedish field studies indicated the resting spores have a half
life of at least 3.6 years and as a result some spores may exist for at least 18 years
in the absence of suitable hosts before spore populations are eroded to undetectable
levels (Wallenhammar 1998). In an attempt to contain the current outbreak of

clubroot disease in Canadian canola infested fields are legally prohibited from
returning to this crop for 5 years (S. Strelkov, 2007 personal communication).
Resting spore longevity perplexed early researchers such as Gibbs (1939), not least
because of bona fide reports that cruciferous crops grown on land previously carry-
ing permanent grassland leys1 could become rife with clubroot disease after 1 or 2
years of arable cropping with brassicas, frequently swedes (B. napus) grown for
stock feed. Temperature, moisture content and position in the soil profile will influ-
ence spore longevity (Monteith 1924; Fedorintschik 1935).
Soil pH apparently affects the rate of release of primary zoospores from the
resting spores. Zoospore numbers increased in acidic compared with alkaline soils
(Bochow 1961) but without much change in total numbers germinating. Spore
dormancy and the need for an external germination stimulus are factors in the rela-
tionship between P. brassicae and the soil environment (e.g. Honig 1931). At the
release of new generations of resting spores from decaying clubs only a few germi-
nate immediately (Ogawa et al. 2001), forms of external stimulus (Ohi et al. 2003;
Hata et al. 2002) are needed which initiate the process. The state of ‘readiness for
germination’ in spores released from host roots was examined by a consecutive
string of researchers (e.g. Humphrey 1892; Chupp 1917; Naumov 1925; Honig
1931; Colhoun 1958); generally they concluded that bacteria and other organisms
disintegrate the diseased host tissues and ‘condition’ spores for more effective ger-
mination. But these secondary microbes are not essential for the germination pro-
cess itself. Undefined mechanisms within the resting spore initiate germination and
control its speed. It appears that these mechanisms within individual spores operate
quite separately from others in the population since not all germinate in synchrony.
Rotating Brassica2 crops with Allium spp. such as leeks or bulb onions reduces the
disease causing ability of P. brassicae (J. Robak, personal communication 2000).
Resistant forms of B. oleracea and B. rapa were advocated by Yamagishi et al.
(1986) as inducing the germination of resting spores of P. brassicae but inhibiting
the full completion of the pathogen life cycle thereby diminishing soil inoculum
content. A similar contention was proposed by Hsieh and Yang (1985). Perennial
ryegrass was suggested by Rod and Robak (1994) as a non-brassica crop capable
of reducing P. brassicae possibly because the pathogen may complete parts of its
life cycle in the root hairs but does not progress any further.
Changing the crop rotationally reduces opportunities for root invasion by soil-
borne pathogens, exposes their resting bodies to extended periods of predation and
enzymatic breakdown from antagonistic flora and fauna and to adverse physical
and chemical environments. Hence rotation suppresses inoculum development and
reduces inoculum potential. For example, pycniospores and chlamydospores of
1
ley = English agricultural term for grassland pasture providing grazing over an extended number
of seasons.
2
In this chapter the latin form ‘Brassica’ is used botanically and vernacular form ‘brassicas’ is
used horticulturally.
Phoma medicaginis var. pinodella (foot rot) survive on buried crop debris for up to
3 years (Carr and Smith 1988); rotations longer than this are key management
strategies for avoiding damage to pea crops. By contrast Fusarium spp. often have
extended host ranges and several are natural soil inhabitants surviving as competi-
tive saprotrophs on crop debris or as various forms of resting spore (Fehrmann
1988a, b, c), so that growing several crops in rotation does not necessarily reduce
the inoculum potential of Fusarium induced diseases (Gair et al. 1972). Similarly
V. dahliae affects a very large number of hosts and there are reports that it perennates
on non-host roots (Rowe and Powelson 2002). But as McKay (1926) demonstrated
in some of the earliest studies of rotation there are benefits which diminish
Verticillium wilt of potato (McKay 1926). Detailed field trials by Emmond and
Ledingham (1972) (Table 1) amply supported this contention.
Valuing rotation as a husbandry tool demands careful analysis, none more so
than with take-all disease of cereals caused by G. graminis var. tritici (anamorph
Phialophora-like sp.). Disease severity increases during the first cropping year and
in subsequent wheat crops even after periods of break crops (Fig. 1).
Disease in mature plants during July is greatest in third and fourth wheat crop-
ping seasons, after that severity declines to a stable lesser intensity. Although this
still exceeds that than found in first year wheat crops. This phenomenon is termed
“take-all decline” and is attributed to the increasing populations of antagonistic
microbes (Hornby 1992). In the decline period ascospores and phialospores of G.
graminis var. tritici may develop, but are devoid of a role in field infection (Hornby
1981). But mycelium, in the declining saprotrophic phase, present on crop debris
provides a source of viable inoculum for up to 3 years depending on soil type
(Lemaire 1988). Even with such a relatively short survival time, rotations may be
of limited use in the management of take-all if short (2–4 years) sequences of sus-
ceptible cereals form part of the cropping regime. Take-all susceptible cereals
(wheat and barley) are the foundation of arable agriculture in Europe. Oats are
resistant to G. graminis var. tritici, but their market is very limited. Cereals can be
used to diminish soil-borne diseases in non-cereal crops. In wheat and/or barley
however, following periods of continuous cropping of up to 4 years duration take-
all severity increases. Some nitrogen fertilisers used in non-cereal crops may
Table 1 Percentage of potato stems infected with Verticillium albo-atrum wilt and black dot
caused by Colletotrichum coccodes in samples from a series of crop rotations (Adapted from
Emmond and Ledingham 1972)
Stems infected (%)
Rotation components V. albo-atrum C. coccodes
A. Continuous potatoes 75 20
B. Potatoes/sweetclover/hay/hay/fallow 5 15
C. Potatoes/potatoes/alfalfa/crested wheatgrass/hay/hay/hay/ 25 25
hay/fallow (sampled year 1 potatoes)
D. Sampled year 2 potatoes 75 25
Fig. 1 Representative First

epidemiology of take-all disease
in successive winter wheat crops
under moderate to severe disease
pressure
Second
Third
Disease severity
Fourth
Fifth et seq.
Sowing Anthesis Harvest

Crop development
encourage G. graminis var. tritici by sustaining the saprotrophic survival phase.

Take-all in cereals imposes significant agronomic constraints on cropping sequences
(Hornby et al. 1998).
Soil Type
The effects of soil structure and texture on the biology of pathogenic microbes are
well established at least in outline. Well structured freely draining soils normally
harbour less plant pathogens. By contrast, badly abused and waterlogged soils tend to
be associated with disease epidemics particularly damping-off, root, foot and collar
rots. Recent research has highlighted the extent of this interaction. Aspergillus flavus
the cause of aflatoxin contamination of cotton seed is a natural soil inhabitant.

Aflatoxins are potent toxic and carcinogenic fungal metabolites that contaminate
human and animal food and feed. Two strains of A. flavus are known; the S-strain
which produces most toxin and the L-strain which generates lesser quantities (Jaime-
Garcia and Cotty 2006). Incidence of S-strain is positively correlated with clay con-
tent and negatively correlated with sand content of soils. Studies in South Texas, USA
related increased S-strain with successive cropping with cotton. There may be further
complicating factors relating to the types of crop suited to the heavier soils and their
abilities to retain a higher relative humidity within the canopy and encourage propa-
gation of the fungus. A further example comes from Africa where in Cameroon
andosols are suspected of suppressing cocoyam (Xathosoma sagittifolium) root rot
caused by Pythium myriotylum compared with the effects of ferralsols. Andosols have
higher contents of organic matter, calcium, potassium, magnesium and nitrogen
which are negatively correlated with disease incidence (Adiobo et al. 2007). The clay
and sand fractions were higher in ferrasols and correlated with disease severity.
Suppressiveness is thought to relate to the organic matter content of andosols which
improve soil structure, increase nutrient content, microbial biomass and activity.
Particles of a larger size may contain a greater proportion of residual cellulose due to
a smaller surface area. The larger size of particulate organic matter and therefore,
comparatively smaller surface area will mean that the state of decomposition is less
and that there may be smaller amounts of recalcitrant substrates.
The particle size distribution in soil has a significant bearing upon the volume of the
interstitial spaces and hence the number of potentially habitable locations available for
colonization by both microflora (Russell 1968; Van Elsas et al. 1986) and meiofaunal
predators. For pathogens with motile zoospores the size of the interstitial spaces has
important implications permitting or restricting the movement of these propagules
through soil, the zoospores of Phytophthora spp. require water-filled pores of at least
40–60 mm in diameter before they can move through soil (Jackson 1975). Chemistry
influences the interactions between particles and microorganisms with fluorescent
pseudomonads being associated with particulate organic matter (Sivasithamparam et al.
1979a). Diseases too, can be influenced by soil texture, although for G. graminis var.
tritici the effects are contradictory; coarse-textured soils of the Pacific North-west of the
U.S.A. favour take-all (Cook et al. 1968) but silty soils in Brittany, France, are more
favourable to the disease than are sandy soils (Lucas and Nignon 1987).
Take-all (G. graminis var. tritici) severity is partly dependent on certain soil types
e.g. crops grown on fenland peats are at very high risk of take-all, but those on well-
structured calcareous clay loams are at lowest risk. Soils with high organic matter
content also favour the development of severe take-all as there is great potential for
accumulation of nitrate due to mineralization of soil nitrogen, so fields of recently
ploughed old pasture or old cleared wood land should be avoided (Hornby et al.
1998). Take-all severity in crops sown in fields known to be at risk can be reduced
if firm seed beds, prepared from ploughed soil, are drilled at moderately low seeding
rates and the crop provided with adequate amounts of nitrogen (especially as ammo-
nium) and phosphate (Hornby et al. 1998). Another means of controlling take-all
may be exploited where wheat is cropped continuously until take-all decline is
achieved; decline can also be triggered more quickly by a severe outbreak of take-all.
Soil in which take-all decline occurs could be described as suppressive (Simon and
Sivasithamparam 1988). During the years preceding decline, however, the crops
grown on that land will not make significant contributions to the farm’s gross mar-
gin. Once take-all decline is established then its loss through planting of another type
of crop, or possibly by liming (Simon et al. 1988), will produce a loss of both agro-
nomic and financial investment (Hornby et al. 1998). The stability of take-all decline
is also dependent on soil fertility, being unstable on soils of low fertility (Prew and
McIntosh 1975), similarly the persistence of introduced antagonists is also strongly
influenced by soil type (Capper and Higgins 1993).
Soil characteristics account for much of the variation in severity of soil-borne
diseases found occurring between sites (Ball et al. 2005). Here it is necessary to
distinguish between pathogen suppression and disease suppression. The former
refers to the capacity of soil to limit the inoculum density of a pathogen and its
saprophytic activity; the latter indicates a restriction in disease development even
when the environment, the host and quantity of inoculum appear conducive.
Disease suppression is related to the microbial characteristics of soil itself because
this effect may be reduced or eliminated by soil sterilisation (Peters et al. 2003).
Production of antibiotics, siderophores, nutrient competition, niche exclusion and
an induction of systemic acquired host resistance (SAR) may affect the level of
disease suppression. In turn these factors relate to total microbial activity in soil
(Höper and Alabouvette 1996). Modifying soil conditions through the use of
organic amendments, fertilisers and tillage practices, or the addition of microbial
antagonists as biocontrol agents influence the degree of disease suppression.
Pathogen suppression is suggested by Ball et al. (2005) to be a direct function of
soil structure. An availability of oxygen and free-draining abilities are essential for
the spread of many fungal pathogens and for the growth plant roots.
Soil structure affects the causation of soil-borne diseases. Otten and Gilligan (2006)
clearly demonstrated that soil-borne fungal mycelia respond to soil structure. Where air
filled pores are large then fungi form big masses of hyphae in an exploitive mode
whereas where the pores are small and constrictive mycelia growth is limited to an
explorative mode. They noted that even small changes in the physical soil environment
resulted in “large and abrupt changes in fungal morphology”. Soil compaction which is
a function derived from the abuse of soil structure and texture encourages disease epi-
demics. These may result from primary infections developing from inoculum capable
of saprophytic survival. The increase in the severity of soil-borne fungal diseases result-
ing from soil compaction (Höper and Alabouvette 1996) arises from an impact on the
dynamics of microbial colony growth. The size of colony determines the distance over
which a pathogen is able to make contact with hosts, the probability of infection is then
determined by the density within the colony itself (Otten et al. 2001). Soil compaction
alters the composition of root exudates and the structure of rhizosphere microbial com-
munities; possibly such changes affect the ability of fungal pathogens to infect roots via
for example, antibiosis or competitive interactions and hence the disease suppressivity
of soils. Rotation and tillage practices both alter soil structure and then affect disease
suppressivity and subsequent host–pathogen relationships.
Primary Cultivation: Tillage3
Physical disturbance of a soil is the primary effect of cultivation. Wetter, denser and
cooler soil conditions typically resulting from reduced tillage systems increase the
amounts of organic matter, encouraging greater microbial activity and biomass
expansion, principally in the upper layers of the soil (Young and Ritz 2000).
Disturbance of a soil results in the redistribution of pore networks and the resultant
changed architecture permits the utilisation of newly exposed substrates and preda-
tion of freshly exposed organisms until an equilibrium is re-established or further
disturbance maintains the dynamic nature of these processes.
The size of soil aggregates was generally found to be larger in no-till compared
with tilled soil. In the upper-most 10 cm of soils, round pores were twice as abun-
dant in no-till soils compared with tilled (Pagliani and De Nobili 1993). The tilled
soils were characteristically platy at their surface and heterogeneous beneath that
with numerous irregular pores and large compact aggregates separated by large
planar pores. By contrast no-till soil had a more homogeneous subangular blocky
structure. This is the reverse of the findings of Drees et al. (1994), hence the peda-
logical effects of tillage and no-tillage regimes vary from site to site and the hus-
bandry regimes employed. Such effects are microbiologically important since Otten
et al. (2004) demonstrated that the spread of fungal pathogens through soil is not a
random process. When spreading through soil and encountering a large continuous
pore volume, fungal (in this case Rhizoctonia solani) spread is enhanced as it effec-
tively by-passes the more tortuous smaller pathways through soil. Enhancing
spread increases ‘colonisation efficiency’ which is the saprophytic equivalent of the
pathozone (Gilligan 1985) and which describes the relationship between successful
colonisation and the distance between inoculum and the target host(s).
Direct-seeding or no-till is defined as planting directly into the residues of previ-
ous crops without cultivation that mixes or stirs soil prior to planting. No-till is
claimed to reduce soil erosion, improve soil structure, organic matter content and
reduce fuel used by agricultural machinery (Paulitz 2006). No-tillage methods are
widely used for cereal production in Australia, Argentina, Brazil and Canada but
not in Europe nor in the Pacific Northwest of the USA. One limitation is that root
diseases caused by species of Fusarium, Pythium or Rhizoctonia may increase with
reduced tillage. This is a cause for alarm certainly in Europe because of the resul-
tant increasing concentrations of fusarial toxins present in harvested grain. This
situation is exacerbated by the removal from the market of several fungicides
capable of controlling this pathogen. A 10-year study of conventional ploughing
and no-tillage (Chervet et al. 2005) recognised the potential environmental advan-
tages of reduced tillage but there remained a major human and animal health prob-
lem resulting from mycotoxin production in no-tilled winter cereal crops especially
those which follow maize (corn). Issues of public health and safety which are
3
Tillage is used here synonymously with cultivation.
c onsequential on changes to crop agronomy identify the need for an holistic

approach carefully divorced from transitory and fashionable land-use policies.
The incorporation of crop debris into soil was once thought to affect the emer-
gence of corn seedlings adversely of corn seedlings (McCalla and Duley 1949) and
the injurious effects of phytotoxic substances extracted from wheat straw on germi-
nating seeds and the growth of seedlings was demonstrated by Guenzi et al. (1967).
Damage to seedlings or reductions in yield, following the incorporation of cereal
residues, are most probably due to the increased activities of Fusarium spp. (Lipps
and Deep 1991), Pythium spp. (Cook et al. 1990; Cook and Haglund 1991) and R.
solani AG8 and G. graminis var. tritici (Cook and Haglund 1991). Yet in crops
sown using direct-drilling techniques where crop residues are not buried by deep
ploughing, the incidence of take-all can be reduced by 50%, even though inoculum
levels are unaffected (Brooks and Dawson 1968). Lampkin (1990) noted that G.
graminis var. tritici is not a major problem where direct drilling and conservation
tillage practices are used and attributes this to the crop rotation and high microbial
activity in the upper soil layers.
More generally, (Otten and Gilligan 2006) surveyed the effects of the use and
the avoidance of tillage on disease incidence and found that conservation tillage
can variously increase (54%), decrease (42%) or occasionally have nil effect (4%)
on plant diseases. These authors note that unstable soil structure (as in silts) results
in wetter, less well aerated and compacted soils in which pathogens such as
Phytophthora and Verticillium spp. are encouraged while R. solani is discouraged
(Alabouvette et al. 1996). But there is no simple relationship between soil
conditions, pathogen vitality and disease incidence. Soil compaction reduced
foot rot of winter wheat caused by Microdochium nivale and Fusarium spp.
(Colbach et al. 1996) and sharp eyespot (Rhizoctonia cerealis) in winter wheat
(Colbach et al. 1997). But had the reverse effect on common root rot of peas
(Fusarium solani f. sp. pisi) (Fritz et al. 1995), crown and root rots in a crop rota-
tion in spring barley (Bipolaris and Fusarium spp.) and soybean (Sturz and Carter
1995) and cereal root rot in humid climates (Sturz et al. 1997). Primary tillage
using a chisel plough reduced root rot of peas (Aphanomyces euteiches) when
associated with prior cropping with oats. Chisel ploughing retained much of the
oat root and shoot tissue in the upper 15 cm of the soil profile whereas mouldboard
ploughing deposited only 10% of these residues in that zone (Staricka et al. 1991).
Prestes et al. (2002) noted that under crop rotation and nil-tillage husbandry as
compared with monoculture and nil-tillage there was a reduced incidence of leaf
blotches in wheat caused by Drechslera (Pyrenophora) tritici-repentis, B. soroki-
niana and Stagnospora (Phaeosphaeria) nodorum. Direct drilling augments the
build-up of organic carbon and microbial biomass in the surface layers of soil and
raises the suppression of G. graminis var. tritici and R. solani (Pankhurst et al.
2002). Encouraging vesicular-arbuscular mycorrhizas (VAM) (see elsewhere in
this book) by nil or limited tillage for low-input agriculture failed to increase
maize growth or yield compared with conventional mouldboard ploughing systems
(Galvez et al. 2001). Hence this fails to exploit the use of VAM for the reduction
of soil-borne pathogens as frequently claimed.
Rhizoctonia spp. cause substantial yield losses in direct-seeded cereal crops

compared with conventionally tilled ones. The strain R. solani AG-8 grew more
rapidly through soil with a long-term history of direct seeding compared with tilled
soils. This effect was ascribed to alterations in soil structure resulting from limited
tillage whereby the residues left on the soil surface adversely affected soil moisture
content (Schroeder and Paulitz 2008). No such effects were found with R. oryzae.
Disease incidence and severity were examined in oilseed rape in Germany under
varying cultivation systems such as ploughing and drilling, cultivator and drilling
and rotory cultivation (rotavation) and drilling. The incidence of V. dahliae was
reduced with ploughing and drilling compared with cultivator or rotavator and
drilling, but the incidence of black leg (Leptosphaeria [Phoma] maculans) was
unaffected. Increasing nitrogen fertiliser applications had no effect on black leg but
reducing nitrogen resulted in an increase in S. sclerotiorum (white mould) on aerial
parts of the plants and pods at later growth stages. Yields and protein contents of
the seed rose with increased nitrogen when associated with inversion husbandry
(ploughing and drilling); oil content was reduced with that system (Sochting and
Verreet 2004). Epidemics of early leaf spot (Cercospora arachidicola) on peanut
were reduced in strip tilled or no tillage land compared with conventional full field
cultivation in Florida, USA. The size of the disease reduction equalled that achieved
by three to five applications of the fungicide, chlorothalonil (Cantonwine et al.
2007). Inoculum of C. arachidicola overwinters on crop debris from where primary
infections spread onto the lower leaves. Strip tillage appears to reduce the inoculum
potential which delays the build-up of epidemics and hence routine spraying is not
required until later in the season.
Secondary Cultivation
A detailed 4-year study in southern England aimed to reduce the unit costs of
growingconsecutive wheat crops without reducing output, by focusing on the inter-
actions between lower-cost establishment and the minimum of subsequent machin-
ery passes through the crop (Knight 2003). On chalkland soil, minimum tillage was
associated with the highest yields and biggest financial margins in all years. On
medium loam, ploughing resulted in the highest yield in 1 year, but overall differ-
ences were small and direct sowing was the most cost-effective husbandry. No
single technique was consistently better on heavy chalky clay-loam, but direct sow-
ing was associated with lower yields in the second and third years, and minimum
tillage produced the highest average financial margin over the three seasons. Direct
sowing had the lowest energy costs for establishment, but also resulted in the lowest
plant populations. Minimum tillage had the lowest total energy costs per tonne of
grain harvested on both the light and heavy soils. Direct sowing resulted in less
broad-leaved weeds compared with ploughing cultivation, but higher blackgrass
(Alopecurus myosuroides) and brome-grass (Bromus spp.) populations. There was
also a lower incidence of eyespot disease (T. yallundae) and incidence of Septoria
tritici (Mycosphaerella graminicola) following direct sowing compared with after

ploughing. The largest yield response to seed treatment was obtained in 1999, when
it reduced take-all disease (G. graminis var. tritici) incidence by half. Despite hav-
ing had the highest take-all disease levels in that first year, direct sowing showed
the smallest average yield benefit on the light and medium soils. Reducing the
number of machinery passes from 7 to 3 decreased yields at all sites in 2000. This
was associated with inferior disease control, only one fungicide having been
applied. There was no disadvantage associated with minimum pass cultivation in
2001, when disease pressure was lower. Where application and establishment costs
were included, however, the minimum pass strategies were the most cost-effective.
There were some interactions between the method of establishment and number of
machinery passes. After ploughing, the 4-pass (with split applications of nitrogen
fertiliser) was as cost-effective as the 3-pass system, whereas after direct sowing
this treatment was no better than the 7-pass. At Biggleswade, Bedfordshire, the
direct-sown blocks benefited more from the 7-pass than the other establishment
methods. With consecutive wheat crops, minimum tillage or direct sowing can
produce yields equal to or higher than ploughing on a range of soil types. The great-
est benefit from ploughing is apparent on medium or heavy soils after a wet
autumn, or where grass weeds are a problem. Adopting minimum pass husbandry
can improve profitability, when the cost of each machinery movement is included
in the accounts. For a single fungicide spray programme strategy to be effective,
however, accurate timing of the application (at flag leaf) is vital. Low cost estab-
lishment does not dictate that a minimum pass approach will be less successful, and
the combined benefits could range from £36 to £120 ha−1. Although the single-
sprays did not often result in significant yield penalties compared with the three-
spray programme, they almost invariably were associated with inferior disease
control, even in 2001. For example, at Andover, Hampshire, in 1999, the incidence
of S. tritici on leaf 2 increased from 12% in the three-spray programme to 63–83%
at the growth stage 32 (GS32) in single-spray treatments, but the associated penalty
of yield losses was only between 1% and 6%. The absolute levels of disease were
lowest, however in 2001, with almost all treatments recording less than 5% S. tritici
on leaf 2. At Andover in 2000, the single-spray timing that was associated with the
greatest control of S. tritici on leaf 2 (GS39–45) was also the one that gave the
highest yields. Applications at GS32 gave the poorest disease control on leaf 2 in
all 3 years at Andover, whereas at Biggleswade the GS39–45 single spray was
equally ineffective. These findings help to explain why applications of fungicide at
GS37 appeared to be the highest-yielding single spray timing at Biggleswade,
whereas at Andover it was at GS39–45. At Biggleswade in 2000, control of brown
rust (Puccinia recondita) was penalized more than the control of S. tritici by the
change from a three-spray programme to the single-spray strategy.
In other broadacre crops, secondary tillage practices have a significant effect on
the vertical distribution of residual stubble which carries disease. For example, oil
seed rape crops and associated subsequent epidemics of soil and stubble-borne
blackleg-canker caused by Leptosphaeria (Phoma) maculans (Schneider et al.
2006). Secondary cultivation and seed drilling raise previously buried infested
stubble residues to the soil surface resulting in opportunities for the transfer of
infection into autumn sown crops. Modelling tillage practices contributes towards
formulating robust forms of sustainable disease control by integrating techniques to
best advantage.
Soil Acidity and Alkalinity (Hydrogen Ion Content, pH)
Microbes in the group Plasmodiophorales are probably the pathogens most noted
for their reaction to changing acidity and alkalinity in soil. The most vexed issue
relating to clubroot disease (P. brassicae) is soil calcium content and the associated
hydrogen–ion content (pH) of soil. Calcium emerges as a fundamental factor in the
life cycles of both P. brassicae and its hosts. Datnoff and others (2007) summarised
the involvement of calcium in host metabolism, physiology and signalling of many
host–pathogen interactions indicating a relationship with expression of resistance.
From the earliest studies of P. brassicae and clubroot onwards the disease was
associated with acidic soils and claims that it was alleviated by the use of various
forms of agricultural lime. Much of the work is, however, contradictory in terms of
the forms of lime used, their sources, rates applied, date of application, recipient
soil types and the measurement used to evaluate efficacy. It is now possible
following 5 decades of research to conclude that clubroot disease incidence is not
limited at pH 7.0 as is still claimed especially in much farm advisory and home
gardening literature. As commented by Colhoun (1958): “results obtained by field
experimentation show the difficulty encountered in determining the exact upper
limit (our italics) of the soil pH at which infection can (still) occur”. This begs the
question as to whether there is an exact upper limit. Colhoun (1958) goes on to
argue that “observations have been made without due attention to the variety of
other factors which also influence infection” are of little if any value. He advocated
the use of potted seedling tests which could be completed in ‘controlled’ condi-
tions. As he also indicates pot tests have been undertaken at high soil moisture
content but have failed to control spore load for example and they are much affected
by seasonality. Glasshouse experiments running through a winter are far less
acceptable because of the weaker host growth at low light levels and shorter day-
lengths compared with those made in spring or early autumn, while summer-time
experiments are likely to suffer from excessive lifts in air temperature. The chemi-
cal and physical forms and quantities of calcium used also affect the results and add
further levels of variables to each experiment. Here again Colhoun (1958) rein-
forces, as with moisture and temperature, lessons from the classic studies of Samuel
and Garrett (1945) related to the impact of spore load, inoculum potential and
intensity. Theirs was one of the earliest scientific validations that the effects of pH
and of calcium could be separated and quantified individually as factors influencing
the environmental success of P. brassicae.
Subsequent to Colhoun (1958) practical studies indicated that the impact of the
balance of nutrients in the soil is significant while the actual content of individual
ions is still important. For example, Myers and Campbell (1985) suggested that
clubroot disease expression depends on a balance between pH and the amounts of
calcium and magnesium in the soil. While Dobson et al. (1983) concluded from their
work using roughly and thoroughly mixed limed soils that if roots and spores occur
within small pockets of low calcium and/or low pH, invasion is possible despite high
overall soil calcium and pH estimations. Fletcher and others (1982) achieved greatest
effects of clubroot disease with field applications of calcium carbonate and calcium
nitrate which increased pH to 7.9 and 8.3, respectively. They also concluded that
although pH was a major factor in reducing disease expression, some other factor
than pH possibly the Ca2+ (calcium) ion itself was involved. Work conducted using
controlled conditions has formed similar conclusions (Hamilton and Crête 1978).
These results still however, beg the question of “where and when is P. brassicae
influenced by the presence of calcium and by pH value?” There is a tendency to
assume that these factors affect the microbe while in the soil but since P. brassicae
spends most of its life cycle within the host it could be fair to suggest that calcium
and pH also affect these environments. A role for calcium in the post-infection
development of P. brassicae is supported by the demonstration that incorporation
into roots is pH-dependent (Myers and Campbell 1985; Campbell and Greathead
1996) contended that P. brassicae is affected at more than one point in the life cycle
between spore germination and the completion of resting spore formation in the
cortical cells by pH and calcium concentration. Numerous detailed long-term
experiments have confirmed this (Webster 1986; Dixon and Webster 1988; Webster
and Dixon 1991a; Dixon and Page 1998; Page 2001). It is evident that the greatest
impact of calcium is when it is present in the period between spore germination to
post-penetration of root-hairs. The latter appears to be when root-hair infection has
the biggest impact on subsequent gall formation. There may apparently be separate
mechanisms since the periods 0–3 and 0–7 days post-penetration seem to be sepa-
rated in the extent of their influence on subsequent disease development. The expres-
sion of effect seems to be cumulative since it took longer when a 30 mel−1 Ca2+
solution was used as compared with one containing 55 mel−1 Ca2+ in order to achieve
similar final results. The host–pathogen response varies also with pH however, that
is a separate factor. But it is worth recording here that calcium at pH 7.2 needed to
be present by day 14 in order to suppress root-hair infection or alter the progress of
galling. The pathogen may be affected by the calcium environment in the root-hair
and this alters subsequent behaviour in the cortical cells. The work of Dixon and
co-workers (Webster 1986; Dixon and Webster 1988; Webster and Dixon 1991a;
Dixon and Page 1998; Page 2001) is supported by results of Donald and co-workers
(Donald et al. 2004; Donald 2005) in Australia. Of major significance is the finding
that high concentrations of calcium at pH 6.2 or 7.2 reduce total numbers of root-hair
infections and the rate of maturation through plasmodial, sporangial and zoosporan-
gial stages as compared with the controls. Raised concentrations of calcium com-
pletely inhibit the later stages of P. brassicae development in the root-hair even
where high inoculum doses are applied. The calcium effect commences in the soil
since Dixon and Page (1998) showed that the germination of resting spores, motility
of zoospores and the composition of benign microbial flora around roots are altered.
High concentrations of calcium could possibly reduce flagellar action as Satir (1982)
and Sleigh and Barlow (1982) reported that changes in calcium of the order of
10−6–10−4 M affected the action of demembranated flagellae, whether this effect
would hold for the flagellae of P. brassicae has yet to be determined.
Recently, Wallenhammar (1999) pointed to the uneven distribution of acidic and
alkaline areas of soil in individual fields with pH ranging from 5.73 to 8.45 in
localised patches. Mattsson (1995) identified that pH values of the subsoil are
frequently more alkaline than the upper horizons in Sweden especially in the
calcareous glacier clay region near Uppsala in eastern central Sweden. This
modernises aspects of Colhoun’s Dilemma related to pH. Earlier Palm (1958, 1963)
had concluded that the effect of pH is not restricted solely to the establishment of
P. brassicae as a parasite because the rate of gall proliferation was markedly sup-
pressed by an alkaline condition of the medium after infection in the host tissues.
It was suggested that changes in the soil reaction may have more drastic effects on
gall development than on the number of infections by zoospores. The use of organic
buffers to adjust pH and calcium content separately from each other and showed
that at 10 mel−1 Ca2+ and a pH of above 7.1 reduced the numbers of primary zoo-
sporangia in root-hairs thus inhibiting galling (Myers and Campbell 1985). Webster
and Dixon (1991a) demonstrated that the effects of pH are independent of calcium
concentration and found that alkaline pH reduced total root-hair infection number
and retarded the maturation of plasmodia, sporangia and zoosporangia. The pH
effect on the maturation of root-hair infections is activated by exposure to alkaline
pH within 3 days of penetration. Prolonged exposure beyond 3 days gives no
additional effect.
There may be a dual effect in that alkaline pH increases sensitivity of the host
and/or P. brassicae to calcium effects as well as increasing the efficiency of calcium
uptake. The effects of pH and calcium are remarkably similar but this does not
necessarily mean they are one and the same as has been suggested by some workers.
They may regulate the pathogenic potential of an inoculum quite separately. Since
pH regulates the response to calcium, intracellular function may be modified in
addition. A high concentration of H+ ions in plant tissues is potentially antagonistic
to calcium. Membrane permeability is lowered by both alkaline pH and by high
calcium. This environment could affect the growth and reproduction of P. brassicae
as it proliferates within the host root-hair and epidermal cells or within the cortical
cells. Alkaline environments could influence primary and secondary invasions,
cortical migration and cell hypertrophy. An involvement of Ca2+ ions in the growth
and reproduction of P. brassicae ultimately leading to induced cell death or hyper-
sensitivity is suggested by Takahashi et al. (2006). At the agronomic level promot-
ing high alkalinity linked with continuous cropping is suggested by Shinoda et al.
(2005) as a means of reducing the soil inoculum load.
In contrast to P. brassicae another Plasmodiophorales, Streptomyces scabies, the
cause of common scab in potatoes is encouraged by alkaline soil conditions.
Manipulating pH towards acidic values considerably reduces disease incidence.
Adding sulphur to soil increases soil acidity and helped reduce the impact of
S. scabies on the marketable yield of potato crops (Pavlista 2008).
Cavity spot disease of carrot (Daucus carota) has been another intractable
p roblem where soil hydrogen ion and calcium content moderate incidence and sever-
ity. Carrots are rejected at grading with one or two visible lesions, and when disease
incidence passes a relatively low threshold it becomes uneconomic to harvest crops
(Hiltunen and White 2002). Early studies attributed cavity spot incidence to interac-
tions with the presence and absence of calcium in soil classing the syndrome as a
‘physiological disorder’. The first solid indication of the involvement of a pathogen
was when three different fungicides with activity against Oomycete fungi all reduced
disease. Very quickly thereafter, the causal agents Pythium violae and P. sulcatum
were isolated from cavity spot lesions and Koch’s postulates satisfied. Calcium
carbonate is known to have significant effects on cavity spot, probably by inducing
a soil microflora inhibitory to filamentous fungi. The management of agronomic
aspects such as irrigation, soil cultivation, and the length of time for which crops are
grown may all be used, while carrot cultivars with some level of field resistance may
be beneficial. One of the most significant factors is disease avoidance by not
selecting fields with high inoculum levels. One serology-based risk assessment test
has been produced and commercialized, and molecular probes which could be the
basis of more sensitive tests are available for both pathogens.
Investigations have been made of the relative importance of primary and second-
ary infections (auto- and allo-infections) in the development of a carrot cavity spot
(CCS) epidemic caused by Pythium spp. Three cropping factors: fungicide applica-
tion, soil moisture and planting density, were selected as the key variables affecting
the disease syndrome (Suffert et al. 2008). Their effects on: (i) disease measure-
ments at a specific time, (ii) the areas under the disease progress curves (AUDPCs)
and (iii) a time-dependent parameter in a pathometric incidence-severity relation-
ship, were studied. A deficit of soil moisture limited the movement of Pythium
propagules towards host tissue, and thus reduced primary infections in the field; it
also promoted the healing of lesions, limiting lesion expansion and the potential for
allo-infections (6.8–7.5 lesions per root in irrigated plots compared with 2.4 lesions
in non-irrigated plots). A negative relationship between the mean root-to-root dis-
tance and the rate of allo-infections was established in microcosms; a reduction in
mean planting density was also effective in limiting CCS development (0.5, 1.6 and
2.0 lesions per root in microcosms containing 8, 16 and 31 roots, respectively). Free
calcium in soil has been demonstrated by Heyman et al. (2007) as substantially
contributing to reductions in the effects of A. euteiches a cause of root rot in peas.
These authors established a negative correlation between calcium content and dis-
ease severity in Swedish soils. Part of this effect may result from an inhibition of
zoospore release from oospores present in these soils.
Nutrient Status
Emphasis in the management of crop diseases is returning towards the concept of

enhancing plant or crop health as compared with previous objectives of eradicating
or diminishing pathogen populations. This approach was favoured until the advent
of extensive numbers of synthetic agrochemicals in the 1970s, with little research
interest from then until the late 1990s. Nutrient management is now increasingly
gaining stature as a practical and environmentally safe approach (Adhilakshmi
et al. 2008) towards crop health which is associated with the stimulation of benefi-
cial soil microbes as opposed to exploiting directly fungitoxic effects. Mineral
nutrition may also directly affect the ability of plants to resist harmful pathogens
(and other soil inhabitants) by up-regulating genetic mechanisms. The literature on
the effects of nitrogen influencing the outcome of both pest and pathogen invasion
is very extensive but requires careful analysis, understanding of the factors involved
and subsequent interpretation. For example, the effects of nitrogen (N) nutrition on
pests of onions, carrots and cabbage were evaluated on organic and mineral soils in
Ontario, Canada in 2000 and 2001 (Westerveld et al. 2002). The damage caused by
onion thrips (Thrips tabaci) was lower on cabbage that received higher than recom-
mended nitrogen. This effect may have been due to delayed maturity of plants that
received high nitrogen levels. These field trials also suggested that leaf blight
(Cercospora carotae) severity decreased with increasing nitrogen. In both instances
it might be expected that an increase in succulence could predispose these crops to
predation. Elsewhere the use of high levels of nitrogen fertilisation has been associ-
ated with increased disease severity, but the formulation of nitrogen used may be at
least as important as the rates used (Huber and Watson 1974).
Arguments for the use of ammonium and nitrate based nutrition were reviewed
by Barker and Mills (1980) and in great depth by Wild (1988). In general ammonium–
nitrogen appears associated with increased disease severity more often than
nitrate–nitrogen, but this is not always a hard and fast rule. Early research tended
to be confused due failures to adjust the rates of nitrogen applied relative to molec-
ular content, an array of formulations used and the huge diversity of environments,
crops and host–pathogen combinations studied. A brief syntheses of results are
provided by Marschner (1990) and Engelhard (1996), while earlier Huber (1978)
considered the impact of pathogen infection on host nutritional metabolism. The
concept that forms of nitrogen might influence the course of disease was pioneered
at the University of Wisconsin from the 1930s onwards by J C Walker who showed
for example, that vascular wilt of tomato (V. albo-atrum) increased with raised
levels of nitrate–nitrogen (Walker et al. 1954), but cabbage yellows (Fusarium
oxysporum f. sp. conglutinans), tomato wilt (F. oxysporum f. sp. lycopersici) and
pea wilt (F. oxysporum f. sp. pisi) decreased as the concentration of nitrate-nitrogen
increased (Gallegly and Walker 1949). While bacterial wilt and canker of tomato
(Ralstonia (Pseudomonas) solanacearum and Corynebacterium michiganense)
increased in severity concomitantly with increased nitrate-nitrogen. Nitrogen fer-
tilisation, as ammonium but not nitrate, was thought to stimulate fluorescent
pseudomonads within the rhizosphere (Smiley 1978). It is now apparent that plant
nutrition must be considered as a component part of integrated control management
for diseases (Dixon 2002, 2009c) in which combinations of nitrogen and calcium
offer the most effective means of countering pathogenic microbes through balanced
host nutrition.
In some instances, acid-forming fertilisers, such as ammonium sulphate or

p hosphate help to reduce disease incidence as with potato common scab (S. scabies)
(Lazarovits et al. 2007). In other instances, a reverse effect is obtained, as with
clubroot (P. brassicae) (Webster and Dixon 1991a). In general the literature on the
influence of soil chemical constituents on common scab severity for example, is
best described as inconclusive. Reviewing the effects of ten plant nutrients on scab
Keinath and Loria (1989) concluded that some of this confusion could be resolved
by examining interactions on the tuber surface between edaphic factors and disease
intensity. The influence of pH is seen as the most consistent factor associated with
potato common scab which is generally most serious with pH ranges of 5–8.
Long-term crop protection can be achieved by modification of the physico-
chemical properties of the soil in favour of the development of an antagonistic
microbial population. This can be achieved by the manipulation of some of the
macro-nutrient and micro-nutrient balances within the soil. Disease suppression
was not associated with the conduciveness of a soil to take-all, but rather to the
supportiveness of a soil to biocontrol activity. Biocontrol activity was positively
correlated with iron, nitrate-nitrogen, boron, copper, soluble magnesium and the
percentage of clay and negatively correlated with soil pH and available phosphorus.
In Western Australia take-all decline has been attributed to populations of
Trichoderma spp. that suppress the pathogen’s saprophytic as well as parasitic
activity (Simon and Sivasithamparam 1989). Soil treatment with T. koningii the
most abundant anatagonist in these suppressive soils reduced saprophytic growth of
G. graminis var. tritici and increased survival of wheat seedlings by about 50%. In
glasshouse experiments with natural soil, grain yield was increased about 10% in
three out of six field trials. A strain of T. koningii originally isolated in Western
Australia reduced take-all severity and increased yield as much as 65% when added
to the seed furrow in field trials in Washington State, USA (Duffy et al. 1996). Both
the take-all pathogen and indigenous antagonists are sensitive to soil pH and min-
eral nutrition. Microbially mediated take-all decline occurs after three wheat crops
in slightly acidic soils but is delayed until the sixth or seventh crop when soil pH is
elevated to 7.0 by liming (Cook and Baker 1983). Ammonium-nitrogen fertilisers
reduce the rhizosphere pH of wheat and increase the proportion of rhizosphere
bacteria that are antagonistic to G. graminis var. tritici; the reverse is true of nitrate–
nitrogen (Smiley 1978). Similarly the take-all suppressive activity of indigenous
populations of Trichoderma spp. is increased by soil acidification with ammonium
fertilisers, but this effect is lost with liming (Simon and Sivasithamparam 1989). An
adequate supply of micro nutrients such as copper, iron, manganese and zinc allevi-
ates take-all and their increased availability under acidic conditions is due in part,
to metabolic activity of certain indigenous microorganisms. In this study there was
a negative interrelationship between boron, copper, iron, soluble magnesium,
nitrate-nitrogen, percentage of clay and take-all severity in the presence of T. koningii
on eight silt loams.
An exceptional diversity of mechanisms contribute to the biocontrol activity of
Trichoderma spp. including the production of antibiotics, volatile organic com-
pounds and lytic enzymes; mycoparasitism and competiveness in the rhizosphere,
bulk soil or crop residues. While each of these has been proposed in different
reports to be primarily responsible for biocontrol, the relative importance of indi-
vidual mechanisms more than likely fluctuates depending on the strain, environ-
mental conditions and pathosystem. Soil nitrate levels have been positively
correlated with cellulase production and may favour competitiveness of the bio-
control agent with the pathogen. While nitrate fertilisers have been identified as
increasing take-all, partially at least because they raise rhizosphere pH, which is
favourable to the pathogen and unfavourable to the biocontrol agent (Simon and
Sivasithamparam 1989). This contrasts with the findings that nitrate–nitrogen
levels were positively correlated with T. koningii biocontrol activity. This suggests
that pathogen suppression in the bulk soil with an acidic pH is less affected by
nitrates (Smiley and Cook 1973) and may have been a major component of bio-
control during early plant growth and other factors may become important as
biocontrol shifts from the infection court to the rhizosphere. Understanding which
abiotic soil factors have most influence on the biocontrol activity of T. koningii
and how these factors interact may provide clues to biocontrol mechanisms and
their regulation in situ. This is especially important with regard to micro-elements
such as iron, pH, boron, copper and soluble magnesium, which affect the produc-
tion of anti-fungal compounds by T. koningii and competition with the pathogen.
Some key soil factors favourable to fungal antagonists (acidic pH and high iron)
contrast with those factors favourable to bacterial fluorescent pseudomonads
(alkaline pH and low iron).
To solve the problem of decline in land productivity and spread of soil-borne
disease such as Fusarium wilt (Fusarium oxysporum f. sp. cubense) in banana
orchards caused by the degradation of land quality, some integrated measures
including soil disinfection by application of modified lime-nitrogen (calcium
cyanamide) fertiliser, decontamination of irrigation water, continued soil disinfec-
tion and field experiments were carried out in an abandoned banana orchard in
China (ZhiYong and XiaoLin 2008). The results showed that the incidence of the
disease and disease index of Fusarium oxysporum f. sp. cubense at key growth
stages were significantly lower with treatment of integrated measures than those
with the aim of using a single control treatment. The sterilization of irrigation
water, to cut the route of the Fusarium wilt distribution, produced a significantly
positive result. The disease incidence was 67.58% in the control treatment, and
13.75% in the integrated treatment. Harvest area increased from 32.42% in the
control to 86.25% in the integrated treatment. Fruit yield per tree was 24.4 kg in the
control and 26.9 kg in the integrated ones. The yield increased more than 10%. It
could be concluded that integrated Fusarium wilt management might be the best
method of control for this disease. More widely, benefits accruing from the use of
calcium cyanamide are consistently greater than might be anticipated solely from
the calcium (50%) and nitrogen (20%) content of this fertiliser. Use of this chemi-
cal is associated with improved soil fertility in terms of increased benign microbial
activity. This apparently leads to a reduction in the pathogenic activity of several
highly aggressive soil-borne microbes particularly those such as Plasmodiophora
brassicae, the causal agent of clubroot disease in brassicas and Sclerotinia
sclerotiorum, a cause of stem and root white rot in an extensive range of crops which
are discouraged by alkaline pH values (Dixon 2009d).
High potassium levels were believed to reduce infection by some wilt inducing
pathogens, such as Fusarium wilt of tomato (Walker and Foster 1946). But they
were without effect on Verticillium and bacterial wilts of tomato (Walker et al.
1954; Gallegly and Walker 1949). For foot and root rots the influence of potassium
is rarely noted, but high potassium, especially if associated with high nitrogen,
reduced Fusarium culmorum on wheat. The reaction with Gibberella zeae (stalk
rot) on maize is more complex and depends on nitrogen level; an increase in potas-
sium reduced stalk rot when nitrogen was low, had no effect at medium nitrogen,
and increased it at high nitrogen; with low potassium then nitrogen level had no
effect. Root rots are generally reduced by high phosphate nutrition; in Canada the
reappearance of browning root rot of cereals due to Pythium spp. was attributed to
insufficient use of phosphate fertilisers. The relationship of calcium to disease
severity is even more tantalising since there various side effects such as the action
on pH, causing in turn imbalances of trace elements. Also related to calcium is the
influence of sodium. Increased sodium in the nutrient solution increased the sus-
ceptibility of tomato to wilt (Fusarium oxysporum f. sp. lycopersici). This may be
connected with the lowering of calcium induced by sodium since high concentra-
tions of calcium are related to reductions of disease severity in tomatoes infected
with Verticillium albo-atrum.
Disease management strategies, such as the application of fungicides (Bateman
1993) can also modify the severity and composition of the disease burden of a
crop. Control of eyespot (Tapezia yallundae) can lead to significant increases in
sharp eyespot (R. cerealis), possibly due to the loss of the exclusive capacity of
the W-type pathotype of T. yallundae (Murray et al. 1998). Prior stem coloniza-
tion by Fusarium spp., (especially F. avenaceum) is able to suppress development
of lesions of T. yallundae caused by the more virulent W-pathotype. If, however,
infection by Fusarium spp. occurs after that by T. yallundae, then foot rot severity
may be increased (Bateman and Munnery 1995); possibly because the primary
colonizer weakens or alters stems increasing their suitability for colonization by
secondary colonizers (Bateman 1993). The incidence of F. culmorum has also
been observed to increase at the same time as decline in G. graminis var. tritici
severity (Vojinovic 1972). With regards to Fusarium spp. alone, supplementary
nitrogen has been reported to increase the incidence of infection by up to 125%
(Martin et al. 1991). Parry et al. (Parry et al. 1995) conclude, however, that the
effect of nitrogen applications and Fusarium spp. are unclear. As with take-all it
could be another example of regional variation due to prevailing climatic condi-
tions, soil types and agronomic practices. The situations in America and the U.K.
are very different and successful strategies imported from other countries often
fail to give the same level of control elsewhere (Hornby et al. 1998).
Potassium phosphonate was found to have fertiliser properties which are linked
to increasing plant growth and vigour and hence reductions in pathogen incidence
for example with root rots caused by Phytophthora palmivora in pawpaw in
Northern Queensland, Australia. This treatment was successfully integrated with
the use of plastic sheeting and organic mulches and growing the plants on 0.75 m
mounds (Vawdrey et al. 2004). Relationships between sugarcane rust (Puccinia
melanocephala) and soil nutrients and pH have been suggested (Johnson et al.
2007). Phosphorus content correlates positively with rust incidence soil organic
matter, sulphur and potassium appear to have similar effects whereas magnesium
is negatively correlated with disease. Additives such as phosphonate (potassium
phosphite) protect trees against infection, increase tree (coconut and durian)
survival and yields and play an important role in integrated control systems
(Guest 2002).
Increasing soil phosphorus level is suggested to accelerate plant growth and this
may become more susceptible to this pathogen. High nutrient levels are associated
with vigorous crop growth, denser canopies and earlier canopy closure creating an
environment conducive to rust development. Results suggest there is a threshold
value for nutrient concentration, below the threshold there is no stimulation and
indeed there may be reductions. This is demonstrated with pH where at values of
£6.0 reduced and >6.0 disease is stimulated.
Availability of micro-elements to microbes and plants is involved in suppressiveness
towards several diseases. High concentrations of iron, manganese, copper and zinc were
associated with suppressiveness to Streptomyces, Aphanomyces, Gaeumannomyces
and Thielaviopsis spp. A high level of micro-nutrients should contribute to a more
diversified microbial flora and hence a more complex and efficient antagonism to
the pathogen. Micro-nutrients are also probably involved in host defence reactions.
In the case of black root rot (Thielaviopsis basicola) of tobacco, iron appears to be
a prerequisite for the antagonistic activity of fluorescent Pseudomonas spp. and the
efficiency of the bacterium in controlling the pathogen. The suppressive effect of
Pseudomonas results from the high production of hydrogen cyanide which is toxic
to the pathogen. Cyanogenesis is regulated by the availability of iron to the bacte-
rium. Diseases are suppressed in acid soils where iron, manganese, copper and zinc
form water soluble salts that are readily available. In alkaline conditions these ele-
ments are insoluble. Thus adding micro-nutrients to alkaline soils does not neces-
sarily lead to disease control. Soil factors influencing the availability of
micro-nutrients include the following:
• Micro-nutrient content of soil minerals
• Age of the soil which affects the degree of alteration of soil minerals
• Leaching of microelements from the soil
• Redox potential
A low redox potential increases the availability of some micro-nutrients especially
iron and manganese which form water soluble salts in the reduced state. The redox
potential is low in fine-textured soils under conditions of high soil moisture and
high microbial activity such as after the incorporation of large quantities of organic
matter. The combination of high soil moisture or low redox potential and high level
of micro-nutrients has been demonstrated to be effective in the suppressiveness of
potato scab (which is suppressed in moist soils in which the availability of
manganese and iron is high). By comparison fusarium wilt is enhanced by impeded
drainage and high soil moisture that is conditions which increase the availability of
iron for the pathogen.
The application of manganese has been associated with the stimulation of soil
bacteria (Casida 1968) and in conjunction with nitrogen (ammonium) fertilisation,
reductions in take-all severity (Brennan 1992a).The reduction of soil pH following
ammonium fertilisation would favour manganese-reducing organisms and would
increase the amount of available (to plants) manganese (Mn2+). Plants deficient in
manganese may have impaired abilities to synthesize phenolics and lignin
within the root system and thus are more susceptible to physical penetration by
G. graminis (Rengel et al. 1994). It is under conditions of both manganese and zinc
deficiency that populations of fluorescent pseudomonads are increased, but within
these populations, the incidence of both manganese-oxidizers and reducers is low
(Rengel et al. 1996). Manganese can also have direct effects on G. graminis since
in its reduced form manganese is toxic, but G. graminis and other rhizosphere
microbes are able to oxidize manganese to its non-toxic and unavailable form
(Mn4+). The manganese oxidizing ability of G. graminis is partly dependent on the
virulence of the pathogen, with virulence being correlated positively with manga-
nese-oxidizing ability (Pedler et al. 1996). Manganese is one heavy metal upon
which there has been a significant G. graminis var. tritici-based research effort,
particularly in Australia (Rengel et al. 1994) and America (McCay-Buis et al.
1995). The effects of manganese are also dependent on plant health and nutrition,
which is again a subject of considerable work in Australia by Brennan (1989,
1992b). Many mineral ions e.g. phosphorus, nitrogen, manganese, zinc, copper and
chloride, have been shown to play significant roles in the responses of plants to
attack by G. graminis. In the majority of this work the ion was applied in isolation,
or perhaps in combination with nitrogen, and until a greater understanding of the
inter-relationships within nutrient and host–parasite-wider microbial community
interactions are obtained, greater exploitation of nutrient provision in the manage-
ment of G. graminis will remain anecdotal.
The interactions between the inhibitory effects of fungal and bacterial
a ntagonists and nutrients have been tested under in vitro conditions against
the wilt pathogen of alfalfa (lucerne) Fusarium oxysporum f. sp. medicaginis,
Trichoderma harzianum and Pseudomonas fluorescens (PI 5) and were found to
be effective. Manganese sulphate at 500 and 750 ppm inhibited the mycelial
growth of F. oxysporum f. sp. medicaginis under in vitro conditions. In pot culture
studies, manganese sulphate at 12.5 mg kg−1 reduced the wilt incidence (23.33%).
Combined application of manganese sulphate 12.5 mg kg−1 T. harzianum 1.25 mg
kg−1 of soil significantly reduced the wilt incidence accompanied by improved
plant growth and yield in pot culture. The mixture of manganese sulphate (25 kg
ha−1) T. harzianum (2.5 kg ha−1) significantly reduced the wilt incidence when
applied as a basal dose in the field conditions. The average mean of disease reduc-
tion was 62.42% over control.
Soil boron content has been associated with adveresely affecting the activities of
P. brassicae since the 1930s (O’Brian and Dennis 1936) onwards. One of the first
controlled studies was that of Palm (1963) who investigated the effect of boron on
P. brassicae in sand cultures and recorded maximum root-hair infection at a

concentration of 0.3 mel−1 boron or less. He further demonstrated that in the absence
of boron the inhibitory effect of calcium on root-hair infection is suppressed, he sug-
gested that lime may fail to diminish clubroot disease in boron deficient soils.
Extended research (Dixon and Wilson 1983, 1984a, b, 1985; Dixon 1983, 1984a, b,
1985) achieved significant reductions in disease index with sodium tetraborate
applied to acidic granitic soils in 3 successive years of field studies. More recent
studies showed that environments with elevated boron concentration there are sig-
nificant effects both on the root-hair and cortical phases of P. brassicae. Throughout
the in planta stages of the life cycle of P. brassicae boron has an impact on the
microbe. There appears also to be a relationship with the quantity of boron in the
plant which is moderated by uptake over time and space as determined by the size
of the plant root system and its capacity to absorb boron. It is likely that there are
interactions with other ions. For example, lime applications in the forms of calcium
carbonate or oxide may alter the nutrient environment in soil to the detriment of
P. brassicae and therefore, make the host–parasite association more affected by
other factors such as boron. Alternatively, boron may have a primary effect because
Webster and Dixon (1991b) found that the effects of boron interact with both the
primary and secondary stages of development of P. brassicae ultimately affecting
the intensity of symptom expression. The environment induced by boron in cells
where membrane permeability and wall structure are altered may be to the detriment
of P. brassicae. It could also make for conditions less conducive for nuclear division
by the microbe. Quite possibly boron effects are distinct from those of calcium and
pH. Dixon (1991) identified that boron affects the progress of P. brassicae by retard-
ing the rate of sporangial maturation. The correlation of diminished intensity of
disease expression and boron suppression of root-hair infection and gall formation
appears related to host exposure. Long exposures to low concentrations seem to
equate with the effects of shorter exposures to higher concentrations. Field and con-
trolled laboratory studies (Craig and Dixon 1993a, b) identified that boron has a
substantial effect on the ability of P. brassicae to invade root-hairs and establish
colonisation in the field. Raising the boron content of the rhizosphere prior to the
availability of a susceptible host to infested soil limited the subsequent ability of
P. brassicae zoospores to penetrate, colonise root hairs and cause symptoms.
Water Management
High levels of soil moisture are often associated with increased disease severity.
This may be due to the presence of increased moisture films through which
motile spores move. Also, water-logged soil or compost will weaken and
asphyxiate root systems, thereby giving portals for the entry of pathogenic
organisms. Californian studies of the incidence of soil-borne Sclerotinia minor
(lettuce drop) as influenced by type of irrigation and the form of tillage indicated
that disease incidence remained significantly higher under furrow irrigation
compared with subsurface drip and was significantly greater on autumn as

opposed to spring crops (Wu and Subbarao 2003). With furrow irrigation the
number of sclerotia at the end of the crop season increased significantly over that
at the beginning of the year, but no significant changes were detected over-years.
By contrast, the number of sclerotia within a single season did not increase
significantly under subsurface drip irrigation, nor was the year-on-year accumu-
lation of sclerotia significantly increased. The degree of aggregation of sclerotia
increased significantly during a cropping season under furrow irrigation but not
under subsurface drip irrigation. The conventional tillage after harvest under
furrow irrigation decreased the degree of aggregation of sclerotia after each
season but the distribution pattern of sclerotia under subsurface-drip irrigation
changed slightly by association with minimum tillage. Spatial pattern analyses
suggested that the aggregation of S. minor sclerotia occurred at a scale of not
more than 1 m and the distribution of diseased lettuce plants was random at a
scale larger than 1 m. The combination of fewer sclerotia produced by each crop
and its unaltered distribution under subsurface drip irrigation and associated
minimum tillage makes it a valuable cultural practice for lettuce drop disease
management. Wet ground (Brenchley 1968) and areas where soil structure has
deteriorated (Yarham 1981) have been associated with increased take-all
(G. graminis var. tritici) severity, however it is more likely that the poor root
growth in such areas predisposes plants to severe take-all (Gair et al. 1972; Parry
1990). Where soil moisture rises from 50% of maximum water holding capacity
up to saturation (Dixon 1981, 1984b) clubroot disease (P. brassicae) severity
escalates. Lange and Olson (1983) emphasised the dependence of zoosporic
microbes on free water existing between the soil crumbs for the movement of
zoospores. Free water is critically important for the formation, discharge and
dispersal of zoospores and may influence the encystment and penetration pro-
cesses at the root-hair surface. The distances travelled by soil-borne zoospores
are relatively short, probably between 10 and 20 mm judging by information for
Olpidium brassicae or Synchitrium endobioticum both relatives of P. brassicae.
Invasion of root hairs occured up to 75 mm from the source of P. brassicae infec-
tion in soils where water mass movements were minimised (Watson 1967).
Mathematical modelling by Yang et al. (2004) demonstrated a relationship
between soil moisture and host invasion. Similarly a soil moisture deficit limits
the movement of propagules of Pythium violae, the cause of cavity spot in car-
rots (Suffert et al. 2008) reducing primary infections in the field, promotes
lesion healing, limits primary infections and subsequent spread. Integrating ele-
ments of husbandry such as harvest date, soil moisture content, sowing density
and crop geometry linked with appropriate fungicide use offers means for con-
trolling this previously intractable disease (Suffert et al. 2008).
Irrigation monitoring and scheduling are imperative as water becomes increas-
ingly scarce producing a drive for greater water-use efficiency which in part at least
aims to control those plant pathogens which are favoured by excessive application
(Passioura 2002). Australian wheat yields have increased by improvements to
health and nutrition of crops and effective water management ensuring that it is
effectively turned into photosynthate used for grain filling. A steady annual yield
increase of 1% can be achieved through plant breeding linked with greater under-
standing of the relationship of roots and soil microbes and knowledge of how roots
utilise soil resources such as water (Passioura 2002). To sustain production of
annual crops in Australian Mediterranean environments, for example, both agro-
nomic and genetic options have been used. An analysis of the yield increases in
wheat in Mediterranean climatic regions shows that there has generally been an
increase in the yields over the past decades, albeit at a lower rate than in more
temperate regions. Approximately half of this increase can be attributed to agro-
nomic and half to genetic improvements. The agronomic improvements that have
been utilised to sustain the increased yields include earlier planting to match crop
growth more closely with rainfall distribution, use of fertilisers to increase early growth,
minimum tillage to enable earlier planting and increase plant transpiration at the
expense of soil evaporation, rotations to reduce weed control and disease incidence
and use of herbicides, insecticides and fungicides to reduce losses from weeds,
pests and pathogens. Genetic improvements include changing the phenological
development for better matching with rainfall patterns, increased early vigour,
deeper rooting, osmotic adjustment, increased transpiration efficiency and improved
assimilate storage and remobilisation. Mediterranean environments that are sub-
jected to annual terminal drought can be environmentally and economically sustain-
able but to maximise plant water-use efficiency while maintaining crop productivity
requires an understanding of the interaction between genotypes, environment and
management (Ashcroft et al., 2003).
Irrigation may enhance sustainable crop production but only if applications are
made as part of an integrated management package. Water productivity (WP)
expresses the value or benefit derived from the use of irrigation (Singh et al. 2006).
Substantial variations appear between water productivities of varying crops such as
wheat, rice and cotton, within crops each relative to the manner of use, prevailing
husbandry practices such as the use of mulches and soil physical and chemical
compositions. Soil moisture content affects the relationships between of R. solani
and its antagonist Trichoderma harzianum (de Paula and Hau 2002). The antago-
nistic ability of T. harzianum was greater in soils held at intermediate moisture
content as compared with very wet or very dry conditions. Soil water logging
appeared to aid the establishment of a new pathogen (Phytophthora botryose) in
plantations of Elmerrillia tsiampacca and E. ovalis, whereas previously this patho-
gen had been known on Heavea braziliensis (rubber) (de Kam and Sukmajaya
2002). Aphanomyces euteiches induced root-rot in pea causes serious crop loss
(Allmaras et al. 2003) and the pathogen remains viable in soil for many years. The
disease is favoured in poorly drained soils, especially when soil compaction caused
by trafficking with heavy axle loaded vehicles impairs drainage. Soil water poten-
tials (SWPs) of P >−5 kPa at the 15 cm depth were associate with pea crop failures
especially where impaired drainage was in the upper 50 cm of fields. Soil–water
retention and field observed SWP indicated <10% air-filled pores frequently
occurred in the 15–30 cm layer, symptoms of anoxia were absent from the crops.
Poor soil drainage influences the mode of infection. The virulence and inoculum
potential (Ulacio et al. 1999) of R. solani propagules affecting rice plants were
related to the period of time where they were exposed to anaerobic conditions.
Glasshouse and field experiments were conducted in Ibaraki, Japan, in 2001,
studied the effects of soil moisture, pH and pathogen resting spore density on the
effectiveness of the biological control of clubroot (P. brassicae) by a fungal endophyte
(Heteroconium chaetospira) in Chinese cabbage cv. Shin-Riso (Narisawa et al. 2005).
Conditions favouring disease development included low pH (5.5) and high soil
moisture content (80%), with significant reductions in the disease observed at a
higher pH (6.3 and 7.2) and lower soil moisture content (40% and 60%). In glass-
house tests, H. chaetospira effectively controlled clubroot (reducing the disease by
90–100%) at pathogen resting spore densities of 104 and 105 spores g−1 soil at all soil
pHs tested (5.5, 6.3 and 7.2). When the resting spore density was 106 spores g−1 soil
however, plants were severely infected, regardless of treatment, and H. chaetospira
had no effect on the disease. At soil moisture content of 40%, disease occurrence was
low, regardless of pathogen spore density, but the disease was significantly lower in
H. chaetospira-treated plants with a pathogen spore density of 105 spores g−1 soil. At
60% soil moisture content, H. chaetospira was significantly impaired the effective-
ness of the pathogen (P. brassicae) at spore densities of 104 and 105 but not on 104 g−1
soil. At 80% soil moisture content, H. chaetospira had no effect on pathogen density.
In situ soil moisture contents were constantly adjusted to relatively low to moderate
values (pF 2.2–2.4 and pF 2.0–2.2, respectively) and high values (pF 1.6–1.8). Other
environmental conditions, i.e. resting spore density and soil pH, were maintained at
constant levels. Control plants (not treated with (H. chaetospira) showed uniformly
high disease levels and proportions of diseased plants across all moisture treatments
(disease index = 72–80, proportion of diseased plants = 85–97%). In the field, H.
chaetospira-treated plants at low soil moisture (pF 2.2–2.4) had 68% disease reduc-
tion compared with the untreated controls and 49% reduction at moderate moisture
(pF 2.0–2.2). Heteroconium chaetospira had no effect on the disease at high soil
moisture (pF 1.6–1.8).
Sowing and Planting Times
Autumn sowing favours the development of take-all (G. graminis var. tritici) for
climatic and temporal reasons (Hornby et al. 1998) and early infections cause the
greatest degree of physiological problems. Ninety per cent of total nitrogen and
phosphorus content of mature cereals is absorbed before the plant has achieved
25% of its final dry weight (Ayres 1984), therefore the rate and volume of soil
explored in the early growth-stages has long-term implications for the subsequent
performance of the plant. This is of particular importance to the uptake of phospho-
rus, which in combination with potassium increases cell wall thickness and tissue
hardiness thus providing additional mechanical resistance to pests and pathogens
(Maloy 1993). Though following infection with G. graminis var. tritici phosphorus
uptake may not be impaired as uptake of phosphorus can occur along the entire
length of the root (Fitt and Hornby 1978; Hornby and Fitt 1982) and transport is
unaffected by the presence of well-developed lesions on seminal roots (Clarkson
et al. 1975). A reduction in the volume of soil colonized by the root system may
limit phosphorus uptake because it diffuses slowly through soil and does not move
more than the width of a root hair, so zones of depletion develop in the vicinity of
roots (Harper 1977).
One of the most dramatic examples of the changing interaction between sowing
date and disease incidence is the soil-borne microbe P. brassicae and clubroot in oil
seed rape (B. napus). Previously, this pathogen was held in check in the British crop
because it was a predominantly sown in the autumn. This meant that it was drilled
in late August to early September into cooling soil. The seed germinated and pro-
duced rosette structured plants by November which formed their components of
yield before growth recommenced in mid to late February. The pathogen was inac-
tive in the cold winter soils remaining as dormant resting spores. Consequently, the
crop grew away and yielded in early summer with little damage from P. brassicae
in contradistinction to the spring drilled crops of Continental Europe (France,
Germany and Scandinavia) which succumbed to clubroot as both host and pathogen
developed as the soils were warming. Now the British crop is being sown in late
July to early August and soils are retaining heat and moisture through the increas-
ingly milder autumn periods as a consequence P. brassicae remains actively caus-
ing damage throughout the year. Greater soil moisture content in the autumn and
winter because of increased rainfall has only served to offer the pathogen addition-
ally improved opportunities for spread and multiplication (Dixon 2006). Cabbage
and other vegetable cole crops which are grown in the colder seasons traditionally
suffer less from Plasmodiophora brassicae. The pathogen is less active in cold soil
whereas summer cabbage and cauliflower are at high risk from it.
Disease may be avoided by early or late sowing or planting so that the crop and its
pathogen are out of phase with each other and an epidemic cannot develop. A tradi-
tional method for reducing Erysiphe cruciferarum (powdery mildew) infection on
brassicas is to sow late so that the foliage is immature and cannot support the patho-
gen during the main period when the crop is at risk. A heavy price is paid, however,
in terms of yield. Mid- and late-season Brussels sprouts in Bedfordshire and the Vale
of Evesham are less badly attacked by E. cruciferarum, perhaps because the environ-
mental conditions are such that the pathogen is at a disadvantage compared with early
season crops grown for processing in Lincolnshire (Dixon 1981).
Soil Solarisation
Solarisation is the practice whereby soil is covered with polyethylene sheeting

capturing heat and raising the temperature substantially such that the surface lay-
ers are sterilised. Obviously, such techniques are only feasible in countries where
intense sunshine is converted rapidly to solar gain such as parts of southern
Europe, Israel, states such as California, USA and parts of Australia. In these areas
solarisation has been used successfully in order to remove pathogens such as

Verticillium and Fusarium spp. The difficulty lies in that most other microbes are
also killed leaving soil populations disbalanced and vulnerable to rapid
recolonisation by the more aggressive forms which are usually pathogenic. In
which case if severe disease epidemics are to be avoided following solarisation the
process must be linked in with other measures encouraging the re-entry of more
beneficial forms. Columns of moist sandy loam soil inoculated with several
soil-borne pathogens were inserted into fields and then subjected to solarisation by
covering with transparent polyethylene sheets (Porter and Merriman 1983).
Temperatures achieved were within the range 38–55°C, this was sufficient to
eradicate: V. dahliae, S. cepivorum and S. minor. Less success was achieved with
F. oxysporum, P. irregulare and P. brassicae. The soil was penetrated by high
temperatures to 6–11 cm with sufficient effect to destroy most propagules to the
point where they could not be subsequently detected. Mechanisms for this effect
were examined by Horiuchi et al. (1982). They suggest that temperatures in excess
of 45°C were sufficient to eradicate P. brassicae and the effect was enhanced by
the presence of organic matter or the fertiliser calcium cyanamide. Their labora-
tory experiments suggested that a relationship between temperature and the time
of exposure could exist since extending treatment at lower temperatures achieved
similar effects to shorter periods of more intense treatment.
Solarisation was examined by Stevens et al. (2003) who found that soils
amended with high nitrogen organic fertiliser (chicken manure) contained signifi-
cantly increased soil rhizobacteria such as Bacillus spp. and fluorescent pseudomo-
nas in the rhizospere, rhizoplane and root interior of sweet potatoes and possibly
suppressed soil pathogenic organisms such as Fusarium root rot (Fusarium solani)
and Java black rot (Diplodia tubericola). Soil solarization offers a means for killing
teliospores of the soil-borne fungal wheat pathogen Tilletia indica (Peterson et al.
2008). A rapid decline in teliospore viability occurred at all treatment depths over
38 days. Initial viability rates of 43%, 71%, and 82% germination were reduced to
0.1%, 7.7%, and 0.2% after 38 days (across all depths) over 3 years – 2003 to 2006.
Mean daily maximum soil temperatures at 5 and 20 cm under clear plastic in 2003,
2005, and 2006 were 67°C, 53°C and 60°C and 43°C, 38°C, and 43°C, respectively.
Soil solarisation was evaluated for the control of Phytophthora root rot of rasp-
berry (P. rubi) (Pinkerton et al. 2009). Cumulative time with soil temperatures >29°C
at 30 cm soil depth in solarised plots exceeded 200 h. Results indicated that soil
solarisation can be an effective component of integrated management of root rot in
the Pacific Northwest, especially when combined with using raised beds and gyp-
sum (calcium sulphate) amendments.
Combining soil solarisation with the addition of organic amendments derived
from the cruciferous plant wild rocket (Diplotaxis tenuifolia) or thyme (Thymus
vulgaris) raised the control of the wilt Fusarium oxysporum f. sp. radicis-lycopersici
(Klein et al. 2007). Adding lucerne (alfalfa) leaves to soil increased the content of
the nematode-trapping fungus (Dactylellina candidum) and microbivorus nema-
todes (Jaffee 2006), but the inter-relationships between these events remains
obscure. Soil solarization in combination with the introduction of biocontrol agents
was evaluated as a potential disease management strategy for tomato damping-off

caused by Pythium spp. (Jayaraj and Radhakrishnan 2008). A rifampicin resistant
Pseudomonas fluorescens strain (PfT-8) and a carbendazim resistant Trichoderma
harzianum strain (ThM-1) were introduced into soil following solarization. Tomato
seeds were planted into treated field plots. Damping-off incidence was significantly
reduced in solarised plots compared with controls. Soil inoculation of biocontrol
agents into solarised plots resulted in the highest suppression of damping-off inci-
dence (PfT-8 up to 92%; ThM-1 up to 83%), and increase in plant biomass (PfT-8
up to 66%; ThM-1 up to 48%) when compared with un-solarised control plots.
Rhizosphere populations of introduced biocontrol agents gradually increased
(PfT-8 up to 102% and ThM-1 up to 84%) in solarised soils when compared with
unsolarized controls. The population of Pythium spp. in rhizosphere soil was
reduced up to 55% in solarised plots; whereas, application of bicontrol agents to
solarised soils reduced the rhizosphere population of Pythium spp. by 86% and
82% in P. fluorescens and T. harzianum applied plots respectively.
Cover, Trap and Biofumigation Cropping
Cover crops are used primarily as a means of protecting uncropped land from
erosion but they may have an effect on the survival of soil-borne pathogens.
The use of cover crops such as hairy vetch (Vicia villosa) and rye (Secale cereale)
in combination with nil tillage husbandry produced beneficial effects by reducing
the incidence of blight (Plectrosporium tabacinum) and the need for fungicidal
sprays on pumpkin crops (Everts 2002). Antifungal effects of intercrops, e.g. pep-
permint and isothiocyanate production by brassica crops like mustard have been
examined. Use of crops to stimulate pathogen germination and then exterminate the
propagules produced (so called trap crops) has long been sought with little really
practically effective effects. The phasing-out of methyl bromide brings the need to
develop a scientific basis for these effects into particularly sharp focus (Matthiessen
and Kirkegaard 2006). Biofumigation is the beneficial use of brassica green
manures that release isothiocyanates chemically similar to methyl isothiocyanate,
the active agent from the synthetic fumigant metam sodium, which is used as a
substitute for methyl bromide in some systems. A systematic approach to research
into biofumigation, specifically aimed at overcoming a long history of empiricism,
has started to see significant advances in both basic and applied knowledge. A key
development has been achievement of maximal biofumigation potential through
greatly enhanced release of appropriate isothiocyanates into soil. These advances
have led to commercial adoption, demonstrating that biofumigation, when applied
to appropriate production systems, can have efficacy and offer cost savings. Despite
this success, biofumigation is not yet seen as being sufficiently powerful or practi-
cal in its implementation to be a realistic alternative to methyl bromide.
Enhanced microbial biodegradation is a cryptic phenomenon that can enhance
or diminish the efficacy of soil-applied pesticides, including isothiocyanates
and most other currently available methyl bromide substitutes. This has been
aided for some particularly vulnerable environments by recent clarification of
key risk factors associated with soil type, soil pH, and calcium content. The
hydrolysis of glucosinolates contained within the tissue of Brassica juncea
releases volatile isothiocyanates and these have been shown to inhibit mycelial
growth of G. graminis and R. solani but not B. sorokiniana (Angus et al. 1994;
Kirkegaard et al. 1996). The toxicity of aliphatic isothiocyanates to fungal
pathogens is correlated inversely with side chain length and of the pathogens
tested by (Sarwar et al. 1998), G. graminis was the most sensitive and R. solani
and Fusarium spp. were more tolerant. Fluorescent pseudomonads are also able
to produce a related compound: hydrogen cyanide (Défago et al. 1990), which
may be the active component of the volatile substances produced by some of the
isolates in this study.
In Germany, where a high value is set on the maintenance of good soil condi-
tions (H. Toxopeus 1972, personal communication), considerable use is made of
green manuring crops such as mustard and radish. These have anti-fungal proper-
ties due to their mustard oil content which reduce the populations of soil-borne
pathogens. Brassica juncea cv. Vitasso grown as an intercrop for strawberry cv.
Elsanta provided biofumigation. This resulted in a significant reduction in the
inoculum potential of V. dahliae microsclerotia (Steffek et al. 2006). Soil-borne
organisms vary greatly in their response to brassicaceous soil amendments. For
example, while the Citrus nematode was consistently suppressed the effects on
Fusarium spp. and weed survival were variable. Zasada et al. (2003) contend that
consistency of effect will only come from an analytical understanding of the active
principles involved.
Combining cover crops with the incorporation of compost increased soil quality in
irrigated, intensive lettuce and green broccoli (calabrese) production in the Salinas
Valley, California, USA (Jackson et al. 2003). There were beneficial effects increasing
soil microbial biomass, increasing total soil carbon and nitrogen, reducing surface bulk
density and decreasing the potential for groundwater pollution as a result of nitrate
leaching below the root zone. Yields were comparable to traditional husbandry and
occasionally resulted in fewer weeds and lower lettuce corky root disease (Sphingomonas
suberifaciens). Although surface minimum tillage reduced yields there was less nitrate
leaching below the root zone. The use of conventional tillage, cover crops, and com-
post produced high vegetable yields and acceptable net economic returns over a 2-year
period, but broccoli was more profitable than lettuce under this regime. Cover cropping
has potential benefits especially where producers are in transition between conven-
tional and organic systems (Baysal et al. 2008). Comparisons of husbandry-business
systems included: tilled fallowing, mixed-species hay production, low-intensity vege-
tables and intensive vegetable production using high tunnels. Biologically most advan-
tageous was mixed-species hay production where percentage carbon and total
phosphorus in soil increased by two- and fivefold and potassium by between sixfold
and 12-fold. Damping-off (Pythium ultimum and R. solani) in soybean and tomato was
reduced by 2–30% in pot tests and in the field. The suppressiveness of soil was
enhanced by cropping with mixed species hay systems.
Organic Additives
The overall effects of mulching with organic materials are beneficial (Quarles
2008), they conserve water and reduce the need for pesticide use. They also reduce
soil compaction and erosion; buffer soil against temperature extremes; reduce plant
diseases, insect pests and weeds; and encourage beneficial insects. Green manuring
is a traditional method for improving soil fertility and eradicating weeds, pests and
pathogens using uncomposted plant materials grown and incorporated in situ. For
example, it formed an integral part of the ‘Scarlett System’ developed in the
Scottish Lothians for land restoration which eradicated perennial weeds and enhanced
soil fertility in combination with the use of the nitrogenous fertiliser calcium cyana-
mide which stimulates the beneficial microbial flora and is only leached-out slowly
through soil (Scarlett 1937). Soil amended with organic additives such as green
manures, composts and animal manures suppresses some soil-borne microbes
(Millard and Taylor 1927; Sanford 1926) with other benefits including less surface
crusting, increased water infiltration and increased activity by benign microbes.
Organic matter additions are thought to suppress the cause of potato early dying
syndrome (V. dahliae and V. albo-atrum) indirectly through increases in soil micro-
bial activity or by release of toxic compounds (Rowe and Powelson 2002). More
recently green manuring (Wiggins and Kinkel 2005), of buckwheat, canola or fal-
low linked with three crop sequences: alfalfa (lucerne)–potato, corn–potato and
potato–potato has been evaluated. When compared with fallow treatments potato
tubers grown in buckwheat treated soil had significantly lower Verticillium (V.
dahliae) wilt ratings and tubers grown in buckwheat or canola treated soil had
greater yields. Potatoes grown in soil planted to corn or alfalfa (lucerne) the previ-
ous year had significantly lower Verticillium wilt and potato scab (S. scabies) rat-
ings as well as higher yields than potatoes grown in soil previously planted to that
crop. Other Streptomyces spp. taken from soils collected from green-manure treated
plots tended to have greater in vitro pathogen inhibitory activity than Streptomyces
spp. from fallow-treated plots. Streptomyces pathogen inhibitory activity was fre-
quently negatively correlated with plant disease and positively correlated with
potato yield. These results offer opportunities for the management of soil-borne
Streptomyces content with a view to reducing the impact of soil pathogens such as
Verticillium. A potato-scab suppressive soil developed in Minnesota, USA, following
long-term potato monoculture and had greater densities of Streptomyces, greater
proportions of antagonistic Streptomyces spp. and Streptomyces isolated from
these soils had greater mean inhibition zone sizes compared with nearby conducive
soil (Lorang et al. 1989 and L. L. Kinkel unpublished data cited in Wiggins and
Kinkel 2005).
Composts affect soil fertility and plant health beneficially or detrimentally
depending on their quality. Their organic substance and nutrient content varies
greatly the origin of materials was the major factor influencing these values (Fuchs
et al. 2008). While the majority of composts protected cucumber plants against
Pythium ultimum, only a few suppressed R. solani infecting basil. The incidence of
damping-off caused by R. solani was significantly and consistently suppressed in

soils amended with residues of clover, peanut or Brassica rapa spp. rapifera cv.
Saori (Kasuya et al. 2006). Tests using antibacterial antibiotics indicated that a
viable microbial community is needed. That contention is supported by Mazzola
(2007) and their studies of apple replant disorder caused by R. solani AG-5.
A composite of Brassica napus and B. rapa seed meal produced the highest disease
suppression. Suppression was a result of the proliferation of Streptomyces spp. not
the glucosinolate content of the meal.
A general or long-term effect of organic soil amendments is the enhancement of
microbial activity including biocontrol agents which enhances natural disease sup-
pressive conditions (Abbasi et al. 2009). Additionally short term effects may be
derived from some amendments such as fish emulsion which these authors found
capable of substantially reducing the viability of microsclerotia of V. dahliae,
effects attributed to the presence of organic aliphatic acids such as glycolic, acetic,
formic, n-butyric and propionic. Damping-off caused by P. ultimum damaging
cucumber declined over time following the addition of fish emulsion as a result of
the erosion of inoculum concentrations.
Research which investigated the relationship between propagule numbers and
genetic diversity of Trichoderma species and Southern blight of tomato caused by
soil-borne plant pathogen Sclerotium rolfsii in soils with long-term organic, sustain-
able, and conventional farms is reported by Liu et al. (2008). Dilution plating was
used to quantify the propagule numbers of Trichoderma, denaturing gradient gel
electrophoresis (DGGE) and DNA sequence analysis were used to identify
Trichoderma species, and glasshouse assays were used to test for soil suppressive-
ness to Southern blight. The propagule numbers of Trichoderma tended to be higher
in soils from conventional farms. There was no clear separation for the propagule
numbers of Trichoderma based on different management systems using canonical
correspondence analysis (CCA). There was, however, general separation for total
microbial communities based on organic and conventional management systems
using CCA. That suggests that the differences in soil suppressiveness to disease from
organic, sustainable, and conventional farms is due to the effects of the total micro-
bial diversity but not directly due to the Trichoderma populations in each farming
system. The propagule numbers of soil Trichoderma did not significantly correlate
with the disease suppressiveness, although individual species such as Trichoderma
harzianum were shown to be related to disease suppressiveness. Moreover, several
Trichoderma species were found in the soil tested based on DGGE and DNA
sequence analysis. Trichoderma hamatum, T. harzianum, Trichoderma virens, and
Trichoderma erinaecem were the most abundant species in tested soil.
Control of Allium white rot caused by Sclerotium cepivorum (Coventry et al.
2006), was developed through the incorporation of onion waste compost into soil.
This reduced the viability of sclerotia of S. cepivorum and the incidence of white rot
disease in the field and was as effective as using the fungicide tebuconazole. Two
mechanisms of suppression were suggested: firstly, a reduction in the soil population
of viable sclerotia which may result from the action of volatile sulphur compounds
present in the composted onion waste, and secondly, prevention of infection of onion
plants from sclerotia. Allium white rot attacks the root system resulting in either death
before harvest or rotting of stored bulbs. The pathogen survives in soil as sclerotia
which may remain dormant in the absence of a host for more than 20 years. Sclerotia
are stimulated into germination by thiols and sulphides released by soil microorgan-
isms from alk(en)yl cysteine sulphoxides secreted by root of Allium spp.
Greenwaste compost has proven broadacre activity against G. graminis var.
tritici (Tilston et al. 2005) and based on experiments performed under protected
conditions, activity against many soil-borne pathogens of cultivated crops.
Pathogens suppressed by greenwaste compost include Sclerotium cepivorum,
Fusarium oxysporum f. sp. dianthi and Phoma medicaginis var. pinodella (Pitt
et al. 1998; Tilston et al. 2002). Composts provide a source of nutrients and when
applied in conjunction with biological control agents can increase the efficacy of
introduced microorganism, especially this is done in a non-competitive environment
(Deacon and Berry 1993). Other organic soil amendments also stimulate the activ-
ity of soil microbes and suppress the survival and activity of Phytophthora spp.
Natural products such as clove oil, neem oil, pepper extract with mustard oil, cassia
extract and synthetic cinnamon oil were tested for the control of Phytophthora
nicotianae affecting periwinkle (Catharanthus roseus). With the exception of neem
oil all these substances appeared to reduce disease expression (Bowers and Locke
2004). Claims have been made that fish oil emulsion added to soil will reduce soil-
borne pathogen damage.
Among the most potent organic extracts are those from the various seaweeds
which inhabit marine continental shelves around the world. In Europe the prime
source is from Ascophyllum nodosum harvested from Scandinavian and British
waters. Such extracts may alter the mode of activity of microorganisms as to
whether they operate as parasites, mutualists or as saprophytes and whether they
enter states of imposed passivity or inherent dormancy. This may lead to these
organic extracts, when placed in the soil environment either altering directly or
indirectly, characteristics such as: root colonisation and penetration, competition
and microbiostasis and antibiosis. The growth in vitro of P. putida and the abilities
to produce siderophores in the presence and absence of organic extracts are illus-
trated in Figs. 2 and 3 (Dixon and Walsh 1998).
The production of siderophores, which are low molecular weight iron binding
compounds, has been associated with the fungal pathogen suppressive properties of
several fluorescent Pseudomonas spp. (Leong and Winkelmann 1998). It is sug-
gested that the iron sequestering abilities of siderophores may deprive a pathogen of
available iron (Kloepper and Schroth 1981). Hence, stimulation of siderophore pro-
duction by the presence of organic extracts correlates with increased soil suppressive
properties. Increasing biological activity by benign organisms in the soil may be
correlated with rising production of extra-cellular enzymes. These degrade organic
materials within the soil providing mutualists such as P. putida with energy supplies
which in turn aids their capacity to inhibit the activities of pathogenic organisms.
Samples of compost were treated with organic extracts derived from seaweed and
this increased activities of the enzymes, amylase, cellulase (b-1–4-glucanase) and
glucanase (b-1–3-glucanase) detected within 48 h of incubation at 25°C (Fig. 4).
Fig. 2 The effect of organic extracts on the in vitro growth of Pseudomanas putida Key: LFe = low
iron content medium; LSE = liquid seaweed extract (Dixon and Walsh 1998)
Fig. 3 The effects of organic extract on the in vitro production of siderophores byPseudomonas
putida Key: LFe = low iron content medium; LSE = liquid seaweed extract (Dixon and Walsh
1998)
In an extended study Abbasi et al. (2006) showed that reductions in potato scab
(S. scabies) and V. dahliae wilt of potatoes using organic extracts were unaffected
by soil type but varied in efficacy from season to season and was ineffective at high
inoculum pressures. Agricultural wastes such as rice straw, rice hull, groundnut
Fig. 4 Extra-cellular enzymes produced by Pseudomonas putida in compost in response to the

addition of organic extracts Key: LSE = liquid seaweed extract (Dixon and Walsh 1998)
husk, maize cob, bagasse, rape seed pomace, castor seed pomace, tree bark,
mushroom growth medium waste and shrimp shell powder are reviewed by
Jennwen (2005) for their use in land reclamation and in the production of horticul-
tural crops. Their effects include improving soil fertility, increasing soil organic
matter and in some instances reducing the incidence of plant diseases. The latter is
attributed to a combination of several effects including direct impact on pathogenic
microbes and improving the vigour of host plants. Amending soils with composts
increased beneficial microbial (actinomycetes, bacteria and fungi) activity espe-
cially where chitin (crab shell) was added (Escuadra and Amemiya 2008) and
raised soil suppressiveness to pathogenic organisms. Alkaline stablised biosolids
produced from dried sludge, cement kiln dust and unspecified fillers are claimed to
reduce the impact of several soil-borne pathogens by changing pH to alkaline val-
ues, adding nutrients and improving structure (Ben-Yephet et al. 2005). Frequently
this requires the use of several factors as with tan spot of cereals caused by
Pyrenophora tritici-repentis which was encouraged by nil-tillage, reduced nitrogen
fertilisation, continuous cropping with wheat or rotation with maize. Integrated use
of nitrogen up to 100 kg ha−1 N, conventional tillage and the use of fungicides
resulted in the highest yields at El Batan, Mexico (Duveiller et al. 2002).
Crop husbandry practices may lead to reduced or improved soil structure and con-
comitantly increased or reduced soil-borne plant diseases (Bailey and Lazarovits
2003). Agricultural practices such as incorporating organic amendments and manag-
ing the type and quantity of crop residue directly affect plant health and crop produc-
tivity. Soil management practices such as tillage, rotation, and stubble (residue)
burning affect the quantities and qualities of organic matter returning into the soil. This
influences pathogen viability and distribution, nutrient availability, and the release of
biologically active substances from both crop residues and soil microorganisms as
illustrated by a model system of B. sorokinia on the development of common root rot
in cereals (Bailey and Lazarovits 2003). The application of organic amendments,
manures and composts rich in nitrogen, may reduce soil-borne diseases as a result of
releasing allelochemicals by microbial decomposition. The form of nitrogen released
can be crucially important when seeking to achieve this effect. Developing disease
suppressive soils by introducing organic amendments and crop residue management
takes several seasons but benefits accumulate as soil health and structure improve.
Characteristics of Soil Microbial Communities
Inoculum Potential
Balanced in the equation of soil factors and husbandry techniques which support
sustainable crop growth are the general traits of microbes which give them advan-
tages as pathogens. Pathogens can only dominate the soil environment and cause
disease when they are present in sufficiently large quantities that they are able to
breakdown the genetic barriers possessed by crop hosts and overcome edaphic bio-
logical, chemical and physical soil environments which are adverse to them. Raising
the microbial diversity of soils is associated for example, with increased suppressive-
ness towards soil-borne pathogens. Grassland soils for example, possess higher
microbial diversity compared with arable ones and are able to diminish the effects of
R. solani (van Elsas et al. 2002). A cardinal characterisitic for successful pathogenesis
by soil-borne organisms is the inoculum potential of a pathogen. This concept
embraces for the pathogen all of those factors which cumulatively lead to successful
invasion, colonisation and pathogenesis. Where inoculum potential is high one or
possibly a few disease-causing microbes form a substantial proportion of the total soil
microflora population and incite ill-health in the host. Despite this concept being
conceived by Garrett in 1956 (Garrett 1956) it remains one of the corner stones of our
understanding of host and pathogen biology in the soil. The concept of inoculum
potential applies to both pathogens and to the benign microbes which antagonise
them and hence may have value in evaluating biocontrol agents.
The biodiversity of benign soil microbes which are antagonisitic to pathogenic
forms is critical in order to maintain soil health and quality (Garbeva et al. 2004).
Diversity is affected by crop type, soil type and their management as discussed
earlier in this chapter. Characterising the diversity of benign microbes is frequently
defeated by sheer scale of soil inhabitants. In the top layers of soil even with only
moderately fertility there are 109 bacterial cells per gram most of which resist
culturing. Less than 5% of soil microbial biomass has been cultured and taxo-
nomically characterised (Torsvik and Ovreas 2002). The relationship between the
size of microbial biomass and the capacity of a soil to suppress pathogens was
demonstrated by van Os and van Ginkel (2001). A Pythium sp. was suppressed
by high microbial biomass which reduced the impact on bulbous Iris. Mechanisms
suggested which underpined suppression included nutrient competition, commen-
salism, microbial antagonism, parasitism and systemic induced resistance. Host
effects which lead to suppressivity have been ascribed to continuous monocrop-
ping leading to: disease decline, cover cropping, compost application, tillage and
rotation. Adding organic amendments such as manure, compost and cover crops
especially when combined with biocontrol agents such as Trichoderma or
Gliocladium, limit pathogenesis as seen with reductions of sheath blight in rice
(Sreenivasaprajad and Johnson 2001). But the position is far from clear as for
example, where minimum or nil-tillage are associated with reduced pathogen
impact in some instances while in others crop damage increases. This lack of clarity
is hardly surprising since there is little evidence with which to understand the differ-
ence between pathogenic and antagonisitic microbes and comprehend the factors
that engender either pathogenicity towards crop plants or antagonism towards the
pathogens. Stukenbrock and McDonald (2008) suggested that pathogens emerge as
a result of cultivation where a homogeneous host population and a conducive envi-
ronment coincide. If this is accepted then it may be reasonable to assert that the
contrary state of suppressiveness also results from the appropriate combination of
microbial populations and soil environmental factors. Understanding the develop-
ment of suppressive soils may be obtained from firstly following the development
of suppressiveness over time, secondly examining naturally occurring suppressive
soils and thirdly using soil treatments which encourage suppressiveness (Borneman
and Becker 2007).
The horizontal and vertical distribution of Aphanomyces spp. causing root rot
disease of field peas is reported by Moussart et al. (2009) and provides clues in under-
standing the distribution patterns of pathogen propagules and the onset of disease.
Measurements of inoculum potential indicated a horizontal distribution of propagules
among several foci in the field. These foci differed in size and disease intensity.
A significant relationship was established between disease severity on maturing pea
plants and total soil inoculum potential. In the vertical distribution of inoculum, maxi-
mum values were found at 10–40 cm but the pathogen could be detected to a depth
of 60 cm. Inoculum potential was least at depths of 0–10 and 50–60 cm. It appears in
this case that the top soil horizon and the lowest one possessed features which vitiated
against the pathogen. Sandwiched between these horizons the pathogen had an
advantage. Similarly the development of arbuscular mycorrhizal fungi and their
inoculum potential in soil varied with different hosts for colonisation such as on prai-
rie little bluestem grass and wild rye. Total soil nitrogen correlated with host root
mass and negatively with the root colonisation (Anderson 2008). Stimulating vigor-
ous host root growth apparently discouraged the pathogen. In another example inter-
cropping apparently aided the inoculum potential of beneficial microbes such
arbuscular mycorrhizas in tropical agroforestry with trees such as Albizzia procera
(safed siris), Ambilica officinalis (aonla) and Eucalyptus teritciornis using black gram
and wheat or green gram and mustard (Kumar et al. 2007). Knowledge of inoculum
concentration is equally important as that for the environment. Thus studies of soil
inoculum densities and Verticillium wilt (V. dahliae) in Acer platanoides and Catalpha
bignonioides indicated that 5% diseased plants occurred at 1–2 microsclerotia per

gram of soil (Gould 2003). Bio-assay techniques used to establish concentrations of
propagules of pathogens in soil have tended to remain empirical and lack analytical
precision. Recently, however, improvements have been achieved using enzyme-based
and PCR-based systems as reviewed by Faggian and Strelkov (2009) for P. brassicae,
the cause of clubroot in brassicas. Methods for the bioassay of soil suppressiveness
to Fusarium wilts, evaluating inoculum potential and the impact of soil fungistasis are
reviewed by Alabouvette et al. (2005).
Disease management may be either largely empirically driven by experience and
practice or it may designed around an epidemiological framework in which the
mechanisms responsible for an epidemic are identified and the knowledge gained is
used to target, improve and deploy methods which reduce yield losses (Gilligan
2002). This requires an understanding of the processes controlling the invasion and
persistence of inoculum and subsequent disease development and expansion. The
spatial structure of susceptible crops affects the spread of diseases across a land-
scape. When susceptible crops are rotated the connectivity between contiguous
fields of susceptible crops changes and transmission between neighbouring fields
alters. Spread by soil-borne fungi such as R. solani is classed by Bailey et al. (2000)
as invasive or non-invasive (finite) when moving from one site to another, as studied
using the colonisation of poppy seeds from agar plugs as a model system. Invasive
spread did not depend on the furthest extent of mycelia growth evident in the tails of
colonisation, but was instead associated with threshold distances which were differ-
ent for low and high nutrient sites. As the probability of colonisation increases above
a threshold value so the likelihood of invasive spread increased. Conversely, below
that threshold finite growth was more common. In nature the colony size, nutritional
state and availability of organic matter are likely to be very variable. Small differ-
ences in soil physical properties, in particular the continuity of air-filled pore space
will have a profound effect on the growth and expansion of a single propagule or
from a colony concentrated round a nutrient source. The colonisation profile is
affected by soil physical properties and this character is analogous to the pathozone
profile for the growth or movement of fungal parasites during primary and secondary
infection. In primary infection the fungus grows saprophytically from a source of
previously colonised host material towards a susceptible host. The analogies between
parasitic and saprotrophic invasion can be extended by considering saprotrophy by
soil microbes as a special case in the general epidemic processes in which colonisa-
tion replaces infection. From this there can be a general interpretation of the dynam-
ics and spread of pathogens and saprophytes, parasites spread horizontally through
a host population (such as roots) and saprophytes move through a discrete series of
nutrient sites. Unlike saprophytes the parasitic fungus could experience a switch
from finite to invasive growth. At low root densities the pathogen is constrained by
the availability of host tissue as this changes there is a switch to rapid invasive
spread. This could explain phenomena such as sudden increases in the rate of infec-
tion during the spread of take-all (G. graminis var. tritici) on wheat as the epidemic
switches from primary to secondary infection in response to changes in the density
of roots (Bailey and Gilligan 1999).
Entry and Colonisation
Root colonisation is an important first stage in infection by pathogens. Early

research by Hiltner in 1904 (Hiltner 1904) suggested the “rhizosphere effect”
whereby microbes are attracted to nutrient supplies exuded by roots. This has been
reviewed by Bais et al. (2006) who contend that in addition to providing a carbon-
rich environment the roots initiate cross-talk signalling with soil microbes which in
turn produce messages initiating colonisation. Motility is an important trait of both
pathogens and beneficial microbes enabling participation in this cross-talk (De
Weert et al. 2002; Lugtenberg et al. 2001). Chemical attraction (chemotaxis) is
involved in cross-talk between microbes and root (Bais et al. 2004a). Electrical
potentials resulting from electrogenic ion transport at the root surface, attracts
swimming zoospores of oomycete organisms to the root surface (van West et al.
2002). Some rhizobacteria create suppressive soils which inhibit the activities of
plant pathogenic microbes. Mechanisms for this form of biocontrol include
competition for nutrients as supplied in root exudates, niche exclusion, induced
host systemic resistance (ISR) and the production of antifungal metabolites. The
best characterised biocontrol agents at the molecular level are species of
Pseudomonas which form antifungal metabolites such as phenazines, pyrrolnitrin,
2,4-diacetylphloroglucinol (DAPG), pyoluteorin and several cyclic lipopeptides
(as discussed elsewhere in this book). Stimulation of acquired resistance to cucum-
ber mosaic virus (CMV) by a strain of Bacillus pumilis in Arabidopsis thaliana
resulted from adapting a signalling pathway for virus protection (Ryu et al. 2004).
Bacillus subtilis strains, on the other hand, assist plants to avoid the Gram-negative
pathogen Pseudomonas syringae pv. tomato by forming a protective biofilm on the
roots of A. thaliana and producing an antimicrobial cyclic lipopeptide, surfactin
(Bais et al. 2004b). Root exudates substantially increase microbial activity in the
rhizosphere (Ohi et al. 2003). Their role in pathogenesis caused by root invading
bacteria and fungi has yet to be fully appreciated. This deficiency is the result of
inadequate technology in the methods of analysis. It is however, recognised that
there is constant “underground warfare” alleviated for host plants by production of
phytoalexins and defense proteins. Sequencing the genomes of crop plants reveals
that they are rich sources of antimicrobial indole, terpenoid, benzioxazinone and
flavanoid / isoflavanoid natural products. This chemical diversity could yield direct
forms of sustainable control of pathogenic microbes and/or indicate indirect means
via the stimulation of their secretion (Bais et al. 2006).
The rhizosheath is the soil adhering directly to the roots and the rhizosphere is
the zone of soil directly under the influence of the roots, the pathozone is the region
of soil surrounding a root, seed or hypocotyls within which fungal propagules must
exist if they are to have any chance of infecting the host (Gilligan 1990). The ability
of a fungus to invade was not determined by the furthest extent of fungal growth
but instead by the distribution and density of fungal biomass as captured by the
pathozone (Otten and Gilligan 2006). The invasiveness of fungal spread can be
predicted from fungal colony morphology together with the geometry of the root or
host population, and small changes in fungal morphology or the distribution of

susceptible hosts (both of which can be mediated by soil physical conditions) and
can make a fungus switch between invasive and non-invasive spread (Otten
and Gilligan 2006). The evolution of infection efficiency (Gilligan 1985; Gilligan
and Bailey 1997) is summarised by a non-linear model for the changing probability
of infection with distance of inoculum of, for example, R. solani from a host in
which certain parameters vary with time. Thus the probability of infection P(r, t),
depends on distance (r) and time (t):
P(r, t) = f(r θ (t))
where q (t) are the time varying parameters.

The rhizosphere is the region of soil influenced most by the presence of roots
(Lynch 1990). The pathozone forms the central environment where propagules of
plant pathogens must lie if they are to have finite chances of infecting host roots
(Gilligan 1985). Both pathozone and rhizosphere are closely related. Dimensions
of the pathozone are dictated principally by the properties of the root and sur-
rounding soil. The major components responsible for the size and shape of the
pathozone also depend on the properties of the inoculum (Gilligan and Bailey
1997). The pathozone is characterised not only by the furthest extent from which
infection can occur but also by a decrease in the probability of infection as the
distance between inoculum and host increases (Gilligan 1985). The probability of
infection equates with infection efficiency relating the spatial distribution of
propagules and roots. The pathozone changes with time as inoculum germinates
and grows or moves towards the host, within this the susceptibility of the host may
also change. These dynamics affect the rate of spread of an epidemic from an
initial inoculum source and could also affect, and be affected by, the presence of
propagules of biocontrol organisms such as T. viride. Bailey and Gilligan (1997)
showed that this biological control organism reduced the extent of pathozone
influence and the infection efficiency of R. solani infecting radish seedlings. From
this relationship the progress of disease could be predicted in a population of
hosts. There are two interacting processes involving the density of mycelium that
reaches the host and the net receptiveness of the host surface which increases
during the period of inoculum challenge (Gilligan and Bailey 1997). High myce-
lial density may be affected by mechanical disturbance arising from host germina-
tion and root growth.
Invasion and persistence by pathogen in host populations are defined by Gilligan
and van den Bosch (2008) in straightforward terms. Invasion they identify as “the
introduction and subsequent increase of the pathogen in a host population” while per-
sistence requires the host population to contain sufficient susceptible individuals such
that pathogenesis continues for a prolonged period. Modifying these characteristics by
forms of control limits the scale and extent of pathogen epidemics. The form of an
epidemic results from the integration of the factors: inoculum, host, environment and
time, environment includes aerial and edaphic meteorological factors and those of the
soil itself such as pH, texture, bulk density and chemistry. The choice then falls to
Population size (Log10 cfu root system-1)

4
0
0 2 4 6 8 10 12 14
−1
Time from sowing (days)
Fig. 5 Fluorescent pseudomonad colonization of winter wheat (cv. Hereward) seedlings grown in
mature greenwaste compost artificially infected (closed symbols) or uninfected (open symbols)
with Gaeumannomyces graminis var. tritici. (After E.L. Tilston, 2000)
which forms of control will yield the most sustainable result in terms of the environment
and crop production. The transition from inoculum to epidemic is driven by fungal
(pathogen) growth, the infection dynamics of a single host and invasive spread of the
disease-causing microbe through a population of hosts (Otten and Gilligan 2006). Like
soil, the rhizosphere and more specifically the rhizoplane, has microsites where the
microbial residents are concentrated. These microsites are often a function of the
architectural complexity of roots and provide an array of potential niches for antago-
nists (Burdon 1987). The number of microsites may limit the size of rhizosphere popu-
lations and account for the colonisation plateau observed in Fig. 5.
Pathogenesis
The invasion and disruption of root tissues following infection of a root system by a
pathogen has a profound effect on the efficiency of the supply of water, ions and
growth regulatory substances and on the rate of root growth (Ayres 1984). The position
and depth of lesions on wheat roots dictate the emergent physiological effects (Fitt and
Hornby 1978). More severe take-all (G. graminis var. tritici) for example, may develop
following infection of newly formed, undifferentiated tissues than after infection of

differentiated tissues where subsequent vascular colonization can by limited by the
presence of an endodermis (Deacon and Henry 1980), particularly if the lesions are
close to the shoot (Ayres 1984). Penetration to the stele effectively excises the distal
portions of roots (Manners and Myers 1981), though this may not necessarily translate
into reduced growth rates (Andrews and Newman 1968) as there is a state of functional
equilibrium in the partition of resources between the roots and the shoot (Brouwer
1977). Low levels of infection can stimulate production of nodal roots (and account
for increases in root-to-shoot ratios), with the result of at least off-setting and possibly
over-compensating for those lost due to penetrating infections (Asher 1972), though
the reverse may also be true (Hornby et al. 1998). The regenerative capacity depends
on the growth stage (Seidel et al. 1981) and of agricultural cereals, barley has greater
regenerative capacity than wheat and this is the reason why wheat is affected more
severely by take-all (G. graminis var. tritici) than barley (Manners and Myers 1981).
Cereal plants are most vulnerable to infection and it is most severe in the early season,
especially where there are high levels of soluble nitrogen available in the soil and a
high population density (Papendick and Cook 1974). Initially crop growth is vigorous
so there is increased rate of water uptake and its rapid depletion consequently water-
stress takes effect earlier, predisposing the plants to attack by G. graminis var. tritici.
The low water content restricts the activities of other microflora, especially bacteria so
levels of antagonism by Pseudomonas spp., for example, are often low. In addition, the
area of the root–soil interface will decrease as the dehydrating roots shrinks away from
the soil particles as its volume decreases (Weatherly 1979). Disease incidences arising
from primary infection (soil-borne inoculum, auto-infection) and secondary infection
(root-to-root passage, allo-infection) are distinguished by Suffert (2007) in modelling
Pythium violae (a cause of cavity spot in carrot) resulting in the confirmation that this
disease syndrome is polycyclic.
Necrotic host tissues, such as diseased roots, are known to support a diverse array
of saprophytic organisms and fluorescent pseudomonads often colonize and domi-
nate in the community arising in take-all (G. graminis var. tritici) lesions (Wilkinson
et al. 1982; Barnett et al. 1999). Within colonized G. graminis lesions, it is suggested
that antagonists prevent pathogen outgrowth from the lesion and limit subsequent
autoinfection (Baker and Paulitz 1996) and infection by other pathogens resulting in
further tissue destruction (Leath et al. 1989). The bacteria present within the rhizo-
sphere are less fastidious with their requirements for pre-formed organic compounds,
vitamins and growth factors than those present in non-rhizosphere soil (Brown
1975). The quantitative and qualitative composition of sugars, organic acid, amino
acids, glycosides, nucleotides and their bases, enzymes, vitamins, indole-derivatives,
phenolic substances and other substances within root exudates changes during plant
growth and root development, and in response to foliar applications of chemicals
(Macura 1968) and environmental and climatic factors (Brown 1975). With increasing
root age cell debris augments the diversity of nutrients within the rhizosphere. The
amount of organic material released by roots can be considerable with 1–2% of
the carbon translocated to the roots being released into the soil as water-soluble
exudate (0.2–0.4%) and 0.8–1.6% as insoluble mucilaginous material (Brown 1975).
If the substances actively secreted by roots, the lysates of root material and gases
released from the roots, referred to collectively as rhizodeposits (Whipps 1990) are
included in such calculations then 12–40% of photosynthate enters the soil in this
manner (Whipps and Lynch 1985). As well as stimulating antagonistic organisms and
other metabolic types of bacteria such as carbohydrate fermenters, cellulose decom-
posers, ammonifiers and denitrifiers; pathogens are attracted towards and stimulated
by root exudates (Burdon and Shattock 1980).
The rhizosphere therefore, presents a suitable microhabitat in which events relat-
ing to disease suppression could occur (Troxler et al. 1997). In suppressive soils, the
zone of suppression around roots extends for up to 1.5 mm from the rhizoplane
(Wildermuth et al. 1985), but the rhizosphere can influence soil up to 2 cm from the
rhizoplane (Brown 1975). For rhizosphere organisms to effect disease suppression,
they must be in place before arrival of the pathogen within the infection court
(Campbell 1989a; Harris et al. 1997). Like roots, germinating seeds exude a rich sup-
ply of nutrients, which also support a diverse microflora (Brown 1975). Microorganisms
of the spermatosphere originate from the testa, and the wider soil, and have the poten-
tial to participate in root colonization. A few bacteria originating from the testa
migrate to and persist within the rhizosphere where colonists are drawn from often
dormant propagules in the soil (Macura 1968). The difference between spermato-
sphere and rhizosphere microbial communities probably being determined by quan-
titative and qualitative differences in the exudates from each organ and the poor
persistence and unimpressive in vivo antagonistic ability probably means that the
contribution to suppression by the spermatosphere community is minor.
In field-grown wheat the seminal roots harbour small, sparse populations of
bacteria located mainly at the junctions of cortical cells. Whereas in old nodal roots
large aggregates of bacteria colonize the troughs created by the extensive longitu-
dinal corrugations and intercellular spaces in the cortex (Darbyshire and Greaves
1971) and may even be found in the xylem (Troxler et al. 1997). The colonization
of furrows on roots has been attributed to increased exudation at cell junctions but
these sites may also offer protection against desiccation and dislocation due to abra-
sion (Mosse 1975). Generally nodal roots exert a greater rhizosphere effect on
antagonistic microorganisms and support larger populations than seminal roots
(Sivasithamparam and Parker 1979b). The difference between nodal and seminal
roots may have a profound influence on the prospects of biological control of take-
all (G. graminis var. tritici) in Britain; where prolonged protection of the nodal root
system of winter wheat during the spring and early summer may be of greater
importance than protection of seminal roots (Hornby et al. 1998).
To suppress, displace or pre-empt a pathogen in the invasion of an host the antago-
nist must be a specialized and aggressive colonist of host tissue. Furthermore an
antagonist must be able to use most of the nutrients needed by the pathogen and
preferably the antagonist should have some means of inhibiting the growth of the
pathogen. Pathogens and their antagonists inhabit ecological niches with a high
degree of equivalence and the only way in which they differ is that antagonists lack
the ability to initiate the flow and transport of food out from the host. Biocontrol agents
can inhibit the growth of pathogens by the following modes of action: pre-emptive
competitive exclusion, antagonism by antibiosis, interference, parasitism or predation,

induction of host resistance and transmission of hypovirulence genes (see else-
where in this book).
Characterising Sustainability Through Suppressive Soils
Much has already been said in this chapter about the occurrence of suppressive soils.
The key objective in developing microbially sustainable soils is the enhancement of
pathogen suppressivity. A healthy soil is defined by van Bruggen and Termorshuizen
(2003) as a stable soil system with high levels of biological diversity and activity,
internal nutrient cycling and resilience to disturbance. This implies these authors
suggest that microbial fluctuations after a disturbance would dampen more quickly in
a healthy than in a chronically damaged and biologically impoverished soil. Soil may
be disturbed by for example, adding a nutrient source, tillage or drying and rewetting.
Disturbance results in the numbers of heterotrophicbacteria and of individual species
starting to oscillate both in time and space. Oscillations appear as moving waves along
the path of an active nutrient source such as a root tip. The phase and period for differ-
ent trophic groups and species of bacteria may be shifted indicating that oscillation
occurs. Analyses of the subsequent populations in oscillations confirm that there is a
cyclic succession in microbial communities. Microbial diversity oscillates in the oppo-
site direction to microbial population size. In healthy soil the amplitude of these oscil-
lations will be small but the background levels of microbial diversity and activity are
high so that soil-borne diseases will face more competitors and antagonists. Soil-borne
pathogens and antagonists alike will fluctuate in time and space as a result of growing
plant roots and other disturbances and the periods and phases of the oscillations may
vary. As a result biological control by members of a single trophic group or species
may never be complete as pathogens will encounter varying populations of the antago-
nistic agent at the root surface. A mixture of different trophic groups occurs at subse-
quent locations along a root. Alternatively regular addition of organic matter to the soil
may increase background levels of microbial activity, increase nutrient cycling, lower
the concentrations of easily available nutrient sources, increase microbial diversity and
enhance natural disease suppression.
The antithesis of avoiding land conducive to disease is the intentional use of
suppressive soils. In suppressive soils disease incidence remains low, despite
opportunities in which the pathogen could have established an inoculum potential
of economic importance, under favourable climatic conditions and in the presence
of a susceptible host. Suppressive soils have been identified for T. yallundae (Baker
1970), R. solani of cereals (Wiseman et al. 1996), G. graminis var. tritici (Cook and
Rovira 1976), P. brassicae (Worku and Gerhardson 1996) and Fusarium wilt of
melon (Alabouvette 1986). Not all suppressive soils are pathogen specific; soil
from a wheat field at Roseworthy Agricultural College in southern Australia was
suppressive to cereal pathogens other than G. graminis var. tritici and included
R. solani, B. sorokiniana and to a lesser degree F. culmorum (Wildermuth 1982).
The presence of an active community of antagonistic microflora and microfauna is

usually proposed as the raison d’être for suppressive soils, and microbial isolates
taken from these soils are often used for further research, e.g. Pseudomonas fluore-
scens strain CHAq (Steffek et al. 2006).
Disease suppression is inhibition of the pathogen when it is growing parasitically
in or on the host (Alabouvette et al., 1996) while pathogen suppression is inhibition
of the pathogen in its saprotrophic growth and, or survival phase(s) in the soil (Cook
and Baker 1983; Alabouvette et al., 1996). Gaeumannomyces graminis var. tritici
has four types of saprotrophic existence within soil viz.: (Pretty 1990) survival on
crop residues, (Houghton 1996) growth in soil, (Britton 1990) colonization of
organic residues and ectotrophic growth on the host root preceding infection (Simon
and Sivasithamparam 1989). It is difficult to measure true pathogen suppression,
particularly for G. graminis var. tritici where spores probably do not perform an
active role in the field-based infection process and because of the absence of a satis
factory selective isolation medium for use with soil; although immunological or
molecular methods may eventually overcome this.
Pathogen suppression may be specific or general. Specific suppression is that
which develops only in the presence of a virulent pathogen (Gerlagh 1968), or is due
to the activities of specific groups or populations of antagonists. The definition of
specific pathogen suppression may be refined further by the inclusion of a clause
relating to transferable suppression. Transferable suppression is that which can be
moved to a conducive medium within a small amount (1% w/w) of test soil. This
implies that the active factor is biological in nature and the amount of soil introduced
is too small to change the nutritional status of the conducive medium (Shipton et al.
1973) and can provide a confirmatory test for a suppressive soil. Greater amounts of
soil have been used to demonstrate transferable suppression, but there is the risk of
obtaining an erroneous result if the nutritional status of the conducive medium is less
than that of test soil (Simon and Sivasithamparam 1989). A general form of patho-
gen suppression arises from the activity of unspecified (generalised) soil microbiota
which can be manipulated by practices such as crop rotation and cultivation (Gerlagh
1968). General suppression is not dependent upon characterized antagonistic abili-
ties but operates by virtue of wider activities such as spatial exclusion. Wider micro-
bial communities have effects that contribute to the phenomenon of general
suppression and the two mechanisms (specific and general) can be complementary
rather than opposed, e.g. the Fusarium wilt-suppressive soils of France which have
been described as showing both general and specific suppression (Alabouvette
1986). Pathogen suppression and disease suppression, however, are not complemen-
tary within ecological niches. Simon and Sivasithamparam (1989) define pathogen
suppression as that occurring outside the rhizosphere, while disease suppression
occurs within the rhizosphere. For microbially-based disease suppression it is impor-
tant that the antagonistic organism is present within the rhizosphere before arrival
of the pathogen. Gaeumannomyces graminis var. tritici has runner (ectotrophic)
hyphae that colonize the root surface and later penetrate the root growing within the
intracellular spaces of the cortex and stele interface, where they could be protected
from the antagonistic activities of other microorganisms.
Naturally occurring disease-suppressive soils have been documented in a variety

of cropping systems, and in many instances the biological attributes contributing to
suppressiveness have been identified. While these studies have often yielded an
understanding of operative mechanisms leading to the suppressive state, significant
difficulty has been encountered in the transfer of this knowledge into achieving
effective field-level disease control. Early efforts focused on the inundative applica-
tion of individual or mixtures of microbial strains recovered from these systems and
known to function in specific soil suppressiveness. The introduction of biological
agents into non-native soil ecosystems however, typically yielded inconsistent
levels of disease control (Mazzola 2007). Of late, greater emphasis has been placed
on manipulation of the cropping system to manage resident beneficial rhizosphere
microorganisms as a means for suppressing soil-borne plant pathogens. One such
strategy is the cropping of specific plant species or genotypes or the application of
soil amendments with the goal of selectively enhancing disease-suppressive
rhizobacteria communities. This approach has been utilized in a system attempting
to employ biological elements resident to orchard ecosystems as a means to control
the biologically complex phenomenon termed apple replant disease. Control of the
fungal pathogen R. solani AG-5 was achieved by cropping with wheat in apple
orchard soils prior to re-planting the site with apple cultivars. Disease control was
elicited in a wheat cultivar-specific manner and functioned through transformation
of the fluorescent pseudomonad population colonizing the rhizosphere of apple.
Wheat cultivars that induced disease suppression enhanced populations of specific
fluorescent pseudomonad genotypes with antagonistic activity toward R. solani
AG-5, but cultivars that did not elicit a disease-suppressive soil did not modify the
antagonistic capacity of this bacterial community.
It has been suggested that fluorescent pseudomonads are unlikely to make sig-
nificant contributions to the suppression of soil-borne pathogens (Sivasithamparam
et al. 1979a; Tuitert et al. 1998). Both groups of authors justify this view with
evidence of low population densities of fluorescent pseudomonads within soil and
compost. Sivasithamparam et al. (Sivasithamparam et al. 1979a) do acknowledge
however, that at certain microsites there may be intense antagonistic activity by
fluorescent pseudomonads. These microsites are highly dispersed in soil and it has
been suggested that the inhabiting colonies of soil bacteria are often small, com-
prising of fewer than ten cells (Gray et al. 1968). Antibiotics are organic com-
pounds of low molecular weight, produced by microbes and at low concentrations
have deleterious effects on growth and, or other metabolic activities of other
microorganisms (Fravel 1988) and may be the cause of the inhibition zones. Under
in vitro conditions fluorescent pseudomonads have been shown to produce a range
of phenolic polypeptide antibiotics, e.g. pyoluteorin and pyrrolnitrin, phlorogluci-
nols, e.g. 2,4-diacetylphloroglucinol and monoacetylphloroglucinol (Howell and
Stipanovic 1980), and phenazines, e.g. phenazine-1-carboxylic acid and 2-hydroxy-
phenazine-1-carboxylic acid (Thomashow and Weller 1988). The ability to synthe-
size antibiotics does not ensure efficacy as a biocontrol agent. Non-antibiotic
producing mutants may exert suppression equal to that of their wild type (Fravel
1988); suggesting that general environmental competence and, or other activities
may be limiting or augmentative (Campbell 1989b; Ryder and Rovira 1993).

Exposure to ecological stresses is probably necessary for the conservation of anti-
biotic production, as isolates maintained on culture media of high nutrient value
often lose the ability to produce antibiotics over time (Bruehl et al. 1969). The
production and activity of antibiotics in soil has been subject to debate for many
years, with evidence that antibiotics are frequently bound to clay and organic mat-
ter, which reduces their effective concentrations. But in turn this may serve to
increase the concentration within microsites (Fravel 1988). With newer techniques
evidence is emerging that antibiotic molecules such as, phloroglucinol are pro-
duced in vivo by rhizosphere colonizing fluorescent pseudomonads and that they
do have activity against pathogens such as G. graminis in the field (Raaijmakers
et al. 1999).
The lack of continuation of the suppression is due to poor longer-term persistence
of antagonistic microbes and the failure for the initial protection to establish a
permanent presence under less suitable physico-chemical conditions (Capper and
Higgins 1993). Limiting physico-chemical conditions could arise in the summer
when soil water potential declines and restricts the growth of antagonists;
G. graminis var. tritici is able to grow over a wide range of water potentials and
would therefore dominate (Campbell and Clor 1985). The problems of known bio-
logical control agents namely longevity of suppression and of applying sufficient
inoculum are common for all externally applied agents.
Forms of crop husbandry such as the addition of soil amendments have a clear
capacity to enhance disease suppression. This may as Mazzola (2004) notes differ
from the manner by which suppressivity operates in natural soils. Crop plants
themselves will affect the forms of suppressivity by altering the characteristics,
activity and composition of the soil microbial community. Opportunities for com-
parative studies of microbial communities have increased significantly with the
advent of molecular diagnostic and analytical tools (Rondon et al. 1999). All
soils, with the possible exception of the most badly degraded, have some capacity
to suppress pathogenic microbes as identified by the rapidity and intensity of
disease development in plants exposed to them in sterilised and non-sterile states.
Such simple comparisons identify the presence of generalised suppressivity in
soils resulting from their natural microbial soil flora. Raising this status from
generalised to specific and obtaining the suppression of particular pathogens
requires the development of viable disease management strategies (van Elsas
et al. 2002) which integrate a panoply of agronomic, biologic, genetic and chemi-
cal techniques.
It should be apparent that soil management practices can affect the population
dynamics of soil microbial communities. Cultural practices can be adequately
combined to benefit natural populations of microorganisms that may have a role in
biological control (Trichoderma spp., and Gliocladium spp.), thus for example
contributing to the management of peanut fungal soil-borne diseases in a sustainable
manner within ecological boundaries (Vargas et al. 2008). During six agricultural
cycles, rhizosphere soil samples were taken from a field subjected to crop rotation
(soybean, peanut, and maize), peanut being under two tillage systems (no till,
reduced tillage) with the aim of quantifying populations of soil microorganisms.

The incidence of diseases caused by soil-borne fungi in peanut was determined at
harvest. The highest amount of Trichoderma spp., and Gliocladium spp. were
recorded when maize was the preceding crop. Regarding tillage systems, the popu-
lations of these groups of microorganisms were higher in peanut under no tillage
than under reduced tillage. Under these conditions, the lowest incidence of peanut
blight (Sclerotinia minor) and root rot (strains of F. solani) was observed, suggesting
a possible natural control of peanut soil-borne pathogens. The quantification of
Trichoderma spp., and Gliocladium spp. was used as a tool to explore the impacts
of different management systems on microbial groups that may be involved in the
biological control of soil-borne diseases, with the aim of combining those practices
that improve native populations of possible beneficial microorganisms. This manip-
ulation can provide sustainable management strategies in the control of soil-borne
diseases, avoiding the use of artificial inoculations of microorganisms, and reducing
agrochemical application.
Integrative Pathogen Management
Plant pathologists have traditionally viewed soil as a hostile environment, harbour-

ing microbial opponents that have adverse effects on plant health (Raaijmakers et al.,
2010). Increasing the efficiency of crop disease control is an essential prerequisite
towards the provision of adequate food supplies for the world’s burgeoning popula-
tion. Currently, at least 15–20% of all crops are lost to the activities of pathogens
either in the field or during preparation, processing and presentation to the ultimate
consumer. Unfortunately, conventional agriculture has had major environmental
impacts, in particular with respect to soil degradation. Soil structure, fertility,
microbial and faunal biodiversity have declined, and root diseases are common
unless genetic resistance, soil fumigation and, or seed treatments are used (van
Bruggen and Termorshuizen 2003). Primarily for environmental reasons and
increasing demands for safe and healthy food from the public, farmers must switch
to more sustainable systems. This chapter has identified from the perspectives of
crop husbandry and pathogen ecology means by which diseases can be diminished
and yields increased sustainably. Essentially this uses the somewhat wider view
adopted by those microbiologists who approach the soil ecologically and see as it
populated by a mixture of microbes fitted to a variety of niches with some even
capable of exploiting more than one lifestyle. Approaching disease control with this
vantage point accepts the emerging concept that sees the need to increase soil qual-
ity and health and approaches pathogens as components of the total soil biological
diversity. Disease is seen as resulting from a disturbance to the balance between
functional groups in soil. An experimental platform for this concept is beginning to
emerge. Molecular methods based on polymerase chain reaction (PCR) of DNA
extracted from soil allow rapid assessment of genetic diversity and increasingly will
be used to measure functional diversity as well taxonomic placing. This enables the
relationships between diversity and disease suppression to be characterised.

Suppressiveness of soils to pathogenic organisms is biological in nature, although
modified by abiotic factors and is of two types. General suppression depends on
overall diversity and activity of the soil biota and acts against a broad range of
pathogens. Specific suppression is due to particular antagonists or functional
groups and acts against single pathogens. Studies on the effects of management
practices on pathogen suppression are still limited in scope, and are often difficult
to interpret because most practices have direct effects on pathogen populations as
well as on suppressiveness. Continuous cropping of a plant species selects for
microflora adapted to its rhizosphere, which may suppress the activities of some
pathogens while increasing those of others. Rotations and reduced tillage should
increase microbial diversity and increase suppressiveness but evidence for these
causal links is hard to find. Treatments that increase soil organic matter such as,
residue retention and application of manure or compost may increase general sup-
pression, and certain types of manure may also increase specific suppression.
Soil biodiversity, pathogen suppression and management practices are yet to be
fully synthesised into models that have general applicability. This means that man-
agement thresholds remain speculative in most instances (Alabouvette et al. 2004).
Soil agroecosystems can be modified through rotation, conservation tillage, nutri-
tional management and other agronomic measures to improve disease suppression
by enhancing the antibiosis abilities of endophytic and root zone bacteria (Peters
et al. 2003). Recognition is emerging that soil suppressiveness is a quantitative
property which may be enhanced by agricultural practice as equally it may be
destroyed by adverse husbandry systems. Sustainable control of soil-borne patho-
gens demands an integrated approach in which husbandry forms the foundation of
a systematic approach over which other factors are added. The extent to which this
pyramid is developed is moderated by the inoculum potential of the soil-borne
pathogen(s) and the capacity of benign (beneficial) microbes to exert natural
mechanisms of control. Integration is becoming the key to sustainable pathogen
control. Sieling et al. (2007) report on extensive studies of wheat grown continu-
ously and in rotations with break crops such as oil seed rape, nitrogen fertilisation
and the use of seed treatment with the chemical fluquinconazole for control of take-
all (G. graminis var. tritici). The preceding crop whether it is wheat or a rotational
component change the inoculum density of G. graminis var. tritici and soil struc-
ture, weed infestation and population composition, soil nutrient status and the
amount and composition of crop residues returning to the soil. This is the type of
multifactorial analysis which is required if reliable integrated systems are to be
made financially attractive to farmers.
Undoubtedly the intensification of agricultural practices will continue driven by
diminishing land available for cropping and an almost exponential increase in
demand for food. This, as experience of the last century of agricultural expansion,
has demonstrated encourages pests and pathogens to thrive. It is now understood
that soil cultivation techniques interact with species antagonistic to crops in differ-
ent ways and hence the intensity of tillage adopted (from full inversion to no-till)
may result in greater or lesser pest or pathogen pressure depending upon the
o rganisms involved (Leake 2003). Selecting one husbandry factor for change such
as tillage alone cannot provide the solution. The requirement is for integration of
the full range of methods including cultural, biological, genetical, mechanical and
where appropriate chemical methods.
The objectives of research must be to evaluate the impact of organic, sustainable,
and conventional management strategies in growers’ fields on soil physical, chemi-
cal, and biological factors including soil microbial species and functional diversity
and their effect on plant pathogens. This type of approach has been adopted with
Sclerotium rolfsii, causal agent of Southern blight in the USA. Soils from ten field
locations including conventional, organic and sustainable farms were sampled and
assayed for disease suppressiveness in glasshouse assays, and for soil quality indi-
cators. Soils from organic and sustainable farms were more suppressive to Southern
blight than soils from conventional farms (Liu et al. 2007). Soils from organic farms
had improved soil chemical factors and higher levels of extractable and microbial
biomass carbon and nitrogen and net mineralizable nitrogen. In addition, soil
microbial respiration was higher in soils from organic than sustainable or conven-
tional farms, indicating that microbial activity was greater in these soils. Populations
of fungi and thermophiles were significantly higher in soils from organic and sus-
tainable than conventional fields. The diversity of bacterial functional communities
was also greater in soils from organic farms, while species diversity was similar.
Soils from organic and sustainable farms had improved soil health as indicated by
a number of soil physical, chemical and biological factors and reduced disease.
A clear example of the manner by which cropping systems have a substantial
effect on the microbial competence of soils has been shown by Alvarez-Solis and
Anzuetto-Martinez (2004). The effects of agricultural intensification on the num-
bers of microbes, microbial respiration, organic carbon mineralization, mycor-
rhizal inoculum potential and biological nitrogen fixation and their relationships
with soil fertility were assessed in Mexican maize cropping systems using long-
fallow, short-fallow, pasture-cultivation rotations and annual continuous cropping.
Microbial respiration was 38% lower in plots with corn under annual continuous
cultivation or pasture-cultivation rotation than in long or short fallow, irrespective
of their condition in cultivation (corn) or fallow (tree and shrub vegetation).
Organic carbon mineralization was 31% and 43% lower in plots with corn under
annual continuous cultivation and pasture cultivation rotation than in cultivated
plots of long and short fallow, respectively. Mycorrhizal colonization of corn was
3.1 times higher in grassland than in cultivated soils with long fallow. The decrease
of microbial respiration was related to the decline of soil organic reserves, the
increase in soil acidity and decrease of basic cations. These results indicate the
importance of the periodic addition of organic and mineral amendments that
should return the soil organic reserves and basic cationic reserves to improve het-
erotrophic microbial activity in these acid soils with intensive agricultural use.
Also results suggest the importance of grassland to maintain mycorrhizal inocu-
lum potential in soils.
Currently, since the adoption of organic systems correlates with substantially
reduced yields the prime demand is for research to develop means for utilising
its environmental advantages while increasing productivity. Sustainable control

changes the environment that is encountered by the pathogen either inside or outside
the host, thereby providing conditions conducive to host growth but adverse to
those of the pathogen. Some microbes are antagonistic to the growth of patho-
gens while others enhance the growth of crops by benign activities. Antagonism
between microbes utilizes factors such as competition, antibiosis and antagonisms.
Relatively recently it has been realised that biological control, for example, is most
effective when mixed populations of microbes are used by comparison with the
previously prevailing approach of using single strains taken from a specific species.
Breeding for genetic resistance is probably the most sustainable form of disease
control. It aims to produce plants which are either unaffected by or withstand the
pathogen per se. Developing new higher yielding and pathogen resistant cultivars is
one of the major factors whereby crop yields can be raised to match rising human
demand. The requirement now is not only to produce pathogen resistant cultivars
but to understand the ecological implications of their use on soil microbial
ecosystems. Chemical control which prevents either invasion, colonisation of spo-
rulation of a microbe by destroying the pathogen either immediately before or after
penetration into the host still has a continuing role to play. The agrochemical indus-
try has matched society’s demands for environmental care with the continuing need
for increased production by designing molecules which possess greater efficiency
in their modes of action, enhanced sprayer operation and much reduced volumes.
Successful production of the large areas of broadacre crops demanded by the
world’s population will only be feasible if this industrial capacity continues in the
long-term. The demand now is that as with resistant cultivars the soil ecological
implications of using pesticide molecules is understood in advance of their application
in practice. But the nub of this Chapter demonstrates diseases in agriculture are
often iatrogenic, so therefore it is possible to alter management practices to reduce
disease severity and incidence. Husbanding soil health recognizes that economic
management of diseases is not achieved by the using one single procedure but
results from the accumulation of multiple strategies which cumulatively constrain
diseases below economic thresholds (Katon 1981). The aim of integrated pathogen
management is to prevent diseases reaching economic thresholds rather than through
curative actions once economic losses might occur. Integrated pathogen management
systems exploit the benefits of organic agriculture within essentially conventional
systems in a way that will enable agricultural production to be extended beyond
present levels with less reliance upon external inputs (Anon 1997). But it will not be
possible to exclude using external inputs. Integrated (Sustainable) Crop Management
(ICM) unifies all of the field processes and procedures with the objective of growing
top quality, healthy, visually attractive crops. Husbandry management starts with
site selection, avoiding land which because of aspect or for edaphic reasons may be
conducive to weed, pest or pathogen problems. The aim is then to diminish the
inoculum potential of organisms that compete with crops for resources by use agro-
nomic knowledge and expertise. The one key resource which is missing in the drive
for sustainable crop production is agronomic knowledge and expertise. Around the
world governments have neglected to sustain their educational capacities for the
continuing supply of personnel capable of viewing soil microbiology and sustain-

able crop production as a continuum from the laboratory bench to the farmers’ fields
(as identified by Dixon (2007) in a Report of a Survey of Members Qualifications,
Careers and Employers Needs prepared for the British Society for Plant Pathology
and available on “www.bspp.org.uk” (accessed 2010) and in the United States
(Martyn 2009). This critical deficiency is probably the biggest single impediment to
achieving a sufficiency of nutritious and attractive food which satisfies the needs
and aspirations of the burgeoning world population.
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Chapter 7
The Impact of Land-Use Practices
on Soil Microbes
Emma L. Tilston, Tom Sizmur, Geoffrey R. Dixon, Wilfred Otten,

and Jim A. Harris
Introduction
The extensive use of land resources for food production, fibre for construction,
wood pulp for paper, removal for extractive industries, sealing for urban and indus-
trial development and as a receiver (either deliberate or accidental) of polluting sub-
stances has wrought huge changes in the chemistry, structure and biology of soils,
away from their natural state.
These pressures are likely to continue, if not increase in the coming century due
to the ineluctable increase of human population; in the order of an additional two
to four billion in the next half century (Cohen 2003). All of these people will
require food, shelter, goods and services, as well as anticipating an increase in mate-
rial wealth through the development of their nation states.
E.L. Tilston (*)

Scottish Universities Environmental Research Centre, Scottish Enterprise Technology Park,
East Kilbride, G75 0QF, UK
T. Sizmur
Department of Soil Science, University of Reading, Whiteknights, Berkshire,
RG6 6DW, UK
G.R. Dixon
Centre for Horticulture & Landscape, School of Biological Sciences, Whiteknights,
University of Reading, Berkshire, RG6 6AS, UK
W. Otten
SIMBIOS Centre, University of Abertay, Bell Street, Dundee, DD1 1HG, Scotland, UK
J.A. Harris
Natural Resources Department, School of Applied Sciences, Cranfield University,
Cranfield, Bedfordshire, MK43 0AL, UK
274 E.L. Tilston et al.
The central challenge this offers is how we can continue to maintain soil health
whilst managing the trade-offs between optimization of agricultural production and
the other ecosystem goods and services provided by plant-soil systems (Kibblewhite
et al. 2008). These authors indicate how the various flows of a range of ecosystem
goods and services are dependant upon the activities of the soil biological commu-
nity (see Fig. 1).
The effects of this increased pressure on soil resources will be manifest in a
number of areas, notably hydrology and soil organic matter. Work by Bellamy et al.
(Bellamy et al. 2005) has demonstrated substantial losses of soil organic matter, in
some cases up to 50% in a 25 year period, probably driven by agricultural intensi-
fication with an element of climate change. The management of these carbon stocks
to mitigate, and adapt to, climate change and increased pressures for food produc-
tion, will be essential to maintain a civilized society (Lal 2007). Even before this
there were calls for institutional changes to increase focus on soil biology, rather
than traditional approaches based on the manipulation of chemistry and physics of
soils (Sherwood and Uphof 2000).
Measuring the Soil Microbial Community
In order to detect the effects that differing land uses and management strategies
undoubtedly have on soil microbial communities it is necessary to have techniques for
measuring their size, composition and function. There are two principal ways of orga-
nising the way in which this can be done and we can adopt a typology based on:
• Size – how much living microbial biomass there is
• Composition – what that biomass comprises of in terms of different “taxonomic”
groups
• Activity – at what rates the microbial community processes materials and energy
• Physical arrangement of the community – using a variety of visualisation techniques
Or we can look at the levels of organisation deriving from the evolutionary history
of soil organisms
• Genotype – the blueprint of the community
• Phenotype – the expressed parts
• Functional capabilities – the potential for the extant community to process
substrates
For a fuller review of the methodologies available see Harris and Steer (2003).
Size
This is the amount living biomass and is usually expressed as biomass-C and may
be determined in a number of ways, directly and indirectly. Fumigation of soils
7 The Impact of Land-Use Practices on Soil Microbes
Fig. 1 Relationships between the activities of the soil biological community and ecosystem services (From Kibblewhite et al. 2008). Reproduced with
permission from the Royal Society
275
with chloroform followed by extraction and determination of soluble carbon, in

comparison with a non-fumigated control is commonly used (Bloem et al. 2005), as
is determination of the soils adenosine tri-phosphate content (ATP), e.g. (Bentham
et al. 1992). This latter method is less commonly used, but has the advantage of
being exquisitely sensitive to low concentrations of biomass, and being absolutely
dependant on the presence of living cytoplasm within a viable cell – it is not likely
to produce artefacts as it is metabolised almost instantaneously on cell death.
Composition
The composition of communities has been commonly used in the sense of species
numbers and their abundance. This is not easy to determine, as the community is
incredibly diverse, with estimates of 10,000 species of soil bacteria alone (Torsvik
et al. 1996), with in excess of 5,000 species in a single gram. Traditional methods
of culturing “plate-counts” seriously underestimate this diversity, with as little as
0.1% of soil microbial species being culturable (Ritz 2007). Modern approaches rely
on biochemical methods such as the extraction of phospholipid fatty acid profiles
(Peacock et al. 2001a; Steer and Harris 2000), which provide a community “finger-
print” which can be used to ascertain the effects of management, pollution, ecosys-
tem health, and plant growth; or molecular and approaches which indicate the
genotypic diversity of the microorganisms in a sample (Torsvik and Ovreas 2002).
Activity
Some measurements are carried out in situ and are particularly useful when
considering changes over short time spans. However, most measurements on
restored sites are those carried out on samples returned to the laboratory. Common
measurements include enzymatic assays and respirometry. Enzyme assays are
particularly versatile and can indicate both non-specific (e.g. dehydrogenase – DHA)
and specific activity within the soil microbial community. Assays for phosphatase,
sulphatase and urease are of particular interest because of their importance to
phosphorus, sulphur and nitrogen cycling (Speir and Ross 2002).
Physical Arrangement
Determining the physical arrangement of the microbial community, particularly with

respect to organic matter resources and soil physical architecture, can be very
rewarding in terms of elucidating the recovery of biotic-abiotic linkages and structural
stability. It is, however, difficult and time consuming but continues to be very useful
in a research context.
7 The Impact of Land-Use Practices on Soil Microbes 277
There is a gap between the scale at which we observe and manage soil ecosystems
and the scale at which processes involving microorganisms take place, and it is
becoming increasingly clear that the emergent behaviour of soils can only be fully
understood if we account for the underlying heterogeneity. Much of our understanding
of spatial heterogeneity in soils relies heavily on destructive sampling and the concept
of soil aggregates. These techniques exert physical forces in order to produce and
break aggregates. Whilst having brought great insight, such approaches do ignore
much of the spatial heterogeneity which may be key to soil functioning. Mineralogical
and biological thin sections of soils revealed the first clear quantitative data of the
enormous heterogeneity of soils at microbial scales (Tippkötter et al. 1986). These
methods still provide the highest spatial resolution to study the distribution of soil
microorganisms (Nunan et al. 2002), and when combined with catalysed reporter
deposition and fluorescence in situ hybridization (CARD-FISH), enable phylogenetic
staining of microorganisms in situ (Eickhorst and Tippkötter 2008). Advancements
of microbial techniques to the 3-D environment where bacteria can be active in rela-
tively thin films adhering to surfaces are however slow.
In contrast, the ability to visualize and quantify the physical heterogeneity at
microscopic scales has advanced tremendously in recent years with the develop-
ment and application of neutron scanning (Schaap et al. 2008), nuclear magnetic
resonance (Pohlmeier et al. 2009) and X-ray tomography (Carminati et al. 2008;
Deurer et al. 2009). This has brought about a revolution in the way soil can be
characterized with a shift in emphasis from destructive analysis of the solid phase
to non-destructive characterization of the geometry and connectivity of the pore
volume. Carminati et al. (2008) demonstrated the intimate relationship between the
distribution of soil water and the micro structure using synchrotron X-ray tomog-
raphy. The distribution of water within a physical structure determines the con-
nectivity of physical habitats and contributes to the great microbial diversity in
soils (Or et al. 2007). Nowadays, micron scale resolutions can be achieved for
small samples, but larger samples (plant-root microcosms) can also be readily
scanned at high spatial resolutions (Fig. 2). This provides great insight into the
complex space within which water, air and resources move, and microorganisms
live and interact.
In addition to the physical heterogeneity, soils are chemically heterogeneous, with,
for example, the distribution of carbon intimately linked to soil structure. Conventional
chemical analyses often take place after homogenizing relatively large soil samples,
and even studies with small samples of soils are effectively bulk analyses (Wan et al.
2007). Recently, microscopic and micro-spectroscopic analyses are beginning to
address this. Examples include the use of stable carbon isotope ratio measurements
by laser combustion with a typical resolution of a few hundred micrometres, soft
X-ray techniques such as STXM (soft tissue X-ray microscopy) and NEXAFS (near
edge X-ray absorption fine structure) spectroscopy which enable the study of soil
organic carbon distributions at a spatial resolution of 30 nm (Wan et al. 2007). To
date, these techniques are restricted to small samples (few millimetres in diameter)
and require access to synchrotron facilities, but more accessible micro computed-
tomography (CT)/micro X-ray fluorescence (XRF) scanners for non-destructive 3-D
Fig. 2 Visualization of the internal structure of soils: Transect through a 3-D volume showing the
internal structure of an aggregate (approx. 2 mm in diameter) scanned at the Advance Photon
Source (APS, Chicago, Dr. M. Rivers) with a spatial resolution of 5.5 mm (left), and the internal
structure of a soil core (approx. 4 cm) from the top layer of an arable field after ploughing (30 µm
resolution). Pores are visible as dark areas (black), particulate organic matter as dark grey, and
denser minerals are presented by lighter grey scale values.
Images courtesy of the SIMBIOS Centre of the University of Abertay Dundee (P. Dello Sterpaio,
D. Grinev, R. Pajor and W. Otten). The image on the left was performed at GeoSoilEnviroCARS
(Sector 13), Advanced Photon Source (APS), Argonne National Laboratory with Dr M. Rivers.
GeoSoilEnviroCARS is supported by the National Science Foundation - Earth Sciences (EAR-
0622171) and Department of Energy-Geosciences (DE-FG02-94ER14466). Use of the Advanced
Photon Source was supported by the U. S. Department of Energy, Office of Science, Office of
Basic Energy Sciences, under Contract No. DE-AC02-06CH11357
chemical analysisare making rapid progress (Sasov et al. 2008), and will soon deliver
major advancement in our understanding of the chemical heterogeneity of soils.
Future efforts should address how to integrate these novel developments and how
to assess its impact on larger scale processes. Such a holistic approach to soil sys-
tems is currently not exploited to its full potential as the technological advances are
predominantly made within separate disciplines. Some difficulties may be overcome
readily via an integrated experimental design whereas others are advanced via
modelling and statistical approaches. This offers challenges for soil scientists as well
great opportunities to deepen our understanding of soil microhabitats and deliver
novel insight into soil ecosystem functioning.
Genotype
This approach looks at the soil microbial community in two ways: firstly, by deter-
mination of gene sequences identified with particular groups or traits; secondly
by considering the soil microbial “genome” as a whole – here it is possible to

examine differences in soil communities caused by differing management
regimes, vegetation types, and external stressors and perturbations by determina-
tion of diversity based on sequences reflective of the fungal or bacterial compo-
nents of the community. Examples of this approach include terminal restriction
fragment length polymorphism (T-RFLP) analysis, and have proven powerful
methods of analysis.
Analysis of particular functional groups in this way also remains as a strong
presence in the field – particularly examination of groups associated with functions
related to nitrogen cycling, such as ammonium oxidising bacteria.
Phenotype
The determination of the soil microbial community “phenotype” has produced a

large body of work in the field. One approach stands out as a powerful, reliable
and discriminatory tool – that of phospholipid fatty acid (PLFA) analysis.
Phenotypic profiling based upon PLFA analysis is used to provide a “fingerprint”
of the extant community composition. Since this analysis is based upon the mem-
brane composition of soil organisms, it reflects the status of living and intact
cells. Typically, an adaptation of a well-established method for extraction of
neutral, glyco- and polar lipids with a single phase mixture of chloroform,
methanol and water, followed by fractionation according to polarity, by adsorp-
tion to silica and selective elution and derivatisation to the fatty acid methyl
esters (FAMEs) prior to analysis by gas chromatography/mass spectroscopy
(GC-MS). This also allows the determination of stress/storage lipids, and, criti-
cally, the ratio of fungal-to-bacterial biomass (Zelles 1999). It is one of the prin-
cipal methods identified by Ritz et al. (2009) for providing clear discrimination
between land-use impacts.
Functional Capability
Recent attempts have been made to determine the ability of the soil microbial
community to process a range of carbon substrates. One common approach has
been the use of the BIOLOG MicroPlate™ bacterial identification system; a
microtitre plate system with 95 carbon substrates – these are inoculated with a soil
suspension and substrate utilisation is detected by a colour change. One drawback
of this approach is that the culturable fraction of the microbial community is
favoured, which may only represent less than 1% of the soil microbial community
(Preston-Mafham et al. 2002). Degens and Harris (1997) have also developed a
catabolic community profiling technique in which the substrate is taken to the com-
munity, and the response measured over the first 4 h, before any cell division is
likely to have occurred and therefore is more likely to represent the response of
the in situ community. This methodology is quite labour intensive and more
recently Campbell et al. (2003) have developed a simpler method to carry out this
assessment, which is capable of discrimination and interpretation when assessing
the impact of land-use and management practice (Ritz et al. 2009).
Conventional Agriculture
Conventional agriculture operates on a simple principle of excluding all but the

crop of interest from a given area. Therefore the microbial community characteri‑
stics reflect that focus on production. As there are clear changes in the microbial
community structure during the course of succession (Harris 2009), it follows that
any management intervention, in terms of land use change, are likely to lead to
significant changes away from that successional trajectory. The major pressures
brought to bear on the composition of the soil microbial community are:
• Cultivation – disruption of fungal networks are significant and long-lasting
• Fertilisation – altering nutrient balance in the soil impacts on the composition of
the microbial biomass
• Pesticides – affect microbial community directly by acting as food sources, or
indirectly as they impact on other components of soil food webs and plant
communities
• Veterinary medicines – in livestock production these compounds have a signifi-
cant effect on the soil microbial community antibiotics
Cultivation
Repeated destruction of stable soil structure by intensive cultivation disrupts fun-

gal networks, and prevents their re-establishment. Alguacil et al. (2008) exam-
ined the effects of tillage practices on arbuscular mycorrhizal fungal (AMF)
diversity in subtropical crops and found that the agricultural systems which
included tillage had significantly lower AMF diversity than non-tilled reference
sites. This effect on AMF is highly significant as they have been demonstrated to
have a central role in the maintenance of plant health and soil fertility (Jeffries
et al. 2003) with impacts on phosphorus-uptake, nutrient uptake generally, water
relations, and above ground plant productivity. These factors are substituted with
specific management intervention strategies in conventional agriculture, but the
potential for the use of AMF in sustainable systems is promising, so their contin-
ued impoverishment in agricultural systems is problematical if we are to secure
effective sustainable farming systems in the future (Gosling et al. 2006).
Fertilisation
There is an extensive literature on the impact of agricultural inputs on the soil

microbial community (Bünemann et al. 2006). Although direct effects of fertilisers are
sometimes difficult to clearly identify, addition of nitrogen in almost any form affects
the carbon-to-nitrogen ratio and increases the degradability of soil organic matter
stocks. Also, an indirect effect of nitrogen addition is that of acidification, which tends
to result in decreased biomass over long periods of time, unless countered with appli-
cations of lime (which in itself may not be a sustainable practice in the long-term).
Nitrogen management is of central importance in productive systems and there
has been serious effort to develop chemical interventions to inhibit nitrification – in
this sense these amount to a pesticide intervention. These have met with variable
success (Subbarao et al. 2006) and there is renewed interest in suppressing nitrifica-
tion using biological control methods, such as using plants known to suppress
nitrogen losses in this way (Subbarao et al. 2007).
Pesticides
The direct effects of herbicides on the soil microbial community would appear to
be minor. There is, however, evidence for impacts of insecticides and fungicides (as
one might expect), as both target important soil groups, with knock-on effects for
other components of the soil food web. Bending et al. (2007) investigated the
impacts of a range of fungicides on soil biology, using molecular techniques, and
demonstrated no measurable impact on bacterial community structure, but in cer-
tain fungicide treatments losses in both fungi and protozoa.
Livestock Management: Pastures
Pastures would, on the face of it, appear not to suffer from the disturbances associ-
ated with aggressive cultivation management. There are disturbances, however,
engendered by the impact of animals on soil structure (Palmer et al. 2006). Wakelin
et al. (2009) have studied the interacting effects of pasture type, liming, phospho-
rus-fertilisation and grazing pressure (and sampling data) on soil microbial
community structure, to identify the most sensitive components, on pasture systems
in Australia. Using molecular tools (denaturing gradient gel electrophoresis, DGGE
and T-RFLP) they found that liming had the biggest effect (mirrored on studies of
pastures in the Scottish Borders; Pawlett et al. 2009), followed by time during
growing season; fungal phylotype richness responded most markedly – capacity for
denitrification hardly at all. This underlines the important general observation that
microbial responses to land-use management at the community level are complex.
Sustainable Agriculture
In sustainable agriculture, the emphasis is on the management of natural resources

so as to maximize production within the constraints of minimizing synthetic inputs
sourced off-farm. By definition, therefore, the ecosystem services of soil microbial
communities are valued more than they are in conventional agriculture and it is
believed that sustainable management practices have positive impacts on microbial
biomass, community structure and function.
Minimal or No Tillage and Organic Agriculture
Minimal cultivation systems probably offer the best prospect for the development
of sustainable production systems based in part on biological control of pests and
pathogens, and biological promotion of fertility (Kennedy 1999). There is some
tentative evidence from a Canadian study that tree-based intercropping systems can
increase microbial diversity, but these are early results and more work needs to be
carried out to confirm this (Lacombe et al. 2009).
The recovery of biodiversity does occur quickly as a result of switching from
conventional to no-till/min-till or organic agriculture, but takes some time for the
complexity of communities to develop – probably due to the time it takes to build
organic matter stocks, rather than a lack of appropriate propagules being available
in the soil or arriving from other sources (Simmons and Coleman 2008). Adl et al.
(2006) demonstrated that even after 26 years in no-tillage management commu
nities of micro-arthropods, nematodes and protozoa had not fully developed the
richness and complexity found in natural communities. All these practices have
been found to result in increased microbial biomass, and a general increase in
fungal contribution to this biomass, leading to improved soil aggregation and stabi-
lisation of soil organic matter (Six et al. 2006). There is also probably a role for the
management of soil microbial communities specifically for the suppression of soil-
borne pathogens in agricultural systems (Watt et al. 2006; Janvier et al. 2007).
Soil Amendments: Biochar
There has been increasing interest recently in the prospects for employing
pyrolysis-derived organic materials, colloquially known as charcoal or “biochar”,
as amendments for degraded soil systems to improve soil nutrient holding and
structural characteristics (Marris 2006). This suggestion arises from studies in the
lowland humid tropics of “Terra Preta” areas found in South America, which
reflect aboriginal practices of returning charcoal to cleared forest areas to provide
for agricultural production on a small scale (typically circa 20 ha) on soils ostensibly
too infertile to support sustainable agriculture (Glaser et al. 2001), see elsewhere
in this Book. This has lead to various proposals for the widespread adoption of the
use of this approach to not only support sustainable agriculture in humid regions,
but more universally as a method for sequestering atmospheric carbon (Lehmann
et al. 2006).
The dynamics of organic matter in systems where biochar has been added are
poorly understood, if at all. Several studies suggest, however, that the addition of
non-native pyrolysed materials may actually result in the accelerated decom‑ posi-
tion of native soil organic matter. Wardle et al. (2008) have recently demonstrated
that addition of fire-derived charcoal to forest humus resulted in a significant loss
of native organic matter over a 10 year period. It is known that addition of charred
plant materials to soil accelerates breakdown of simple carbohydrates (Hamer et al.
2004), and that microbial community activity and metabolic efficiency increases
linearly with the addition of charcoal to soils (Steiner et al. 2008). Several potential
mechanisms for these observations are possible – including the increased retention
of soluble forms of nitrogen by high cation-exchange-capacity of pyrolised materi-
als facilitating the accelerated decomposition of native organic matter by overcom-
ing recalcitrance of the native materials. This suggests that adding pyrolysed
organic materials to soils may result in a net loss, not gain, in soil carbon, and irre-
vocably change soil organic matter dynamics, but such effects may be modulated
by the soil type and its carbon status.
Animal Manures, Sewage Sludge and Other Organic Wastes
The application of animal manures, sewage sludges and composts have the poten-
tial not only to increase soil organic matter stocks, but also to increase the size
(Anderson and Domsch 1989; Guerrero et al. 2007), activity (Bolton et al. 1985)
and diversity (Hassink et al. 1991) of the soil microbial community. Such enhance-
ments to the soil microbial community may have benefits for plant productivity
through increased nutrient cycling rates (Doran et al. 1987; Kirchner et al. 1993)
and reducing the risk of soil erosion through improved soil structure. Promotion of
aggregation (Angers et al. 1992) and the allied properties macroporosity and pore
continuity (Bronick and Lal 2005), also reduce nutrient losses through leaching
(Elliott and Coleman 1988).
Phospholipid fatty acid profiling of soils from long-term agricultural field trials
(10–100+ years) has proved particularly useful in describing the changes in micro-
bial community structure developing in soils amended with inorganic nitrogen and
farmyard manure. In agreement with other biological and chemical parameters
(Kandeler et al. 1999), the relative responses are typically no amendment < inor-
ganic nitrogen < farm yard manure (Peacock et al. 2001b; Wander et al. 1995;
Böhme et al. 2005; Toyota and Kuninaga 2006). The abundance of biomarkers for
Gram-negative bacteria are normally increased in soils receiving application of
farmyard manures (Peacock et al. 2001b; Böhme et al. 2005; Bossio et al. 1998),
but decreased after application of inorganic nitrogen (Peacock et al. 2001b; Böhme
et al. 2005). Applications of inorganic nitrogen (Peacock et al. 2001b) or compost
(Bossio et al. 1998), are however associated with increased abundance of Gram-
positive biomarkers. Sewage sludge applications elicit responses comparable with
those for farmyard manure (Petersen et al. 2003).
One disadvantage of the extensive and repeated application of organic amend-
ments, including farmyard manure, is the accumulation of heavy metals and other
xenobiotic compounds within soil (DEFRA 2009; Hamscher et al. 2005; Stoob
et al. 2006). Antibiotics, copper and zinc are commonly added to livestock feed as
growth promoters, to increase the supply of trace elements or for medicinal pur-
poses (DEFRA 2009). Without changes to upstream animal husbandry and waste
management, greater use of manures and allied materials within a more sustainable
form of conventional agriculture has the potential to produce substantial, long-lived
changes to soil microbial community composition through the introduction of met-
als and antibiotics/other veterinary medicines (Abaye et al. 2005). Elevated levels
of metals in soils decrease soil microbial biomass, activity and diversity, and
change the structure of the microbial community (Bååth 1989). These changes
probably occur at lower concentrations than those at which toxic effects to plants
or animals are detected (Giller et al. 1998). As concentrations are built up slowly
over time it is likely that the changes develop from stress responses rather than
profound disturbance responses. The ratio of cyclopropyl fatty acids to their meta-
bolic pre-cursors is commonly used to indicate stress within microbial communi-
ties (Bossio and Scow 1998) and has been used to indicate long-term metabolic
stress associated with sewage sludge application (Petersen et al. 2003). Applications
of farmyard manure, on the other hand, tend to decrease stress indicators suggesting
alleviation of sub-optimal nutritional status (Bossio et al. 1998; Bossio and Scow
1998). Sulphonamides are broad-range antibiotics commonly used in European
agriculture, they inhibit dihydropteroate synthesis in the folic acid pathway (O’Neil
et al. 2001) reducing bacterial reproduction. They enter the soil either by direct
deposition via livestock, or indirectly through the spreading of manure or slurry
and it would appear that the presence of manure increases the tolerance of soil
microbial communities to sulphonamides antibiotics (Demoling et al. 2009;
Hammesfahr et al. 2008).
Use of Plant Growth Promoting Rhizobacteria (PGRP)
The importance of bacterial symbionts to legumes has long been known, but wider
use of plant growth promoting bacteria has become more commonplace. There are
now several PGPR formulations available which are used commercially in agricultural
production (Lucy et al. 2004). Direct inoculation of soils with microbial prepara-
tions has also been shown to increase the efficiency of fertiliser uptake (Adesemoye
and Kloepper 2009). They are also being used in forest re-establishment and phy-
toremediation of contaminated soils.
Impact of Civil Engineering, Mineral Extraction

and Other Forms of Extreme Disturbance
In response to changes in policy and economic activity, it is not unusual for land
formerly subject to industrial uses e.g. coal spoil tips to be restored to agricultural
uses such as pasture and biofuel production (Davies 1999). The terrestrial civil
engineering programmes for mineral extraction and landscape re-instatement inevi-
tably lead to significant degradation of the soil ecosystem. Principally, there are
large shifts in the functional and phenotypic characteristics of the soil microbial
community, which are consistent and predictable for the type of degradative activity
applied. Similarly, as the system recovers (or not as the case may be) during the
course of reclamation and restoration programmes there are significant shifts in the
community structure towards the type of profile found in the desired target system.
Harris (2009) has suggested that the close relationship between the successional
state of an ecosystem, the fungal-to-bacterial ratio and size of the microbial
community in bulk soil offers a template for assessing the degree of degradation
and the extent of ecosystems affected by civil engineering-based activities (Fig. 3).
The handling and re-instatement of soils and soil forming materials is usually
one of the principal activities of the land reclamation process. There is little consid-
eration given to the soil as a living material, rather it is treated as an engineering
material with some unfortunately recalcitrant characteristics. What is meant by
restoration and reclamation is central to the assessment of the success or otherwise
of such programmes, and still the subject of much debate (van Diggelen et al.
2001). Harris (2003) has reviewed the use of soil microbial community measure-
ments to determine the “state” or “quality” of soils in restoration programmes,
Fig. 3 Relationship between ecosystem successional state and microbial community size and
composition. Copyright: JA Harris. From (Harris 2009)
related to the physico-chemical condition of soil, and their role in facilitating and
assessing restoration success (Harris 2009).
There is a plethora of methods now available for the rapid and reliable assay of
the soil microbial community (Ritz et al. 2009). These allow for assessment on a
time scale fast enough to make management interventions to enhance or correct the
trajectory of soil recovery to a condition being targeted to secure the target state.
This offers the prospect of the development of decision support tools encompassing
the full range of variables associated with soil handling and re-establishment of the
soil-plant system. Eventually this may lead to a reduction in the number of restora-
tion schemes subject to failure.
Changes During Disturbance
Impacts on the soil microbial community during movement and trafficking

operations are significant (Harris et al. 1989). They are not confined to the mineral
extraction and construction industries: Frey and co-workers (Frey et al. 2009)
examined the impact of logging operations on soil bacterial community structure,
with particular reference to the effects of compaction. Using analysis of soil micro-
bial biomass-carbon and T-RFLP community genotypic profiling, they detected a
decrease in microbial biomass, and shifts in the bacterial community composition.
They ascribed these changes to the decreases in soil porosity (up to 17%) and large
decreases in pores of >50 µm, which were associated with higher water-logging and
restricted gas exchange.
Changes During Topsoil Storage
There are large and rapid changes in the soil community as a result of the handling,
and movement of soils during civil engineering activities, with these damaging
effects made worse by storage for long periods in stockpiles (Harris et al. 1993;
Stahl et al. 2002). Bacterial numbers initially increase as a result of the large
amounts of nutrient made available by the death of larger organisms (most signifi-
cantly the fungi and invertebrates, such as earthworms) caused by the shear and
compaction forces applied during the soil lifting and store construction process.
Following this the bacterial component of the soil biomass declines as both nutrients
and oxygen are exhausted at depth in the stockpile, particularly in fine textured
soils, until only anaerobic metabolism persists.
Invertebrates, particularly earthworms, are severely affected by handling proce-
dures. They are liable to be crushed and unable to find physical refuges from the
large compaction and shearing forces being imparted to the soil by the heavy earth-
moving equipment, leading to large scale declines in their populations (Scullion
1994). Eventually the soil microbial biomass in stockpiles declines to values less than
5% of the undisturbed values, and, depending on texture, the microbial biomass in

less than 10% of the volume of stockpiled soil recovers to pre-disturbance values
(Harris and Birch 1990a). This recovery is further destroyed, however, when the
soil is re-instated at the end of coaling operations.
Recovery After Disturbance
Ruzek et al. (2001) demonstrated clear relationships between time since restoration
and increases in soil microbial biomass, in reclaimed sites in the Czech Republic
and Germany. Ruzek and co-workers (2001) indicated that this was related to both
organic matter content as a starting point in new reclamations, and the textural
characteristics of the soils reclaimed, and developed an algorithm which could be
applied to different soil types, with modifications appropriate to regions with differing
soil resources.
Total biomass is not the only characteristic which changes with time. Yin et al.
(2000), showed that the total number of bacterial species on a site recovering from
mining activity increased fairly quickly around pioneer vegetation, but the propor-
tion of these species which were active (as determined by a radio-label incorporation
method) took much longer to increase, and at no point matched the undisturbed
reference forest site This offers a potentially very sensitive approach for determining
the efficacy of treatment strategies for restoring function to systems.
Relationship Between Soil Microbial Community

and Other Parameters
Of great interest to those attempting to restore function to soils after civil engineer-
ing activities is the interaction between the microbial community and soil structural
characteristics.
Edgerton and co-workers (Edgerton et al. 1995) investigated the recovery of soil
structural characteristics and the size of the microbial biomass in a number of soils
re-instated after opencast mine restoration (Fig. 4). This relationship is linear for
the sites shown in Fig. 3, after which time the relationship becomes log-linear. This
development of structural stability, principally in this case the ability to retain structure
when subject to wetting and shaking (quite vigorously) is essential if the soil is to
function and redevelop drainage channels. Otherwise the re-instated soil will
remain compacted and liable to shed rather than absorbing water – causing off-site
pollution. The microbial communities in such degraded soils will never recover
function and will be droughty in the summer and water-logged in winter. This pro-
cess takes longer than the standard 5-year post-mining recovery period normally
allowed. This also illustrates that measurements of the microbial community can
600
11 Years
>2mm agg stab (g / kg) 500
400
6 years
300
6 years
5 years
Compacted
200
100 6 years
waterlogged
0
50 100 150 200 250 300 350 400 450 500 550
ATP
Fig. 4 Relationship between water stable aggregates (>2 mm g kg−1) and microbial biomass
(ATP ng g−1) at five restored sites (Redrawn from Edgerton et al. 1995). Reproduced with permis-
sion of Elsevier
often be used as a surrogate for the recovery of other soil characteristics and func-
tions, with assessments rapidly and reliably available.
Effects of Management
The importance of the type of management employed in restoring sites are illustrated by
the work on restored species rich meadows in the English Midlands around Nottingham,
subject to soil re-instatement after opencast coal mine operations (Harris and Birch
1990b). There were two undisturbed meadows, classified as “wet” and “dry” and two
sites re-instated some 5 years previously, and two 10 years previously. Two management
regimes had been applied – grazing or cutting and leaving the aftermath on the field.
Determinations of soil activity by soil enzyme measurements (dehydrogenase activity
which can be used as a surrogate for respiration) were made at 0–5 cm intervals to a
depth of 30 cm. A clear result emerges from this study – cutting and leaving the after-
math was superior to grazing in encouraging microbial activity leading to a more rapid
recovery of the activity profile. What is also clear, however, is that these soils will take
in excess of 20 years of this type of careful management before they begin to approach
the undisturbed reference sites with regards to microbial community activity. This
gradual development of soil catabolic capabilities and respiration dependant on man-
agement practice has been found in other studies, e.g. (Chodak et al. 2009).
The use of other biomarkers is becoming routine in the determination of ecosys-
tem status with respect to land use change – the lipids are the major group used for
this purpose, and have found widespread application (Zelles 1999). Mummey
and co-workers (Mummey et al. 2002) investigated the application of one group of
lipid biomarkers, the fatty acid methyl esters (FAMEs). Lipids were extracted and
derivatized to FAMEs, from samples taken from a surface mine reclamations of
different ages, and an undisturbed reference site. They were clearly able to distin-
guish between the different stages of reclamation, and identified a trend towards the
undisturbed reference condition. They suggest that the ratio of fungal-to-bacterial
biomarkers is a useful indicator of reclamation progress, and this is a view supported
by other authors (Harris 2003, 2009).
Quite simple measurements of soil microbial lipid characteristics can yield useful
information. Figure 5 shows the effects of varying intensities of military traffic/train-
ing on the soil microbial biomass, as determined by PLFAs (Peacock et al. 2001a).
All levels of traffic caused some decrease in biomass, but heaviest traffic (i.e. tank
training) caused a decrease by an order of magnitude. In an area which had been
remediated (i.e. 10 years after replanting with trees) the microbial biomass had
returned to the levels found in light and moderately trafficked areas but note that the
variability here was very high indicating the patchy nature of the recovery.
Bentham et al. (1992) investigated the effects of time and management by
comparing reclaimed opencast coal mining sites restored to either grassland or
woodland with a number of reference sites, including flood meadow, chalk grass-
land, rough grassland and woodland. The size and activity of the soil microbial
communities were measured by adenosine triphosphate determination (microbial
biomass), ergosterol determination (fungal biomass); and, dehydrogenase activity
(microbial activity). Multivariate statistical analysis showed that the site restored
to grassland clustered with a “rough” (improved) grassland; and, the restored
Microbial biomass PLFA (Pmol/g dry weight)
45000
40000
35000
30000
25000
20000
15000
10000
5000
0
Reference Light traffic Moderate Heavy traffic Remediated
traffic
Fig. 5 The effect of military traffic and remediation on soil microbial biomass as determined by
PLFA (pmol g−1) (Redrawn from Peacock et al. 2001a). Reproduced with permission of Elsevier
woodlands are “moving away” from the 5 year grassland restoration in the direction
of woodland reference sites. A more comprehensive database would facilitate inter-
pretation of data of this type, allowing us to identify the effects of management
interventions.
Concluding Remarks
It is clear that there are profound changes in the soil microbial community in
response to the land uses currently practiced. The evidence comes form a wide
variety of sources and needs to be brought together with physico-chemical and
pedological data and knowledge in a unified model or “theory of soil”, a notion first
suggested by McBratney over a decade ago (McBratney 1998). This is a major
collaborative project for society, without which sustaining both food supply and
environment will be inefficient and costly.
We have, however, a framework for putting this work in the context of society
as a whole in the form of the concept of ecosystem services. Barrios (2007) has
reviewed the role that soil biota plays in underpinning the delivery of ecosystem
goods and services, and particularly its importance in promoting productivity. He
identifies research gaps and opportunities, in particular:
• Integration of spatial variability research in soil ecology and a focus on “hot
spots” of biological activity
• Using a selective functional group approach to study soil biota and function
• Combining new and existing methodological approaches to that link the tempo-
ral-spatial dynamics of soil organisms to the delivery of specific services
• How these relationships might be directly or indirectly manipulated to enhance
particular functions (this will result in trade-offs of function)
• How remote sensing of vegetation condition and composition could inform
understanding of soil microbiological communities at a landscape scale; and
• Developing land quality monitoring systems that inform land users of ecosystem
service delivery to aid policy and decision making
With respect to this latter point Ritz and co-workers (Ritz et al. 2009) have carried
out a sophisticated analysis of the plethora of methods available (184 and counting)
used to determine the size, composition and activity of the soil biological commu-
nity in the context of informing national soil quality and ecosystem assessment
schemes. The results of this work are very promising, and there is a real prospect
of deploying a handful of techniques to capture the entirety of ecosystem level
responses to land-use and climate change, better to inform the policy and decision
making as called for by Barrios (2007). This fits neatly in the concept of sustainable
agriculture, as it provides a context for determining the impact of different farming
practices, and a readily available toolkit of techniques with which to assess their
success or failure.
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Chapter 8
The Effects of Plant Breeding on Soil Microbes
Petra Marschner and Zed Rengel
Introduction
Breeding wheat in the twentieth century, for example has been successful on many
accounts, resulting in increased yields and grain quality (and contributing to the
“Green Revolution”). One of the consequences of breeding for increased yield and
partitioning of biomass into grain (raised harvest index, i.e. the grain-to-straw bio-
mass ratio) has however been a decrease in the root-to-shoot biomass ratio (Rengel
2005). This had significant implications for crop nutrition, since the smaller root
system can only support the relatively larger shoots when provided with large quanti-
ties of inorganic fertilizers. What remains unclear, and needs to be one of priorities
for future research, is whether altered root-to-shoot ratios influence root exudation
and therefore the structure of microbial communities in the rhizosphere.
Soil microbes can play an important role in growth and nutrient uptake by
plants, as well as modifying the tolerance of plants to biotic and abiotic stresses;
so are worthy of stronger consideration in plant breeding. Those microorganisms
colonizing the roots as symbionts and in the rhizosphere are particularly important
in plant–soil relations and some of the observed effects of breeding, e.g. enhanced
nutrient acquisition, probably result not only from changes in plant physiology, root
morphology and function alone but also from changes in the nature of the interac-
tions with soil microorganisms.
Physiological or morphological changes induced by breeding can affect micro-
organisms in the rhizosphere and bulk soil both directly and indirectly (Table 1),
thereby potentially affecting microbial community structure and activity as well as
their effect on plants. In this review, we will focus on the effect of plant breeding
P. Marschner (*)
Soils, School of Agriculture, Food & Wine, The University of Adelaide, Adelaide,
South Australia 5005, Australia
Z. Rengel
School of Earth and Environment, The University of Western Australia, Crawley,
Western Australia 6009, Australia
298 P. Marschner and Z. Rengel
Table 1 Direct and indirect effects of plant breeding on soil microorganisms

Direct Indirect
Root exudates (amount and composition) Nutrient and water uptake
(competition with microbes, water
availability)
Root morphology Changes in nutrient availability
Composition of residues (including genes Soil temperature (more or less shading)
from other organisms)
Susceptibility/tolerance to infection Amount of residues
on soil microorganisms, particularly symbionts and rhizosphere microorganisms.

We discuss how the changes induced by plant breeding could affect microbial diver-
sity and function as well as nutrient cycling in ways that are usually not considered
by plant breeders.
Breeding for Modified Nutrient Uptake
Associative and Symbiotic Nitrogen Fixation
Under certain conditions, biological nitrogen fixation via associative bacteria may
contribute up to 50 kg/ha or 30% of nitrogen needs of wheat and up to 150 kg N
ha-1 year-1 in sugar cane fields in Brazil (Rengel 2002). Brazilian cereal and sugar
cane genotypes may have inadvertently been selected for efficient symbiosis with
associative diazotrophs (Dobereiner 1997), thus significantly decreasing or com-
pletely eliminating the need for inorganic nitrogen fertilization of these crops.
Genotypic differences in capacity to support associative nitrogen fixation have
been found among cereal species and genotypes (see Rengel 2002). Rice genotypes
show more than one order of magnitude difference in the percentage of nitrogen
derived from air (Ndfa) by associative fixation (Shrestha and Ladha 1996). Some
maize genotypes had similar yield when inoculated with Azospirillum and when
artificially fertilized with 100 kg N ha-1 (Garcia de Salamone et al. 1997).
The symbiosis between legumes and Rhizobium is highly specific; inoculation
with a given Rhizobium genotype may increase nitrogen fixation strongly in one
cultivar while having little effect on that process in another cultivar (Hardarson and
Atkins 2003). The higher capacity of certain Rhizobium genotypes to form success-
ful symbioses with a particular plant genotype increases their survival and competi-
tive abilities in soil, hence affecting the composition the rhizobial community in
the soil. Increased nitrogen fixation, either from associative or symbiotic bacteria,
will have indirect effects on soil microorganisms via the higher nitrogen content of
the resultant plant residues. These effects will occur mainly after harvest or decay
of the plants but may also happen during plant growth when roots die and are
decomposed. Effects can include increased activity and/or abundance of microor-
ganisms involved in nitrogen mineralization, increased microbial activity more
generally because of the higher nitrogen supply, stronger decomposition of native
8 The Effects of Plant Breeding on Soil Microbes 299
soil organic matter (priming effect) (Kuzyakov et al. 2000), increased abundance of
microbial species with high nitrogen requirement and reduced nitrogen fixation by
free-living bacteria (see chapters elsewhere in this Book). This may affect micro-
bial community composition, and in the longer term, nutrient cycling itself.
However, to our knowledge, there are no studies which consider the effects of
enhanced nitrogen fixation on soil microorganisms and nutrient cycling. Long-
term field experiments would be required in order to assess this. Nevertheless,
some of the effects induced by the growth of plant genotypes associated with high
nitrogen fixation could be similar to those of high increased availability induced
by high doses of inorganic fertilizer which have been shown to change the bacte-
rial colonization of roots (Marschner et al. 1999), microbial community composi-
tion (Marschner et al. 2004), growth and activity of bacteria (Soederberg and
Baath 2004; Alarcon-Gutierrez et al. 2008) and bacteria-feeding nematodes
(Griffiths et al. 1992) and increase or decrease the density of diazotrophs (Azaizeh
et al. 1996). However, the effect of plant residues with higher nitrogen content
may differ from that resulting from inorganic fertilizer because they also supply
carbon. Since soil microorganisms are primarily limited by carbon availability
(Kemmitt et al. 2008; Hoyle et al. 2008), changes induced by high nitrogen con-
tent in residues are likely to be greater than those caused by the simple addition of
inorganic fertilizer.
Arbuscular Mycorrhiza
Arbuscular mycorrhizal (AM) fungi are less host-specific than rhizobia, but their rate
of colonization and other benefits to the host are plant (host) genotype-dependent.
Zhu et al. (2001) showed that mycorrhizal responsiveness in terms of phosphorus
uptake was lower in modern wheat genotypes than in older cultivars, suggesting
that current plant breeding practices have selected for genotypes that are less
dependent on mycorrhiza for their phosphorus uptake (Zhu et al. 2001). Likewise,
Xavier and Germida (1998) found that wheat genotypes differ in the capacity to
sustain AM colonization. Yield responses varied from zero to both increased or
decreased values. In durum wheat genotypes, the benefits from the AM symbiosis
were not proportional to the extent of the root colonization because the genotypes
differed in degree to which they were dependent on mycorrhiza (Al-Karaki and
Al-Raddad 1997). Similar results were obtained for barley genotypes varying in their
phosphorus use efficiency (Baon et al. 1993).
Differences in the extent of root colonization of plant genotypes may affect the
survival of mycorrhizal fungi. If host roots are poorly colonized, the fungus may
not derive sufficient energy to sporulate and reproduce. Consequently, the coloniza-
tion potential of the soil may be reduced with resultant decreases in colonization of
following crops. Differences in AM colonization can also affect other soil microor-
ganisms either directly via the fungus or indirectly through effects on host plants.
Arbuscular mycorrhizal colonization decreases root exudation in some cases (Dixon
et al. 1989; Graham et al. 1981; Marschner et al. 1997) although in others there may
be no effect (Azaizeh et al. 1995). Mycorrhizal colonization may affect exudate

composition (Marschner et al. 1997; Po and Cumming 1997) and the carbohydrate
metabolism of roots (Buwalda and Goh 1982; Shachar-Hill et al. 1995). Mycorrhizal
fungi themselves may release exudates which selectively influence the microorgan-
isms of the rhizosphere. Thus, differential colonization affects exudates composi-
tion and hence has an influence on other soil microorganisms. Indeed, previous
studies have shown that AM colonization itself and the extent of colonization affect
the structure of microbial communities in the rhizosphere (Amoralazcano et al.
1998; Andrade et al. 1997; Marschner et al. 2001; Marschner and Baumann 2003;
Meyer and Linderman 1986; Posta et al. 1994; Wamberg et al. 2003; Secilia and
Bagyaraj 1987). Additionally, bacterial community structure in the rhizosphere is
affected by interactions between host genotypes and mycorrhizal fungal species
(Marschner and Timonen 2004). Colonization by AM can affect nematode infection
of roots (Cooper and Grandison 1986; Elsen et al. 2001), which is likely to affect
the community structure of the parasite, in turn, this could have a cascading effect
on the types, density and interactions between microbes.
Phosphorus Efficiency
Plant genotypes differ in phosphorus-use efficiency, i.e. capacity to grow with low
phosphorus supply. Differential phosphorus-use efficiency can be due to differences in
uptake and/or internal utilisation. Phosphorus-use efficiency has been studied exten-
sively in cereals, particularly wheat (e.g. Zhu et al. 2001; Osborne and Rengel 2002a;
b; Marschner et al. 2006) and legumes (Bolland et al. 1999; Horst et al. 2001; Lynch
and Brown 2001) but relatively little is known about differences in phosphorus-use
efficiency in Brassica species or in vegetable and oil seed rape genotypes (Greenwood
et al. 2005). Although most studies have focussed on plant growth and yield, a number
have investigated the effect of phosphorus-use efficiency on soil microorganisms.
Comparisons of modern and old wheat cultivars, Zhu et al. (2001) showed that
the greater the phosphorus utilisation efficiency of the non-mycorrhizal plants, the
smaller the growth response of the plant to mycorrhizal colonization. Moreover,
despite similar AM colonization rates, old cultivars were more responsive to
mycorrhizal colonization than modern cultivars.
In a comparison between two wheat cultivars (P-efficient cv. Goldmark and
P-inefficient cv. Janz), Marschner et al. (2006) found that genotypic differences in
phosphorus efficiency is explained by plant traits, including root growth, phospho-
rus uptake and utilization and rhizosphere properties such as acid phosphatase
activity and concentrations of available and microbial phosphorus. The genotypes
differed in their associated microbial community composition, which might indicate
that a differential capacity of the genotypes for growth in soil with low phosphorus
availability is, at least partially, due to differences in microbial community compo-
sition in the rhizosphere. Interestingly, there were no differences in mycorrhizal
colonization between the genotypes. A positive correlation between microbial
phosphorus in the rhizosphere and shoot dry weight, phosphorus uptake per plant
and available phosphorus in the rhizosphere suggested the importance of this
source for plant uptake. These findings supported the previous hypotheses (Oberson
et al. 2001; Seeling and Zasoski 1993) that an active microbial biomass with a high
turnover rate provides a slow-release, sustained source of available phosphorus.
However, while phosphorus-efficient cv. Goldmark appeared to compete effec-
tively with rhizosphere microorganisms for phosphorus and might have taken
advantage of the microbial pool as a source, this was not the case for phosphorus-
inefficient cv. Janz. Root exudates of cv. Goldmark may favour rapid microbial
turnover and/or stimulate the growth of rhizosphere microorganisms with high
phosphorus mobilization capacities and low requirements. The correlation between
microbial community composition and acid phosphatase activity indicates that
different microbial species release varying amounts of phosphatase, and/or that micro-
bial species differ in their capacities to decompose the phosphatase enzyme. Moreover,
microbial community composition was correlated with phosphorus availability in
the rhizosphere. This suggests that microbial species differ in phosphorus uptake,
requirement and/or mobilization capacity (Banik and Dey 1983). Thus in wheat,
phosphorus-efficient genotypes appear to have rhizosphere microbial communities
that mobilize this element but also have high turnover rates, releasing it for subse-
quent plant uptake. By contrast, in the rhizosphere of phosphorus-inefficient geno-
types, rhizosphere microbial communities also mobilize this element, but due the low
turnover rate, it remains locked-up in the microbial biomass.
Interestingly, microbial biomass phosphorus in the rhizosphere was not corre-
lated with plant uptake in three brassicas (two canola, Brassica napus genotypes
and a mustard, Sinapis sp.) (Marschner et al. 2007), which differed in phosphorus
efficiency. In the brassicas, shoot uptake was positively correlated with root length
as well as phosphorus availability and phosphatase activity in the rhizosphere. The
genotypes also differed in microbial community composition in the rhizosphere,
but community composition did not appear to be related with a differential capacity
for growth in phosphorus-limiting conditions. Thus in brassicas, traits such as root
length and the capacity to maintain high phosphorus availability in the rhizosphere
by mobilization via carboxylate or phosphatase release explains the observed differ-
ences in plant growth and phosphorus uptake. It cannot be excluded however, that
some of the observed differences in mobilization between the genotypes were due
not only to exudates released by the roots but also to differential capacities of the
rhizosphere microorganisms to mobilize phosphorus.
Some plants, such as grasses, are unable to mobilize sufficient phosphorus from
phytate (inositol hexaphosphate) under sterile conditions (Richardson et al. 2000;
Hayes et al. 2000a), because they release only very small amounts of phytase into
the rhizosphere (Hayes et al. 2000b). Inoculation with a soil suspension (contain-
ing microorganisms) restores the capacity to utilize phytate (Hayes et al. 2000a;
Richardson et al. 2001). Moreover, some soil microorganisms, particularly fungi can
utilize phytate in vitro (Richardson and Hadobas 1997; George et al. 2007). To increase
the capacity of grasses to grow in soils with low phosphorus availability, fungal
phytase genes have been introduced into wheat and these plants are able to utilize
phytate under sterile conditions (George et al. 2005). There are also wheat genotypes
with an over-expression of genes involved in citrate or malate synthesis and release
from roots (Delhaize et al. 1993; Pellet et al. 1995). It was suggested that engineer-
ing plants with increased synthesis and exudation of organic acid anions might
increase their capacity for growth in phosphorus-limiting conditions (Lopez-Bucio
et al. 2000), but this has yet to be demonstrated (Delhaize et al. 2001).
Plant genotypes that increase phosphorus availability in the rhizosphere would
affect soil microorganisms by preventing deficiency. If the higher phosphorus avail-
ability is associated with higher carbon and nitrogen availability, this may lead to
increased growth and activity by rhizosphere microorganisms, and could favour
microbial species that have high growth rates under conditions of phosphorus suf-
ficiency. On the other hand, slower growing microbial species with high phosphorus
mobilization capacities would be out-competed. If plants with high inherent capaci-
ties for mobilizing phosphorus are grown repeatedly in a soil, this might lead to an
overall decrease in mobilization capacity of the soil microbial community. It remains
to be experimentally verified however if this would really be the case.
Manganese Efficiency and Availability
Yield of cereals on calcareous soils is frequently limited by manganese deficiency

caused by low availability, rather than low total manganese content in soil (Rengel
2000). Compared to inefficient genotypes, manganese-efficient genotypes take up
more of the element from soils with limited availability, but the physiological
mechanisms underlying efficiency are poorly understood (Rengel 2000, 2001a).
As a result of root uptake and poor mobility, the concentration of manganese in
the rhizosphere is lower than in the bulk soil. Such depletion is more prominent in the
rhizosphere of manganese-efficient than inefficient Triticum aestivum genotypes
(Marschner et al. 2003). In another study however, the genotypic difference in effi-
ciency between Triticum aestivum and T. durum genotypes was not due to differences
in chemical mobilization of manganese in the rhizosphere (Sadana et al. 2002).
Reduction and oxidation of manganese by microorganisms are important
components of manganese cycling in soil. Whereas reduction increases manganese
availability, oxidation decreases it. Interestingly, manganese reducers appear to be
more abundant in the rhizosphere of some efficient compared with inefficient
Triticum aestivum genotypes (Rengel et al. 1998).
Differential manganese efficiency in cereals might also affect their susceptibility
to Take-all (Gaeumannomyces graminis var. tritici) (Rengel 2001b). It has been
shown that increasing fertilization of manganese decreases Take-all infection in
wheat and this is explained by two factors: (i) increased defence reaction by roots
to Take-all infection, and (ii) inhibition of the growth of the take-all fungus by high
manganese concentrations in soil (Marschner et al. 1991). The increased defence
reaction in plants that have adequate manganese status is related to its role in the
synthesis of phenolic compounds and lignin which form a mechanical barrier in

roots preventing penetration by the pathogen into cells, thereby inhibiting or limit-
ing infection. The rate of lignin accumulation in the whole root system was lower
in manganese-efficient than in inefficient genotypes (Rengel et al. 1994). In that
study, manganese fertilization did not affect lignin concentration in the whole root
system but still decreased Take-all infection. These apparent contradictions could
be due to localized high concentrations of lignin around infection points rather than
an increased lignin concentration in the entire root system.
Differential manganese availability in the rhizosphere of manganese-efficient
and inefficient plant genotypes may affect microbial community composition
(Marschner et al. 2003); high availability could favour microbial species with a
high manganese requirement, and decrease the density of microbial species sensi-
tive to high concentrations. A higher lignin concentration of the roots may reduce
root colonization by beneficial microorganisms such as AM fungi or pathogens and
could also affect root exudation; and, consequently microbial community composi-
tion and nutrient cycling. Moreover, higher lignin concentration may reduce
decomposition rates of roots and favour microorganisms with capacities to decom-
pose lignin such as fungi and actinobacteria.
Other Plant Breeding Effects
Breeding can affect soil microorganisms by changing the chemical composition of

the plants. Here we discuss the effect of enhanced glucosinolate concentration and
the introduction of the Bt genes. Lastly, we briefly mention an example of breeding
for enhanced tolerance to Rhizoctonia induced disease and how this may affect soil
microorganisms.
Glucosinolates in Brassicas
Brassicas contain glucosinolates (GSL), which are hydrolyzed by the enzyme

myrosinase into glucose, sulphate and biocidal products such as isothiocyanates,
nitriles and ionic thiocyanate (Brown and Morra 1997; Kirkegaard and Sarwar 1998a).
Among these metabolites, isothiocyanates are considered to be the most toxic to
organisms (Kirkegaard and Sarwar 1998a). The toxicity of isothiocyanates is due
to irreversible reaction with sulphhydryl groups, amine groups and disulphide
bonds of proteins, which can lead to enzyme inactivation (Kirkegaard and Sarwar
1998a). Glucosinolates and myrosinase are spatially separated in intact cells, there-
fore isothiocyanates are produced only when cell integrity is disrupted (Kirkegaard
and Sarwar 1998a).
The formation of biocidal metabolites from GSLs is used in ‘biofumigation’,
a suppression of soil-borne pests and pathogens after incorporating green manure
or harvest residues from canola (B. napus L.) or other high glucosinolate brassicas
(Kirkegaard and Sarwar 1998b; Kirkegaard and Matthiessen 1999). The dynamics
of GSL hydrolysis after incorporation of plant material into soil has been studied
intensively (for an overview see (Kirkegaard et al. 1999; Rumberger and Marschner
2003). Isothiocyanates are degraded in soil within a few days and are adsorbed by
organic matter (Gimsing and Kirkegaard 2006; Motisi et al. 2009). Despite the
rapid hydrolysis of glucosinolates and high rates of isothoicyanate degradation in
soil, addition of residues high in GSL may inhibit growth of microorgansims for up
to 2 weeks after addition to soil. This suggests that the inhibitory effect of residues
is due to the release of isothiocyanates during the first days following incorpora-
tion, but that other mechanisms are likely to contribute to lasting persistence (Scott
and Knudsen 1999). Even longer lasting effects of incorporation of residues with
high GSL concentration are reported by Smith et al. (2004), where incorporation
of brassica residues caused changes in the rhizosphere microbial community
composition of the following wheat crop. Daily addition of the isothiocyanate
2-phenylethylisothiocyanate (PEITC) for 5 days affected both bacterial and eukary-
otic community structure (Rumberger and Marschner 2003). On the other hand,
incorporation of high GSL brassica residues did not affect the substrate-utilization
pattern of bacteria in the rhizosphere of pea although it inhibited the growth of the
seedlings (Scott and Knudsen 1999).
In canola roots GSLs are highly localised, the highest concentrations were mea-
sured in two cell layers just below the outermost periderm layer, being up to 100-fold
higher than published concentrations for whole roots; by contrast, primary tissues
contained negligible GSL concentrations (McCully et al. 2008). McCully et al.
(2008) hypothesised that release of GSLs is a normal developmental process during
the secondary thickening of stems with senescence of surface cells leading to release
of GSLs and their biocidal hydrolysates into the rhizosphere of intact roots. These
authors did not detect myrosin idioblasts close to the root surface and suggested that
GSLs released during plant growth are hydrolyzed by myrosinase in the rhizosphere,
ensuring a continuous localized source of biotoxic hydrolysates in the long-lived
components of the root system.
In agreement with the findings of McCully et al. (2008), Rumberger and
Marschner (2003) found in a pot study with different brassica genotypes, that the
concentration of PEITC in the rhizosphere of living canola roots was greater in
first order laterals than in those of the second order. The PEITC concentration
detected in the rhizosphere was in the range that had been shown to affect microbial
community structure when added directly to the soil. Indeed, there was a significant
correlation between the PEITC concentration in the rhizosphere and the commu-
nity structure of the active fraction of eukaryotes and bacteria.
In a field experiment with several canola cultivars, PEITC concentrations in the
rhizosphere ranged between 0 and 12,000 pmol g-1 and this was affected by plant
growth stage and environmental conditions (Rumberger and Marschner 2004).
Maximal PEITC concentrations were found at the boot stage in the winter-grown
cultivars and at flowering in the spring cultivars. PEITC concentrations in the
rhizosphere decreased towards maturity, which agrees with the low root GSL
concentration in mature canola (Kirkegaard et al. 2001b; Potter et al. 2000).

Surprisingly, there was no difference in PEITC concentrations in the rhizosphere
between cultivars with high and low seed GSL concentrations, suggesting that seed
concentration is a poor indicator for release of isothiocynates from roots. As in the
pot experiment (Rumberger and Marschner 2003), bacterial community composi-
tion in the rhizosphere was affected by PEITC concentration. Both pot and field
experiment showed, however, that other factors such as soil moisture, plant devel-
opmental stage and dry matter had a greater effect on the microbial community
composition in the rhizosphere, suggesting a limited effect of PEITC released by
roots on microbial community composition in the rhizosphere. Moreover, isothio-
cyanate release by roots is highly variable. It varies with plant developmental stage
as described above and is also related to growth conditions, with adverse growing
conditions such as low light and temperature or pathogen attack decreasing iso-
thiocyanate release (Kirkegaard et al. 2001a). Potter et al. (2000) showed that root
GSL concentration and thus probably also isothiocyanate release into the rhizo-
sphere can differ widely between otherwise phenotypically similar plants of the
same cultivar.
Thus, brassicas with high GSL concentrations can affect soil microorganisms
during plant growth by release of GSLs from roots and their hydrolysis into toxic
isothiocyanates in the rhizosphere and following incorporation of GSL-rich resi-
dues into the soil. In biofumigation, the aim of the latter is a decrease of pathogenic
microorganisms, however a long-lasting effect, particularly with repeated cultiva-
tion of GSL-rich cultivars, on microbial community composition and nutrient
cycling cannot be ignored.
Transgenic Plants with Increased Resistance Against Damage

by Pathogens or Insects
In order to increase resistance against bacterial pathogens, transgenic potato plants

containing lysozyme, which degrades the murein in bacterial cell walls, were pro-
duced. Bacillus subtilis cells are killed on contact with roots of transgenic potato
(Heuer et al. 1999) and the bacterial community composition in the rhizosphere of
transgenic potato assessed by molecular techniques differs from that of the wild
type (Lottmann et al. 1999). On the other hand, the number of culturable total aerobic
bacteria or phytohormone-producing bacteria in the rhizosphere did not differ
between transgenic and wild-type potatoes (Flores et al. 2005).
The insecticidal Cry protein from Bacillus thuringiensis (Bt) has been intro-
duced in crops such as: cotton, maize, potato, rice and tobacco in order to decrease
insect damage (Stotzky 2005; Saxena and Stotzky 2000). These crops synthesise
Cry proteins in all tissues, and it has been shown that these proteins are released by
roots of transgenic maize (Icoz and Stotzky 2008a; Saxena and Stotzky 2002). The
question therefore arises about the fate of Cry1Ab in soils and if it can affect soil
microbial populations.
There are conflicting results regarding the uptake of Cry proteins by plants.
Saxena and Stotzky (2002) found that the protein is not taken up from soil or in
hydroponic culture by: carrot, maize, radish, or turnip. The Cry protein was recently
detected however, by Western blot in shoots of a range of plant species after its
addition or that of residues of transgenic maize into soil (Fiorito et al. 2008).
Persistence of Cry protein in soil is increased by the presence of clay minerals
such as kaolinite and montmorillonite, probably by adsorption (Saxena and Stotzky
2002). Fiorito et al. (2008) showed that when they are bound to clay, the Cry proteins
were not utilized for growth by soil microorganisms, whereas the microorganisms
readily utilized the free proteins as sources of carbon and energy. Bound Cry pro-
teins remain active however and therefore kill larvae of soil insects (Stotzky 2005),
which via a cascading effect through the soil food web, could indirectly affect soil
microorganisms.
Two to 3 weeks after addition of transgenic maize tissue to soil, the Cry3Bb1
protein could no longer be detected, suggesting a low persistence in soil (Saxena
and Stotzky 2002) however, the persistence of different Cry proteins may vary.
After 4 years of consecutive planting of a number of transgenic maize cultivars
(some expressing Cry1Ab1, others Cry3Bb1) and incorporating the residues,
Cry1Ab1 could be detected in the soil whereas Cry3Bb1 could not (Saxena and
Stotzky 2002). In that field study, there were no consistent statistically significant
differences in the density of different groups of microorganisms and the activities
of various enzymes in comparison of soils planted with Bt and non-Bt maize, sug-
gesting that although the proteins may persist in soil, there is little effect on soil
microorganisms even after 4 years of continuous cultivation.
The decomposition rate of residues of transgenic Bt plants was lower than that of
their near-isogenic non-Bt plant counterparts (Stotzky 2005), which could be due to
the higher lignin content of the residues of the transgenic Bt plants. The density of
culturable bacteria and fungi and the activity of a range of enzymes involved in resi-
due decomposition were not significantly different between soils amended with resi-
dues of Bt or non-Bt maize. The discrepancy between decreased decomposition rate
on one side and no apparent effect on cuturable bacteria and fungi could be due to
the fact that less than 5% of soil microorganisms are culturable (Bakken 1985).
Thus, the decreased decomposition of the residues of Bt maize is probably due to
reduced microbial activity or changes in community composition of the non-cultur-
able microorganisms which represent the vast majority of microorganisms in soil.
In a review, Icoz and Stotzky (2008b) noted few or no toxic effects of Cry pro-
teins on: collembola, earthworms, mites, nematodes, protozoa and woodlice and
the activity of various enzymes in soil. Although some significant effects of Bt
plants on microbial communities in soil have been reported, they were transient and
not related to the presence of the Cry proteins and appeared to be plant genotype
and site-specific (soil type and climate).
Thus, the effect of transgenic plants containing Cry proteins on soil microorgan-
isms remains unclear. Some negative effects have been reported however, such as
the decreased decomposition of residues of Bt maize and the persistence of certain
Cry proteins in soil where they retain their insecticidal activity and could therefore
affect soil microorganisms indirectly via cascading effects within the soil food web.
More studies, particularly long-term field studies, are required to identify the plant
genotype-soil-climate combinations in which effects on soil microorganisms in
terms of community structure and activity occur and to identify the mechanisms
that lead to these effects.
Control of Rhizoctonia
Pathogenic fungi in the genus Rhizoctonia (such as R. solani, or R. cerealis) affect

many cultivated plants causing a range diseases, e.g. sheath blight in rice (Lu et al.
2009), sharp eye spot in wheat (Nagendran et al. 2009), damping-off in sugar beet
(Bokmeyer et al. 2009) and brown patch in tall fescue (Zhang et al. 2009). Chemical
suppression of Rhizoctonia is relatively poorly effective, albeit new chemical prod-
ucts show promise (Gallou et al. 2009). By contrast, biocontrol agents (such as
Trichoderma spp.) were found to be very effective, e.g. Rhizoctonia solani can be
controlled with Trichoderma harzianum (Ruocco et al. 2009). Biocontrol agents as
well as Rhizoctonia and other pathogenic fungi can exude antimicrobial compounds
into the surrounding soil (Vleesschauwer et al. 2009), potentially altering the com-
position of microbial communities. The effects of pathogenic fungi or biocontrol
agents on soil and rhizosphere microflora are poorly understood. Using biocontrol
agents against one pathogenic fungus may increase plant susceptibility to others, as
shown by using Serratia plymuthica to increase resistance of rice plants to blast
disease caused by Magnaporthe oryzae, which resulted in plants becoming more
sensitive to Rhizoctonia solani (Vleesschauwer et al. 2009).
Intensive breeding efforts aiming to increase the resistance of cultivated plants
to Rhizoctonia via traditional as well as transgenic technologies (Nagendran et al.
2009) may result in an exacerbation of antimicrobial effects in the rhizospheres of
new cultivars, which would affect not only the targeted phytopathogenic fungi but
may have significant (and potentially undesirable) effects on the microbial com-
munities in the rhizosphere. Hence, increasing attention needs to be given to the
effects of plant breeding in changing the structure of the rhizosphere microbial
communities as breeders strive for increased release and activity of antimicrobial
compounds in the rhizosphere.
Conclusions
Some plant breeding effects on soil microbes are more obvious than others e.g.
raising the tolerance of genotypes to pathogens will decrease their growth and sur-
vival in the soil or legume genotypes favouring selected Rhizobium genotypes
because they can only be colonized by this particular strain. As discussed in this
chapter however, plant breeding may radically change microbial community
composition and/or activity (Table 2, Fig. 1) in often unpredictable ways. Changes
in microbial community composition may or may not be associated with changes
in function, but changes in microbial activity are likely to change function (nutrient
release or immobilization, release of enzymes); the changes may be transient and
the result (e.g. net mobilization or net immobilization) is often unknown. However,
the high spatial and temporal variability of soil microorganisms and their activity
makes it unlikely that a particular plant genotype will have a consistent effect on
microbial community composition and activity.
Soil microorganisms and their activity are not affected only by plants, plant resi-
dues and root exudates but also by environmental factors such as climate and soil
properties (e.g. moisture, temperature and texture). Thus, a plant genotype may
have a strong effect on soil microorganisms in one set of conditions, but not in
others.
Because of this apparent variability of effect, it is essential to increase the
knowledge about specific plant genotype-microbe interactions so as to enhance our
understanding of long-term effects specific genotypes might have on the structure
and function of microbial communities. Such knowledge will guard against select-
ing crop genotypes that might result in losses to the diversity and/or functionality
of soil microbial communities. This is particularly important because microbial
activity and community composition in turn affect plant growth and nutrient
uptake. In parallel, the long-term negative effects of a plant genotype on soil micro-
organisms could be counterproductive to the undoubted benefits of plant breeding
as a route towards sustainable crop production.
Table 2 Potential changes in soil microorganisms induced by plant breeding (see text for
references)
Microbial community Microbial
Effect of plant breeding composition biomass Potential effects
Increased N and P Species with high N, Increase in Microbial biomass
availability P demand ↑ microbial C, turnover?
Species with low N, P N, P Priming effect?
demand ↓ Increased activity Loss of
Species mobilizing P, Species?
N2 fixers ↓ Diversity?
Inhibitory compounds Tolerant species ↑ Biomass ↓ Functionality?
e.g. isothiocynates, Susceptible species ↓ Activity ↓ Food web structure?
antibiotics
Lignin-degrading Biomass ↓
Higher lignin species ↑ Activity ↓
concentration in Species unable to
residues degrade lignin ↓
Fig. 1 Schematic diagram of effects of plant breeding on soil microorganisms during plant
growth and after harvest/plant death, 1 indicates effects of plant breeding on soil microorganisms,
2 indicates effects of soil microorganisms on plant growth and residue decomposition
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Chapter 9
Utilizing Soil Microbes for Biocontrol
Alison Stewart, Michael Brownbridge, Robert A. Hill,

and Trevor A. Jackson
Introduction
This review focuses on the potential for microbial biological control of soil-borne pests
and pathogens. The range of crops affected by key soil-borne pests and pathogens, and
opportunities for biocontrol using composts, organic soil amendments and/or augmen-
tation with selected bioactive microbes are discussed. Selected examples of biological
control successes, constraints and likely future developments are also explored.
The Role of Soil Microbes in Biocontrol
The benefits of adding organic soil amendments, especially composts and

mulches, to agricultural cropping systems has long been recognised. These
amendments have been shown to increase soil fertility and suppress a range of
soil-borne plant pests and pathogens. Additional benefits include water retention,
increased soil organic matter and enhanced soil aeration. There have been many
research studies on successful compost-related suppression of plant pests and
diseases, particularly plant collar and root rots, although some studies using
organic amendments have resulted in increased disease incidence (Hoitink and
Boehm 1999; Hoitink et al. 1996a). A key factor in disease suppression is the
A. Stewart (*) and R.A. Hill

Bio-protection Research Centre, Lincoln University, P.O. Box 84, Lincoln University,
Lincoln 7647, Canterbury, New Zealand
M. Brownbridge
Vineland Research and innovation Center, P.O. Box 8000, Vineland Station,
ON LOR 2EO, Canada
T.A. Jackson
316 A. Stewart et al.
degree of decomposition of organic matter in compost, which determines the

numbers and kinds of miroorganisms responsible for disease suppression (Hoitink
and Grebus 1994; Hoitink et al. 1996b). In this context, compost is defined as
organic matter that has undergone controlled aerobic biological transformation,
involving a heating cycle, resulting in pasteurisation and maturation. Mulching is
similar to the natural process of organic matter decomposition on the forest floor
and is defined as any organic material, composted or raw, that is placed on the
soil surface (see other Chapters in this Book).
Disease Suppression Using Composts
Under natural conditions, healthy soils are rich in microbes, many of which can sup-
press plant diseases. Provision of organic amendments to the soil, by ploughing-in
green manures or through the application of composts, can further promote disease
suppression. A variety of disease suppressive effects from composts have been
described and these effects comprise a combination of physiochemical and biological
characteristics. One mechanism is thought to be a result of toxic compounds released
from decomposing plant material (Gimsing and Kirkegaard 2006). However, disease
suppression reported in the literature is mostly attributed to the biological activity of
microorganisms, described as beneficial microorganisms or biological control agents.
Species reported to act as biocontrol agents in compost-amended substrates include
bacteria such as Bacillus spp., Enterobacter spp., Flavobacterium balustinum and
Pseudomonas spp., and fungi such as Penicillium spp., Gliocladium virens, and sev-
eral Trichoderma spp. (Litterick et al. 2004). These beneficial microorganisms can be
released from the compost or the compost may provide nutrients that stimulate the
proliferation of antagonistic bacteria and fungi in the rhizosphere (Noble and
Coventry 2005; Green et al. 2006). Four main mechanisms of disease suppression
have been described: competition for nutrients (between the microorganisms from
compost and soil pathogens); antibiotic and enzyme production by beneficial micro-
organisms; parasitism and predation by beneficial microorganisms of pathogens; and
enhanced plant disease resistance (both induced systemic resistance and systemic
acquired resistance). In addition, more vigorous growth on and better use of decaying
organic matter by beneficial microorganisms results in microbiostasis through the
repression of pathogen spore germination and growth. Several or all these different
mechanisms of disease suppression may occur simultaneously through the activity of
one or more beneficial microorganisms present in disease suppressive composts. For
example, several different Trichoderma spp. can compete with pathogens for nutri-
ents and space, exhibit antibiosis, parasitize the pathogen and elicit induced plant
resistance (Harman et al. 2004; Hill et al. 1999).
Natural disease suppression in some composts is due to the succession of
antagonistic microbes that flourish in the organic material. These suppressive
effects have been observed in a wide range of crops and soil-borne diseases,
including: Phytophthora root rot in avocado (Costa et al. 1996); Pythium in cotton
9 Utilizing Soil Microbes for Biocontrol 317
and turfgrass (Craft and Nelson 1996; McKellar and Nelson 2003); snow moulds
in turf grass (Boulter et al. 2002); Fusarium, Pythium and Rhizoctonia in tomato
and cucumber (Kannangara et al. 2004; Trillas et al. 2006), and many others in
cereal, fodder, vegetable and horticulture crops (Litterick et al. 2004; Noble and
Coventry 2005).
Composts can be pre-inoculated with beneficial microorganisms prior to application.
Use of inoculated composts not only promotes disease suppression, but utilises an inex-
pensive waste product, adds value to the composted product, and additionally provides
nutrients to the soil. Nakasaki et al. (1998) reported successful inoculation and growth
of Bacillus subtilis in composted grass-clippings. When applied to turf grass, the
Bacillus-augmented compost significantly reduced the severity of Rhizoctonia large-
patch disease. While it is generally accepted that general pathogen suppression is pro-
vided by a consortia of microbes, Nakasaki et al. (1998) demonstrated the potential to
inoculate compost with a specific bacterium to control a specific plant pathogen.
Bacillus-compost preparations applied against Phytophthora blight of bell pepper pro-
vided a biocontrol efficacy of 60% and increased yield by 200% through disease sup-
pression and growth promotion compared with untreated controls (Jiang et al. 2006).
This study highlights the dual action of some suppressive microbes as plant growth
promoters. Recent research suggests that Trichoderma-amended compost can reduce
the incidence of plant pathogens. Trillas et al. (2006) showed suppression of R. solani
in cucumber seedlings using compost inoculated with T. asperellum. A significant
reduction in bacterial leaf spot of radish, lettuce and tomato has also been observed
using bark compost inoculated with T. hamatum 392 (Aldahmani et al. 2005).
Pest Suppression Using Compost
Composts applied as a mulch not only suppress plant diseases and help protect and
conserve agro-ecosystem health and quality, but also suppress insects, weeds and
nematodes. Control of arthropod pests through mulch application occurs by increasing
densities and diversity of generalist predators in the soil and on plants, by reducing
environmental suitability for herbivore population growth (Brust 1994), or through
biofumigation, the release of toxic compounds from decomposing plant material
into the soil (Gimsing and Kirkegaard 2006). Most research concerning the inocula-
tion of compost/growing media with microbes has focused on the suppression of
insect pests in container-grown greenhouse and nursery ornamentals. Two major
insect pests, black vine weevil, Otiorhynchus sulcatus, and western flower thrips,
Frankliniella occidentalis, have been targeted in particular. The soil-dwelling stages
of both these insect species are susceptible to entomopathogenic fungi, which pos-
sess several desirable traits that make them suitable for development as compost
amendments. Use of the fungus Metarhizium anisopliae has been extensively stud-
ied for black vine weevil control in containerised production, and has provided
efficient control of the pest on a range of plant types (Moorhouse et al. 1992a, 1993a,
b; Bruck 2005, 2006). The fungus is effective in soil and a range of soil-less potting
media (including peat, bark, coir and peat-blends incorporating composted green
waste) whether applied as a drench or pre-mixed into the medium as conidia or
granules, although pre-mixing provides a more homogenous distribution of infective
material throughout the medium (Bruck 2006; Moorhouse et al. 1992b, 1993c;
Kepler and Bruck 2006; Shah et al. 2007). A strain of M. anisopliae active against
black vine weevil was shown to persist at infective levels for >4 months in all media
and considerably longer (approximately 1 year) in peat- and bark-based media
(Bruck 2005, 2006; Moorhouse et al. 1993c). Factors affecting pest and disease sup-
pression by composts include the raw materials used, the composting process, condi-
tions and microbial colonisation, the moisture content of the compost, compost
stability/maturity, compost nutrient content, timing of compost application and rate
of application and the longevity of disease suppression. The use of bioinoculated
composts or composted mulches offers potential in many cropping systems such as
orchards, soft or vine fruits, e.g., kiwifruits, strawberries, grapes. In addition to
improving overall soil health and providing favourable conditions for insect infec-
tion to occur and disease-suppressive organisms to flourish, composts can be seeded
with insect-active or antagonistic microbes and, thereby, provide an effective means
of managing pest and disease problems that would complement environmentally
sustainable production systems.
Suppressive Soils
Disease severity or incidence remains low in suppressive soils, in spite of the pres-
ence of the pathogen, a susceptible host plant, and climatic conditions favourable
for disease development. This suppressiveness is attributable to microorganisms
and has been divided into two main categories: general suppression, which is asso-
ciated with the total amount of microbial activity in relation to the pathogen; and
specific suppression, which is attributable to the activity of one or a few inhibitory
microorganisms (Hoitink and Boehm 1999; Cook and Baker 1983).
Take-All Suppression
Take-all is an important root disease of grasses and cereal crops, especially wheat
and barley, worldwide and is caused by the fungus Gaeumannomyces graminis var.
tritici (Hornby et al. 1998; Asher and Shipton 1981). Significant yield reductions
can occur after two successive crops in the same field as a result of take-all disease
(MacSpadden Gardener and Weller 2001). However, in wheat cropping areas where
monoculture is practiced, a severe outbreak of take-all followed by 4–6 years of
wheat or barley monoculture can result in a natural disease suppression called take-
all decline (MacSpadden Gardener and Weller 2001). Take-all decline is thought to
be due to specific suppression of the disease with research showing that disease
suppression is transferrable and can multiply (Cook 2003, 2007). Indeed, field plots
inoculated with take-all produced good disease symptoms. However, plots treated
at tillering with “starter” soil (0.5% by volume) from soils with 12 years wheat
monoculture produced plants with healthy looking roots and stems with natural
biological control being demonstrated as the cause of this take-all decline (Cook
2007). Specifically, strains of the rhizosphere-inhabiting bacterium Pseudomonas
fluorescens that produce the broad spectrum antibiotic 2,4-diacetylphoroglucinol
(DAPG) are responsible (Cook 2007). The genes associated with this antibiotic
synthesis have been identified allowing the molecular detection and quantification
of these bacterial strains in take-all suppressive soils (Weller et al. 2002). The
threshold for P. fluorescens DAPG producing strains to cause take-all suppression
was shown to be 105 CFU g -1 root (Weller et al. 2002). The elimination of these
bacterial strains from the soil has been shown to stop take-all suppression, but it
can be restored by their reintroduction (Cook 2007). DAPG-producing strains of
P. fluorescens have also been shown to suppress soil-borne diseases in different
host/pathogen systems (Cook 2007). In New Zealand wheat soils, particular species
of actinomycetes, fungi and bacteria (including P. fluorescens) were associated
with take-all suppressive soils (general and specific) (Chng 2009).
Fusarium Suppressive Soils
Soil suppressiveness to Fusarium, in most instances, has been attributed to biotic

characteristics, although soil physiochemical factors are also involved (Amir and
Alabouvette 1993; Hoper et al. 1995). Suppressiveness is lost through sterilisation
and may be restored by the addition of a little untreated suppressive soil (Whipps
1997), while the addition of organic amendments and plant residues resulted in
suppression of diseases caused by Fusarium oxysporum in field soils (Lodha 1995).
A variety of different mechanisms have been demonstrated in this suppression,
including general suppression (Serra-Wittling et al. 1996), specific antagonism
(Trillas-Gay et al. 1986), propagule lysis (Sequeira 1962) and induced systemic
resistance (Pharand et al. 2002; Larkin et al. 1996). For example, conifer litter on
the forest floor induced germination and subsequent destruction of macroconidia
and chlamydospores of F. oxysporum (Toussoun et al. 1969). Competition for
substrate in the soil, often by non-pathogenic fusaria (Toyota et al. 1996), and for
root infection sites (Olivain and Alabouvette 1999) contributes to suppressiveness
in some soil, while antibiosis has been implicated in suppression of Fusarium wilt
in peas (Weller et al. 2002).
Augmentation with Selected Bioactives
The soil can be augmented with selected microbes to bring about the biological
control of soil-borne pests and pathogens. There are numerous comprehensive
reviews on specialised topics describing the biocontrol activities of different
microorganisms against plant pests and pathogens (Van Driesche and Bellows
1996; Vincent et al. 2007; Jaronski 2007). In this review section, we highlight
specific examples of successful augmentation for biocontrol of insect pests, plant
pathogenic nematodes and plant microbial pathogens.
Insect Pests
Entomopathogenic Fungi
Fungi are the predominant pathogens of insects and play a significant role in the
natural regulation of soil-dwelling pests. Among all of the disease-causing microbes
of insects, fungi are unique in their ability to infect their hosts through the external
cuticle. Several hypocrealean entomopathogenic species are important constituents
of the soil biota and appear to be ubiquitous inhabitants of soils worldwide, having
been recovered from a diverse array of geographic, climatic and agro-ecological
zones. Species such as B. bassiana and M. anisopliae are commonly found in both
cultivated and undisturbed soils, although their natural distribution does appear to
be linked to habitat (Meyling and Eilenberg 2006) and soil populations are influ-
enced by agricultural practices (Jaronski 2007; Meyling and Eilenberg 2007).
Fungi have been widely evaluated as control agents for a variety of noxious
arthropods of agricultural and horticultural importance. They have many desirable
traits including; minimal risk posed to beneficial non-target species such as
earthworms and Collembola, which are key service-providers in the soil ecosystem
(Brownbridge and Glare 2007; O’Callaghan and Brownbridge 2009), and host-
specificity, which enhances their potential role in integrated pest management
(IPM). Furthermore, these beneficial fungi can persist in the soil for several years
with new ‘flushes’ of inoculum provided following the successful infection and
colonization of a susceptible host. This leads to high, localized concentrations of
infective conidia and greater opportunities for insect infection to occur. Long-term
survival of entomopathogenic fungi in soil does appear to be reliant upon their
access to susceptible hosts, as they are generally considered poor saprophytes in the
competitive soil environment (Jaronski 2007; Keller et al. 2003). For those species
with relatively narrow host-spectra, this can limit their natural occurrence and lon-
gevity in treated soils (Meyling and Eilenberg 2007; Keller et al. 2003).
A diverse range of subterranean pests have been targeted for biocontrol. The
majority of these initiatives have sought to use fungi as inundative control agents,
an approach which is not reliant upon fungi recycling within the pest population.
The primary candidate organisms have been M. anisopliae, B. bassiana, and
B. brongniartii and several commercial products and proto-products have been
developed (de Faria and Wraight 2007). Here, we will focus on biocontrol initia-
tives targeting insects, which cause economic losses through direct feeding damage
to roots or root crops, although examples of initiatives against other important spe-
cies with a soil-dwelling phase in their life cycle are included.
Fungi have been widely tested against root-feeding Coleoptera in particular,

including pests of agricultural and horticultural importance. Beauveria brongniartii
is used in grassland regions of Austria, Italy and Switzerland to control the
European cockchafer, Melolontha melolontha (Keller et al. 2003; Keller 2000).
Formulated on colonized barley kernels, the fungus is applied to the soil (pastures,
grasslands, forests, orchards, etc.) via modified seed drills, and suppresses
cockchafer populations for several years. In trials to assess effects of B. brogniartii
on non-target organisms, the fungus was considered low-risk for a predatory
carabid, Poecilus versicolor, which was exposed directly to different pathogen
formulations and to mycosed cockchafer cadavers (Traugott et al. 2005). Thus,
while active against M. melolontha, the pathogen does not appear to affect
coleopteran natural enemies in the targeted ecosystem. The red-headed cockchafer
(Adoryphorus coulani), a destructive root-feeding pest of pasture and field crops in
Australia that was recently introduced into New Zealand, has been successfully
controlled with a strain of M. anisopliae specifically selected for its activity at
cooler temperatures and a commercial product (ChaferGuard®; Becker Underwood
Pty Ltd, Australia) was developed (Rath et al. 1995). The fungus is produced on
rice and formulated on the grains for application to soil using a seed drill at the time
of pasture renovation (Rath et al. 1995; Rath 2002). In field trials, the pathogen
regulated cockchafer populations in treated soils for more than 3 years, delivering
a 23% increase in pasture productivity with no apparent impact on non-target
invertebrates (Rath et al. 1995). Control of the New Zealand grass grub, Costelytra
zealandica, may also be possible with M. anisopliae or B. bassiana, as larvae are
hosts to these species and natural epizootics are periodically observed (Glare et al.
1994; Townsend et al. 1995). However, insect infection by a selected M. anisopliae
isolate was inhibited at 15°C, indicating the necessity of matching a candidate
strain to the prevailing environmental conditions in which it will be used (Glare
et al. 1994). Research has demonstrated the virulence of an exotic B. bassiana
isolate for clover root weevil, Sitona lepidus, a pest that has a severe impact on the
productivity of New Zealand white clover-ryegrass pastures (Gerard 2001). This B.
bassiana isolate is easily mass-produced on rice and persists in treated soils with
farm-scale field trials on a prototype grain-based formulation drilled into clover
root weevil-infested pasture significantly reducing larval and pupal populations
without impacting non-target species (O’Callaghan and Brownbridge 2009;
Brownbridge et al. 2006). Metarhizium anisopliae and B. bassiana are frequent
natural pathogens of the black vine weevil, O. sulcatus, and M. anisopliae has
provided excellent control of this pest, as discussed previously.
The larval stages of several dipteran species also cause direct economic damage
through their feeding activity on root- and other field-crops. Few chemical control
options are available for these pests and concerns over resistance, pesticide residues
and environmental impacts have prompted the search for more benign alternatives,
including entomopathogenic fungi. Sugarbeet root maggot, Tetanops myopae-
formis, is a major problem for North American sugarbeet growers. Metarhizium
anisopliae successfully reduced feeding damage and increased yield under low to
moderate root maggot pressure in infested sugarbeet fields (Campbell et al. 2000).
The effectiveness of M. anisopliae treatments was further demonstrated in trials

devised to evaluate effects of formulation and application rate on efficacy
(Campbell et al. 2006). Although higher yields were obtained under an intensive
insecticide program, particularly in areas where root maggot infestation levels were
high, the fungus was seen as being complementary to an insecticide regime in such
situations and integration of the two strategies could reduce overall pesticide use
(Campbell et al. 2006). Larvae and adults of the onion maggot, Delia antiqua, are
also susceptible to M. anisopliae and promising isolates have been identified in
laboratory trials (Davidson and Chandler 2005). These strains were also pathogenic
to cabbage maggot, D. radicum, a primary pest of brassicas in northern temperate
regions (Chandler and Davidson 2005). Drench applications of the fungus reduced
survival of cabbage maggot larvae and pupae by up to 90% and adult emergence
by up to 92% in glasshouse trials. However, when applied to infested cauliflowers
in the field, the fungus did not control D. radicum populations, although 30% of the
larval cadavers recovered from treated plots were infected (Chandler and Davidson
2005). Advances in formulation and delivery techniques were considered critical to
improving the efficacy and cost-effectiveness of this control approach in all of the
above examples (Jaronski 2007; Campbell et al. 2006; Chandler and Davidson
2005).
Several pest species cause no crop damage during the soil phase of their life
cycle. These stages are often targeted with insecticides that could be replaced with
fungal treatments. Ekesi et al. (2005) showed that fungi were effective against soil-
dwelling stages of three fruit-fly species in this pest complex. In field trials,
emergence of adult fruit flies were significantly reduced over an extended period in
soils treated with M. anisopliae formulations with this treatment providing superior
control to diazinon. Furthermore, M. anisopliae had no adverse effects on the non-
target fruit-fly parasitoids (Ekesi et al. 2005) and shows great promise for use in
IPM strategies for this pest (Ekesi et al. 2007).
Western flower thrips (F. occidentalis), a pest of global significance on a wide
range of economically important crops, largely pupate off-plant in the soil. Although
western flower thrips are susceptible to B. bassiana and I. fumosorosea in peat and
other plant-growing media, M. anisopliae has consistently provided the best levels
of control (Ansari et al. 2007, 2008; Butt and Brownbridge 1997). Indeed, under
greenhouse conditions M. anisopliae was effective when applied either as a drench
or pre-mixed into the medium as dry conidia, and was more efficacious than either
fipronil or impidacloprid treatments(Ansari et al. 2007, 2008).
Social insects are often considered poor targets for microbial control owing to
their hygienic behaviour. However, opportunities exist to use fungi against
subterranean termites. Isolates of B. bassiana, M. anisopliae and Paecilomyces
appear to be non-repellent and capable of infecting and being transmitted among
termites, causing rapid mortality in exposed individuals (Milner et al. 1998; Sun
et al. 2003; Wright et al. 2002, 2005). Direct inoculation of termite mounds in
Australia with M. anisopliae successfully controlled termite populations (Milner
et al. 1998), while in Kenya, application of M. anisopliae at maize planting signifi-
cantly reduced termite lodging and increased grain yield (Maniania et al. 2002).
Entomopathogenic Bacteria
The soil-dwelling larvae of the Scarabaeidae often feed on plant roots, causing
significant losses in economically important crops worldwide. These larvae have
proven especially difficult to control with all but the most toxic and persistent of
insecticides. However, they are hosts to a wide range of pathogenic bacteria, some
of which have been used in augmentative biological control (Jackson and Glare
1992). Milky disease, named from the milky colouration that develops after colo-
nisation of the insect haemolymph by strains of the bacterium, Paenibacillus popil-
liae, is unique to scarab larvae. Although milky disease bacteria are frequently
found in scarab populations, the disease rarely controls insect populations under
natural conditions. Resistant spores, which can survive for many years in the soil,
are released from the host after death. To increase the impact of this pathogen,
milky disease bacteria have been cultured and introduced into turf for control of
Japanese beetle (Popillia japonica) in the United States since the 1940s (Fleming
1968). Formulations of P. popilliae have been commercialised, but use of the bac-
terium has been limited by the high cost of in vivo production and the slow progres-
sion of disease. It has been suggested that the bacterium would be best used as an
inoculative agent within wider pest management programmes rather than as a
biopesticide to provide immediate control (Redmond and Potter 1995).
Strains of the non-spore forming bacteria, Serratia spp., cause amber disease in
the New Zealand grass grub, C. zealandica (Jackson et al. 2001). Pathogenic strains
contain a specific plasmid (Hurst et al. 2000), and ingestion brings about a cessa-
tion of feeding, clearance of the gut and eventual death of the larval host. Cultivation
of pastures for cropping and resowing generally kills grass grub and eliminates
pathogenic strains of bacteria, leaving new pastures vulnerable to pest attack. This
provides an opportunity for biological control, where artificially produced bacteria
can be applied to healthy grass grub populations to promote epizootics of disease
and prevent pasture damage. Strains of S. entomophila have been commercialised
as the products Invade™ and Bioshield™ and successfully used for grass grub
biological control for more than a decade (Jackson and Klein 2006). For best effect,
the bacterium is applied to a healthy grass grub population in young pastures.
Recycling of the disease through the pest population provides a reservoir of patho-
genic bacteria that prevent damaging outbreaks of the pest.
Antibiosis/Toxins
Insecticidal toxins are produced by most entomopathogenic fungi during

pathogenesis. After successfully penetrating the insect cuticle, the fungi enter the
haemocoel where they have to overcome insect immune responses in order to
colonize and kill the host. Generally, these toxins are bioactive secondary metabo-
lites secreted during growth inside the insect. The toxins can have a range of
effects, and they are considered important pathogenicity determinants (Strasser
et al. 2000). Toxic (and other) effects of metabolites produced by M. anisopliae
(destruxins), B. bassiana (e.g., oosporein, beauvericin, bassianolide, oxalic acid)

and Tolypocladium spp. (efrapeptins) have been well documented in insects and
other arthropod pests (Vey et al. 2001; Charnley 2003; Kirkland et al. 2005;
Quesada-Moraga et al. 2006; Zimmermann 2007a, b). Attempts to increase the
potency of M. anisopliae through genetic manipulation have recently focused on
production of isolates to express scorpion neurotoxin during colonization of the
insect hemolymph (Wang and St Leger 2007).
The colonisation of plants by endophytic fungi can provide protection from
insect predation via in planta production of metabolites by the endophyte. These
metabolites can be directly toxic to the pest, inhibit insect development, or deter
feeding activity. These associations tend to be beneficial to plant survival and
include provision of protection against herbivorous insects and plant-parasitic
nematodes (Vega et al. 2008). Many economically important turf and forage grasses
are naturally infected with endophytes, and breeding programmes have provided
lines that contain selected strains. For example, Neotyphodium uncinatum produces
loline alkaloids in meadow fescue (Festuca pratensis), which are expressed
throughout the plant (Bush et al. 1993). The alkaloids have bioactivity against a
range of insects conferring resistance to foliar and root aphids, while deterring
larval grass grub feeding and development. Murphy et al. (1993) observed such
effects on scarab grubs in tall fescue (F. arundinacea), while Clement et al. (2005)
found that Neotyphodium-infected wild barley induced a range of responses to
different insect pest species.
Some entomopathogenic fungi are also endophytic (Vega et al. 2008). Beauveria
bassiana for example, is an endophyte in a diverse assortment of host plants. In all
cases, plants have been protected against insect damage yet mycosed insects have
not been recovered. This evidence suggests that the suppressive effects are a result
of feeding deterrence or antibiosis due to fungal metabolites produced in plant
tissues, or direct toxic effects following consumption of these metabolites or
vegetative mycelia. To date, evaluations have concentrated on effects against foliar-
feeding pests. Effects against root-feeding insect pests have not been determined,
but could provide a novel means of protecting crop plants.
Although soil-inhabiting insects appear to be resistant to most strains of Bacillus
thuringiensis (Bt), the organism is the source of a wide and expanding range of Bt
toxins. Resistance to Bt toxins may have evolved through close association of
larvae with bacterial spores in the soil. However, one isolate, the Buibui strain, has
been shown to be effective against some scarab pests, including Japanese beetle
(Alm et al. 1997). It has been suggested that a protein/spore mix is necessary to
induce mortality suggesting a role for the live cells beyond that of the toxin alone.
Strains of B. thuringiensis serovar. israelensis are now being produced for control
of tipulid larvae in pastures suggesting that, as a wider range of strains is discov-
ered, there will be more uses for this bacterium.
The Tc toxin complex is another important toxin group that has been discovered
associated with soil bacteria. This complex is found in strains of the nematode-
associated bacteria Xenorhabdus and Photorhabdus spp., as well as soil bacteria
from the Enterobacteriaceae. While many strains of Tc-producing bacteria are
weakly pathogenic to insects, a novel bacterium, Yersinia entomophaga, has a wide

insect host range and is capable of killing late instars of several soil dwelling pest
species (Hurst et al. 2007). Bacterial toxins applied to the soil face similar problems
to many chemicals by inactivation through adsorption or microbial activity. These
problems may be overcome by formulation of the toxins into resistant granules and
baits (Brownbridge et al. 2006).
Plant Parasitic Nematodes
Plant-parasitic nematodes cause yield losses worth billions of dollars each year.
Currently, crop rotations with resistant or non-susceptible cultivars, combined with
applications of nematicides, are used to regulate these pests. Nematicides can
provide short-term gains in productivity. However, due to the ability of nematode
populations to build up rapidly, the benefits are limited in both space and time. The
ban on the soil sterilant methyl bromide has provided additional impetus to search
for alternative, sustainable management options.
Nematodes are regulated by a diverse array of natural enemies including fungi
and bacteria, as well as invertebrate predators. Considerable research has been
done to evaluate different microbial control agents, yet few commercial products
have resulted and none of these are in widespread use. There are many reasons
for this, but the primary difficulties have been related to development of efficient
mass production systems for the innoculative or innundative use of these organ-
isms, limited host range, poor understanding of host/pathogen interactions, and
lack of good ecological data to develop products that deliver consistent levels of
suppression (Dong and Zhang 2006).
Microbial biocontrol agents of plant-parasitic nematodes appear to have a
cosmopolitan distribution in soils worldwide. Their prevalence is generally corre-
lated with high host nematode populations, but cropping sequence, tillage practices
and ability to survive as a saprophyte also influence the incidence of these infec-
tious or parasitic organisms. Microorganisms investigated for control include a
range of nematode-trapping species, such as Arthrobotrys and Dactylaria spp.
(Davies 2005; Singh et al. 2007) and infectious microbes, broadly divided into
those that are facultative pathogens, such as the fungi Paecilomyces lilacinus and
Pochonia chlamydosporia (formerly Verticillium chlamydosporium) (Kiewnick and
Sikora 2006; Pérez-Rodriguez et al. 2007), obligate parasites, such as the fungi
Hirsutella rhossiliensis and H. minnessotensis (Chen and Liu 2005; Mennan et al.
2006), and bacterial antagonists, such as Pasteuria penetrans and Pseudomonas
oryzihabitans (Vagelas et al. 2007).
Nematode-trapping (‘predatory’) fungi from various taxonomic groups have
been widely studied for control of root-knot and cyst nematodes. Numerous surveys
have been done to document associations with important plant-parasitic species of
nematodes and to determine their prevalence in different crops and soils. These
organisms have a diverse range of trapping devices, which include adhesive hyphae,
adhesive branches and knobs, and more complex multi-dimensional networks and
constricting rings (Davies 2005). Formation of capture organs can be stimulated by
the presence of nematodes, but the fungi generally respond to a limited host spec-
trum in both trap formation and initiation of a trapping response (Davies 2005).
Nematophagous species also produce compounds that attract some nematodes
while being repellent to others; this is related to the developmental and nutritional
status of the fungus, and the nematode species (Davies 2005). All of these factors
influence the efficacy of these fungi and, together with difficulties associated with
the large-scale production and preservation of infectious inoculum for field-scale
application, have restricted their development into reliable biological control agents
for plant-parasitic nematodes.
As with insect pathogenic microbes, many nematophagous fungi and bacteria
produce toxins and enzymes that play a key role in nematode infection and mortality,
which are often primary virulence factors. Other microbes produce compounds that
are directly toxic or show antagonistic effects against nematodes. Although the
broad-spectrum, proteinaceous endotoxin produced by B. thuringiensis is better
known for its insecticidal activity, some Bt proteins are also toxic to free-living and
parasitic nematodes (Crickmore 2005; Tian et al. 2007). Their potential utility
against plant-parasitic species has not been fully investigated. Rhizosphere-
colonizing bacteria and fungi can reduce populations of plant-parasitic nematodes
by regulating nematode behaviour, interfering with plant-nematode recognition, or
by direct antagonism through the production of toxins, enzymes and other metabo-
lites (Tian et al. 2007). Several Bacillus spp. are pathogenic (e.g., B. subtilis), while
metabolites produced by rhizosphere-competent microbes, e.g., Pseudomonas spp.,
have antagonistic effects such as inhibiting egg hatch, and reducing survival and
mobility of infective second-stage juveniles. Suppressive effects have been obtained
with culture filtrates of the rhizo-competent bacterium, Burkholderia cepacia, and
Trichoderma fungi against root-knot nematode, providing additional evidence that
extracellular factors produced by these organisms are responsible for the observed
results (Meyer et al. 2000; Sharon et al. 2001). Although Trichoderma spp. are more
commonly used as biocontrol agents for soil-borne diseases, these findings indicate
potential for a multi-functional role in plant bioprotection. The promise shown by
several rhizobacteria has led to the development of commercial bionematicides
based on Bacillus spp. and B. cepacia for control of root-knot and other nematodes
on vegetables (Tian et al. 2007). Nematode suppression may also be related to the
development of systemic resistance induced by the presence of the bacteria in the
rhizosphere. Endophytic bacteria and fungi can also induce resistance, and secondary
metabolites secreted by the microbes are thought to play an important role in this
phenomenon (Siddiqui and Shaukat 2003; Siddiqui et al. 2005; Dababat and Sikora
2007; Tesfamariam et al. 2009). Interestingly, applications of entomopathogenic
nematodes have also reduced plant-parasitic nematodes; the cause of this effect has
been attributed to allelochemicals produced by the nematodes or their associated
symbiotic bacteria (Somasekhar et al. 2002).
The greatest progress towards development of innundative controls has been
achieved with fungi and bacteria that parasitize female nematodes or infect eggs.
Various Pasteuria spp. are obligate parasites of a wide range of economically

important plant-parasitic nematodes (Davies 2005; Trudgill et al. 2000). High
incidence of P. penetrans has been associated with soils that are suppressive to root-
knot species and control has been achieved in some soils when amended with the
bacterium (Trudgill et al. 2000). However, commercialization of the bacterium as a
bionematicides has been hindered by the inability to culture the organism outside a
nematode host and its host specificity (Davies 2005; Davies et al. 2001). Recent
advances in in vitro production techniques, combined with research to understand the
mechanisms behind endospore attachment and specificity may allow these hurdles to
be overcome in the future (Davies 2005; Davies and Opperman 2006).
Both P. lilacinus and P. chlamydosporia (V. chlamydosporium) parasitize nema-
tode eggs and have been widely studied for use against root-knot and cyst
nematodes. In spite of early concerns around potential human-health risks associ-
ated with P. lilacinus, commercial products based on the fungus are now registered
for use on a variety of field and greenhouse crops in several countries. The fungus
is readily mass-produced on grains, but the high application rates required with
early products, combined with inconsistency of efficacy, have inhibited their wider
adoption (Kiewnick 2004). This prompted investigations into factors affecting host
specificity and surveys for strains with broader host spectra (Kiewnick 2004;
Freitas et al. 1995). Pochonia chlamydosporia is frequently present in nematode-
suppressive soils; it colonizes the rhizosphere of some plants and proliferates in egg
masses produced by root-knot nematodes. The fungus lends itself well to develop-
ment as a myconematicide; optimal growth conditions have been defined on a
variety of substrates and the organism produces resistant chlamydospores that are
easily handled and applied for nematode suppression (Pérez-Rodriguez et al. 2007).
[Sustainable strategies have been developed for the management of root-knot
nematodes in organic vegetable production systems by integrating applications of
P. chlamydosporia with the cultivation of plants that are poor nematode hosts, soil
infestations before planting a susceptible crop in the following season (Kerry
and Hidalgo-Diaz 2004)]. The strain used in these studies has subsequently been
registered as a bionematicide in Cuba, and similar development programmes have
been followed in several other countries (Pérez-Rodriguez et al. 2007).
Hirsutella rhossiliensis is an efficient natural pathogen of nematodes and
has been recovered from many plant-parasitic species. The fungus has effectively
suppressed nematode populations in greenhouse and small-plot studies, but field-
scale trials have provided variable results (Chen and Liu 2005; Jaffee 2000).
Addition of organic amendments to soil generally led to a reduction in nematode
parasitism rates by H. rhossiliensis in spite of the fact that there was an increase
in bacterivorous and fungivorous nematodes in the treated soils, demonstrating
the importance of host species in fungal dynamics (Jaffee et al. 1994). This
observation was borne out by trials to evaluate effects of crop sequence (nematode-
susceptible versus non-susceptible) and tillage on nematode parasitism by the
fungus, which demonstrated that observed effects could largely be attributed to
the density-dependent relationship between the fungus and its host, whereas
tillage practices appeared to have only a minor effect (Chen and Liu 2007).
Hirsutella minnesotensis also has a wide nematode host range and shows promise
as a biocontrol agent for the soybean cyst and root-knot nematodes (Chen and Liu
2005; Mennan et al. 2006, 2007).
Microbial Plant Pathogens
Soil-borne diseases can cause major economic losses on a wide range of fruit, vegetable,
arable, pasture and forest crops. Fungi are the main causal agents with important
pathogenic species found within several genera including Sclerotinia, Sclerotium,
Fusarium, Pythium, Rhizoctonia, Phytophthora, Verticillium and Gaeumannomyces.
Selected bacterial genera such as Agrobacterium, Erwinia and Pseudomonas also
include plant pathogenic species. Soil-borne diseases are often difficult to control
because of the pathogens’ ability to produce persistent survival structures that allows
populations to build up over time. There are few disease resistant cultivars available
to growers and control has relied on the use of soil-based pesticides. However, issues
with loss of efficacy due to fungicide resistance or enhanced microbial degradation
and health and environmental concerns leading to use restriction and/or deregistra-
tion has provided the impetus for the development of biological control alternatives
(Martin 2003). The last decade has seen an increase in the number of commercial
products brought to the market and these are now being integrated into mainstream
production practices (Fravel 2005; Stewart 2001).
In contrast to the predominantly parasitism-based methods of biocontrol of insect
pests and plant parasitic nematodes, biocontrol of microbial plant pathogens can be
achieved by a number of different strategies, both direct and indirect. The direct
approach involves the introduction of specific microbes into the soil where they
interfere with pathogen survival, growth and/or plant infection. There are four direct
mechanisms of biological control of plant diseases; parasitism when the biocontrol
agent feeds directly on or inside the pathogen; antibiosis, which is the inhibition of
the pathogen by a toxic metabolite produced by the antagonist; competition with the
pathogen for limited resources; and hypovirulence, which is the use of less virulent
strains of the pathogen to control the virulent strain (Chernin and Chet 2002).
Indirect methods include induced resistance where the biological control agent
activates physical and/or chemical self-defence responses within the host plant
against a particular pathogen. All biological control agents utilize one or more of
these general indirect or direct mechanisms and the most effective biological control
agents often use two or three different mechanisms. In this section, we will highlight
successful examples of microbes that utilise these different biocontrol strategies.
Parasitism
A parasite is an organism that lives either in or on its host, the latter serving as both
the habitat and the substrate. In this relationship, the parasite obtains nutrients and
energy from the host and the host is usually exploited to such an extent that its
metabolism is adversely affected resulting in inhibition of growth and development,

and in some circumstances, death. Parasitism of one microorganism upon another
is common within the soil environment and the interaction can range from non-
specific to highly host-specific.
In 1932, Weindling (1932) reported that Trichoderma lignorum could parasitize
a number of soil-borne fungi in culture and suggested controlling certain patho-
genic fungi by augmenting soil with an abundance of this parasite. This promul-
gated the idea of using mycoparasites for biological control. Two broad types of
mycoparasitic interactions are recognised; namely biotrophic mycoparasitism,
which is an obligate relationship whereby the parasite obtains nutrients from living
cells with little or no apparent harm to the host, and necotrophic mycoparasitism,
where the parasite destroys the host before, during or after invasion and utilises
nutrients from the dying/dead host. Commercial exploitation of mycoparasites for
biocontrol of soil-borne pathogens has principally been restricted to the latter
category (Cortes-Pentagos et al. 2007). The most intensely studied of these latter
mycoparasites include Trichoderma spp., Gliocladium virens, Coniothyrium min
itans, Pythium oligandrum, Sporidesmium sclerotivorum, Talaromyces flavus and
Laetisaria arvalis. All of these fungi have been reported to have potential for use
in biocontrol against specific pathogens under certain conditions (Adams 1990).
The broad aspects of mycoparasitism have been reviewed recently by Lartley
(Lartey and Conway 2004). Here we cite a number of specific examples to illustrate
the range of parasitic interactions that have been investigated and highlight those
with the most commercial potential.
One of the most studied mycoparasites is the soil-borne saprophytic fungus,
Trichoderma. This genus contains a number of species that act as mycoparasites
against a range of economically important soil-borne plant pathogens and have
been successfully used in the field to control plant disease. The sequential events
involved in mycoparasitism by Trichoderma are described by Chernin and Chet
(2002). Trichoderma can detect its host pathogen from a distance and the
mycoparasite alters its growth pattern to grow towards the host in a positive
chemotropic fashion (possibly related to a chemical gradient). Recognition is
brought about by interactions between complementary molecules present at the
surface of both the host and the parasite. Such recognition processes have been
shown to include lectin-carbohydrate interactions. Lectins are sugar-binding
proteins/glycoproteins that agglutinate cells. Elad et al. (1983a) demonstrated the
role of lectins in the host-mycoparasite interaction between T. harzianum and
Rhizoctonia solani, a soil-borne pathogen that causes damaging root, stem-base and
tuber rots of a wide range of crop species. Barak et al. (1986) further investigated
the role of lectins in the Trichoderma-Rhizoctonia interaction and demonstrated the
presence of L-fucosyl residues on the Trichoderma cell-wall surface that could
serve as receptors for the Rhizoctonia agglutinin. Using a biomimetic system based
on nylon fibres designed to mimic pathogen hyphae, Inbar and Chet (1992)
provided direct evidence for the involvement of lectins in mycoparasitism.
Following recognition, Trichoderma hyphae attach to the host pathogen via hook-
or appressorium-like structures and grow along or coil around the pathogen hyphae.
Elad et al. (1983b), using T. harzianum and R. solani/S. rolfsii model systems,
revealed lysed sites and penetration holes in the hyphae of the host fungus caused
by the antagonist attaching and coiling around it. These observations suggested the
de novo synthesis of lytic enzymes produced during the early stages of interaction
with the pathogen and their secretion at the interaction sites resulting in degradation
of the cell wall. This production of lytic enzymes has been shown to be a crucial
property of mycoparasitic fungi. The role of lytic enzymes, in particular, chitinolytic
enzymes, in fungal mycoparasitism and biocontrol activity are discussed in several
contemporary reviews (Lorito 1998; Manocha and Govindsamy 1998; Steyaert
et al. 2003).
Another successful example of hyphal parasitism occurs between the
mycoparasite Pythium oligandrum and pathogenic Pythium spp. (e.g., ultimum).
With this mycoparasitic fungus, contact occurs at random with no apparent directed
growth of the mycoparasite towards the host. Parasitism involves lysis of the host
hyphae and granulation of the cytoplasm. This can often occur very rapidly within
seconds of penetration of the host by the mycoparasite. The hyphae of the
mycoparasite then grow throughout the mycelium of the host (Laing and Deacon
1991). P. oligandrum has shown biocontrol activity against a number of important
soil-borne plant pathogens in the field. For example, oospore preparations of P.
oligandrum applied as a seed treatment have reduced the incidence of P. ultimum
damping-off of sugarbeet (Veseley and Hejdanek 1984) and several commercial
products have been developed. For example, Polyversum marketed by Biopreparaty
Ltd, Czech Republic can be used as a seed treatment or soil spray to control
clubroot of brassica caused by Plasmodiophora brassicae, Sclerotinia root rot and
Leptosphaeria black leg of canola and damping-off diseases of vegetables (www.
polyversum.cz).
Some fungal pathogens survive for many years in soil by forming black rest-
ing structures called sclerotia. These are long lived (2–8 years) and very diffi-
cult to eradicate once they have infested a soil, thus making them suitable
targets for biocontrol using mycoparasites that are able to attack the sclerotia.
A large number of sclerotial mycoparasites have been reported as potential
biocontrol agents. For example, Trichoderma spp. and G. virens against patho-
gens such as Sclerotium rolfsii, Sclerotium cepivorum, Sclerotinia minor,
Sclerotinia sclerotiorum and R. solani; C. minitans against sclerotia of
Sclerotinia and Sclerotium spp. and S. sclerotivorum against mainly Sclerotinia
spp. (Chet 1990). All of these organisms have been investigated as potential
biocontrol agents with good success achieved in some circumstances (Jeffries
and Young 1994). For example, Coniothyrium can destructively parasitize the
sclerotia of S. sclerotiorum, S. minor, S. trifoliorum, S. cepivorum and
B. cinerea (Jeffries and Young 1994). Coniothyrium was able to parasitize and
kill sclerotia of S. sclerotiorum produced in the soil, on the root surface, inside
the root and within the stem base of sunflower plants resulting in significant
reductions in pathogen inoculum levels (Jeffries and Young 1994). Its use as a
biocontrol agent for control of S. sclerotiorum has been widely investigated
(Whipps et al. 2004; Gerlagh et al. 2003). Two products have been commercia-
lised – Contans® (Prophyta GmbH, Germany), and Koni® (Hungary). In both
cases, the biocontrol agent can be applied directly to soil containing sclerotia
or as a spray on diseased crops. Contans WG, a water dispersable granule
containing 1 × 109 conidia g –1 substrate is used as a soil application to infect
S. sclerotiorum and prevent apothecial production (Luth 2001). Soil application is
made several months before growing the crop, with conidia mixed into the upper
5 cm soil layer (Whipps and Gerlagh 1993).
From this extensive work on sclerotial mycoparasites, a number of key attributes
have been identified that determine how effectively sclerotial numbers are reduced
in the soil, including; the parasite’s intrinsic parasitic ability, its growth potential in
soil, activity over a range of environmental conditions and survival and reproduc-
tion potential. The biggest obstacles to utilising mycoparasites for practical disease
control are the large quantity of the biocontrol agent necessary to achieve biocon-
trol when applied directly to soil in the field, and the biocontrol agent’s ability to
reproduce on the host and redistribute itself within the soil. It is clear that in order
to achieve successful biocontrol with a mycoparasite detailed knowledge of the
density and distribution of the pathogen in the soil and the efficiency of parasitism
is required.
Antibiosis/Toxins
Antibiosis is antagonism mediated by specific or non-specific metabolites of micro-

bial origin, by lytic agents, enzymes, volatile compounds or other toxic substances.
Antibiotics are low molecular weight compounds produced by microbes, which are
active at low concentrations and which inhibit the growth or metabolic activities of
other microbes. Most are produced by soil inhabiting microbes (Fravel 1988). An
extensive range of soil microbes have been reported to produce antibiotics that have
a role in biocontrol including the bacterial genera Agrobacterium, Bacillus and
Pseudomonas, the actinomycete genus, Streptomyces, and fungal genera including
Trichoderma, Gliocladium and Penicillium. A selection of examples is given below
to illustrate this diversity.
Bacteriocins are a subclass of antibiotics produced by bacteria with specificity
towards bacterial strains closely related to the producer. The best-known example
of this is with control of crown gall caused by Agrobacterium tumefaciens by the
related A. radiobacter (Kerr 1980). The production of a bacteriocin, agrocin 84, by
A. radiobacter is primarily responsible for the observed biocontrol of crown gall.
Production of agrocin 84 is controlled genetically on a plasmid and sensitivity to
agrocin 84 is determined genetically on the Ti (tumour inducing) plasmid in the
target bacterium (Murphy and Roberts 1979). A mutant of A. radiobacter, which no
longer produced agrocin 84 (because the plasmid had been removed), could not
control crown gall (Das et al. 1978). The product NoGallTM containing 106 cells g–1
peat of A. radiobacter strain K1026 is marketed world-wide by Becker Underwood
Pty Ltd (Australia). The product is applied as an aqueous solution to seeds, seed-
lings and cuttings before planting to protect the wound sites from infection by the
crown gall pathogen (www.beckerunderwood.com).
Bacillus is a ubiquitous soil organism, and various species and strains are
reported to produce a large variety of antifungal compounds, such as cepacin and
pyrrolnitrin. Bacillus cepacia is an effective biocontrol agent for Pythium-induced
damping-off and Aphanomyces root rot of peas (King and Parke 1993) and
R. solani root rot of Poinsettia (Cartwright and Benson MD (February 1994).
Bacillus subtilis isolate RB14-C produces the lipopeptide antibiotic iturin A. When
the bacterium was introduced into the soil it was able to control R. solani on tomato
(Szczech and Shoda 2006) and various other fungal pathogens (Hiraoka et al.
1992). Shortly after application to the soil, RB14-C was shown to produce iturin A
and this was believed to play a major role in disease suppression. A mutant of RB14
RD1, deficient in the production of iturin showed only low protection of tomato
plants compared with the parental strain (Asaka and Shoda 1996).
Fluorescent pseudomonads are known to produce a wide range of different
antibiotics such as phenazines (Thomashow and Weller 1988), 2, 4-diacetylphloro-
glucinol (Fenton et al. 1992), pyrrolnitrin (Hill et al. 1994), pyoluteorin (Kraus and
Loper 1995) and their involvement in plant disease biocontrol has also been well
documented. For example, production of an unusual lipopeptide antibiotic
(AFC-BC11) was shown to be responsible for the ability of B. cepacia BC11 to
effectively control damping-off disease of cotton caused by the fungal patho-
gen R. solani. Howie and Suslow (1991) used an antifungal minus mutant
of P. fluorescens to show that the antibiotic was of primary importance in the sup-
pression of P. ultimum on cotton.
Strains of P. fluorescens are suppressive to G. graminis var. tritici, the causal
agent of take-all of wheat, and production of a range of antibiotics has been
implicated in this biocontrol (Hoffland et al. 1996). For example, bacterization of
spring or winter wheat seeds with P. fluorescens 2-79 and P. chlororaphis 30-84
isolated from wheat grown in take-all suppressive soils, results in significant sup-
pression of take-all (e.g., 2-79 increased yields by >10% in commercial scale
tests) (Weller 1988). Both strains produced phenazine-1-carboxylic acid (PCA)
and 2-79 also produced anthranilic acid (Weller 2007). Single site Tn5 insertions
were used to make mutants defective in the production of the phenazine antibi-
otic. All of these mutants were less suppressive to take-all in greenhouse tests
(Schippers et al. 1985).
Pyrrolnitrin is an active metabolite produced by a range of strains of
Pseudomonas spp. that is active against Rhizoctonia, Fusarium spp. and other plant
pathogen fungi. Ligon et al. (2000) produced P. fluorescens BL915 mutants that did
not produce pyrrolnitrin to demonstrate the important role of pyrrolnitrin in the
biocontrol activity shown by this strain. The author also genetically modified
P. fluorescens strain BL915 to produce more pyrrolnitrin. The modified strain was
compared to the wild-type in a field trial in 1997 on cotton for control of R. solani
infections. Cotton seed was planted in the furrow and inoculated with R. solani by
spreading wheat bran inoculant over the seed. Prior to closure of the furrow, the
seeds were treated with a lyophilised preparation of the biocontrol agents. The
wild-type strain gave 50% control compared to the chemical, while the genetically
modified strains gave increased levels of control equivalent to those achieved by

chemical (quintozene) treatments (Ligon et al. 2000).
The antimicrobial compounds produced by Trichoderma and Gliocladium
constitute a diverse group with respect to structure and function and the group
contains both volatile and non-volatile compounds (reviewed by Sivasithamparam
and Ghisalberti (1998)). Different metabolites exhibiting antibiotic activity in
Trichoderma can be classified into two main types: Low molecular weight volatile
metabolites such as pyrones and butenoliches, volatile terpenes and the isocyane
metabolites and high molecular weight polar metabolites like peptaibols (Cardoza
et al. 2005). Gliovirin, a diketopiperazine antibiotic produced by G. virens is highly
toxic to P. ultimum, but is inactive against other fungi associated with cotton
seedling disease. A UV induced mutant of G. virens deficient for gliovirin produc-
tion did not inhibit P. ultimum in culture and did not protect cotton seedlings from
damping-off in P. ultimum infested soil indicating an active role for gliovirin in the
control of the pathogen (Howell and Stipanovic 1983). The antibiotic, gliotoxin,
isolated in 1936 (Weindling and Emerson 1936), was first associated with the
biocontrol of R. solani on seed potatoes by G. virens (Aluko and Hering 1970).
Since then there have been numerous reports associating gliotoxin production with
biocontrol (Lumsden et al. 1992).
The pyrone, 6-pentyl-2H-pyran-2-one, is the representative metabolite common
to the Trichoderma genus. This compound is a flavouring agent responsible for the
coconut aroma associated with this fungus. This metabolite was used in plate tests
against R. solani and F. oxysporum f. sp. lycopersici in which the addition of 0.3
mg mL–1 of pyrone to agar medium caused a 69.6% growth reduction in R. solani
and a 31.7% reduction in F. oxysporum after 2 days (Claydon et al. 1987). When
used in spore germination tests, 0.45 mg mL–1 was found to completely inhibit the
germination of Fusarium spores (Claydon et al. 1987). Its antibiotic activity against
a number of plant pathogens has been demonstrated (Ghisalberti et al. 1990).
Competition
Competition occurs when two or more organisms demand more of the same
resource than is available. Competition between a biocontrol agent and a pathogen
may lead to control, if the biocontrol agent’s growth results in reduction of the
pathogen population or inoculum production. Nutrients and space availability are
two of the main limitations in the colonization of a particular ecological niche and
so are often the main resources being competed for. Microbes with a high growth
rate and optimal nutrient utilisation have clear advantages for survival. Many of the
microbes utilised as biocontrol agents fulfil these characteristics (Cortes-Pentagos
et al. 2007). Nitrogen, carbon and iron competition have also been associated with
pathogen suppression by a number of biocontrol agents. A number of examples
illustrating specific resource competition interactions leading to disease control
are given below.
Space and Nutrients
Trichoderma and Gliocladium are common soil-borne saprophytic fungi. Their

rapid growth, prolific conidiation and range of variation in substrate utilization
make them very efficient saprophytes and their success as biocontrol agents is often
linked to success in nutrient competition. They are biologically adapted to aggres-
sive colonization of available nutrient bases and are able to persist as quiescent
chlamydospores and conidia when nutrients are lacking. Optimum biocontrol is
most likely if Trichoderma can rapidly colonize the site or nutrient base before the
pathogen can establish. Rhizosphere competence is an important attribute that will
facilitate a biocontrol agent’s ability to compete for nutrients especially when the
biocontrol agent is applied as a seed treatment (Ozbay and Newman 2004).
Rhizosphere competent Trichoderma spp. have been shown to effectively utilise
root exudates and cellulose substrates on or near the root (Cotes et al. 1996) leading
to disease control, for example, in the control of F. oxysporum in the rhizosphere of
cotton and melon by T. harzarium T-35 (Sivan and Chet 1989).
Idriella bolleyii is common on roots of cereals and grasses and has been shown
to be able to control take-all disease (Kirk and Deacon 1987) and other root rot
pathogens such as the eyespot fungus, Tapezia yallundae (Reinecke and Fokkema
1981). When applied as a seed treatment, I. bolleyii will colonize wheat roots and
produce infective spores on inoculated plants. These spores are carried in perco-
lating water and spread down the root system. The biocontrol agent controls
the cereal root pathogen by competitive exclusion from naturally senescing host
tissues (Allan et al. 1992). In glasshouse experiments, I. bolleyii significantly
reduced infection of wheat roots by the take-all fungus G. graminis var. tritici when
inocula were dispersed in the soil at ratios of 10:1 (Ib:Ggt) or more (Lascaris and
Deacon 1991). Idriella bolleyii has several attributes that make it an effective bio-
control agent. It sporulates readily in liquid culture and retains viability as a seed
coat making inoculum production cost efficient, it grows readily on root and stem
base tissues and is a normal resident colonizer of the root and stem basal tissues of
cereals in the field and it has a reasonably wide spectrum of biocontrol activity
(Lascaris and Deacon 1991).
Iron Competition
Conditions of low iron in the soil are inhibitory to plant pathogens. Iron is
commonly present in the soil in an insoluble form, but some bacterial and fungal
species have developed a system for iron uptake. This involves the production of
siderophores, which are peptides able to bind iron and transport it inside the cell.
Iron chelated with these siderophores is unavailable to plant pathogens so their
activity is thereby reduced (Cortes-Pentagos et al. 2007). Pseudomonas spp. are the
best prokaryotic system studied in relation to siderophore synthesis. They secrete
molecules such as pyoverdines or pseudobactins that bind ferric iron with high
affinity (Raymond et al. 1984). Iron competition, mediated by siderophores, is the
main mechanism by which Pseudomonas putida WCS 358 controls Fusarium

pathogenesis in carnation and radish (Weisbeek and Gerrits 1999). Similarly, Loper
(1988) showed that siderophore production by P. fluorescens was an important
mechanism of Pythium inhibition during seedling emergence in cotton. The role of
siderophores in plant disease expression has been demonstrated using Pseudomonas
mutants lacking the ability to produce siderophores (Buysens et al. 1996).
Non-pathogenic and Hypovirulent Strains
Preliminary exposure of plant tissues to non-pathogens, or avirulent strains of a

pathogen often results in increased resistance to subsequent challenge by the
virulent strain. For example, Nel et al. (2006) reported the potential of non-pathogenic
F. oxysporum and other biological control organisms for suppressing Fusarium wilt
of banana. Glasshouse evaluations showed two non-pathogenic F. oxysporum
isolates CAV255 and CAV 241 reduced Fusarium wilt incidence by 87.4% and
75%, respectively. Similarly, Shishido et al. (2005) reported effective biological
control of Fusarium wilt of tomato by non-pathogenic F. oxysporum Fo-B2 in dif-
ferent environments. Inoculation of Fo-B2 onto tomato roots significantly reduced
the severity of disease and the biocontrol agent was most effective when it colo-
nized vascular tissues intensively. A key observation was that indigenous soil
microbes were a primary factor negatively influencing the efficiency of Fo-B2.
Therefore, early establishment of the antagonist in a non-competitive environment
prior to out-planting could improve the efficacy of biocontrol. The soil-borne
fungus F. oxysporum f. sp. radicis-lycopersici causes tomato foot and root
rot (TFRR), which can be controlled by the addition of the non-pathogenic fungus
F. oxysporum Fo47 to the soil. To examine biological interactions between the two
fungi, they were labelled using different autofluorescent proteins as markers and
visualised using confocal laser scanning microscopy (Bolwerk et al. 2005). This
study revealed that there needed to be at least 50-fold more F. oxysporum Fo47
inoculum propagules than F. oxysporum f. sp. radicis lycopersici to give biocontrol.
The study also showed that Fo47 hyphae attached to the root earlier than the
pathogen and it was postulated that F. oxysporum Fo47 controls TFRR by
germinating more quickly and being a better nutrient competitor on the rhizoplane
than the pathogen.
Hypovirulence refers to the reduced virulence of selected isolates within a popu-
lation of a plant pathogen. Hypovirulence has been associated with the presence of
double stranded RNA (dsRNA) characteristic of fungal viruses (Nuss and Koltin
1990). However, other factors such as mitochondrial mutations, nuclear mutations
and encapsidated fungal viruses have been associated with hypovirulent isolates.
The potential in utilising hypovirulent isolates of fungal pathogens in a biocontrol
strategy resides in the ability to transfer hypovirulence from hypovirulent isolates
to virulent isolates and thereby reduce the mean disease severity of the population
through overall reductions in virulence, growth, sporulation and/or survival (Harris
2000). Most work on hypovirulence has been conducted on Rhizoctonia spp.
The genus Rhizoctonia includes some of the most aggressive soil-borne plant
pathogens, for example, the polynucleate R. solani that attacks solanaceous plants.
Many Rhizoctonia isolates are avirulent or hypovirulent on plants, even isolates
belonging to the same R. solani anastomosis group (AG) show a continuum
between high virulence and avirulence (Herr 1995). Several hypovirulent strains
have been shown to have biocontrol potential against virulent R. solani (Cardinale
et al. 2006). Similarly, hypovirulent isolates of S. sclerotivorum, S. minor and
S. homeocarpa have been evaluated for their role in reducing virulence in populations
of these pathogens (Boland 1992). Transmission of the hypovirulent phenotype and
dsRNA occurs through hyphal anastomosis and was shown to occur in culture and
on lettuce tissue (Melzer and Boland 1996). With S. sclerotivorum, transmission
did not occur consistently between isolates from the same or different mycelial
compatibility groups and so this could represent a significant barrier to the use of
hypovirulence as a biocontrol strategy. However, the number of mycelial compati-
bility groups in S. minor is relatively small and so transmissible hypovirulence may
have more promise as a control strategy for this fungus. Mycelial suspensions of a
hypovirulent isolate of S. minor applied to leaf lesions initiated by virulent isolates
on lettuce suppressed lesion expansion by up to 100% and significantly reduced the
development of sclerotia on diseased tissues (Melzer and Boland 1996). The earlier
in lesion development the hypovirulent isolate was applied the more it suppressed
lesion development by the virulent isolate.
Induced Resistance
Numerous biocontrol agents have been shown to be able to induce host plant
defences. Induced resistance has been investigated more fully for control of foliar
pathogens, but there are several examples of successful control of root pathogens
including root parasitic nematodes (reviewed by Agrawal et al. (1999)). Examples
of biocontrol agents shown to induce systemic host plant resistance include
Paenibacillus polymyxa (Timmusk and Wagner 1999), Pseudomonas (Meera et al.
1994), rhizobacteria (Harman et al. 2004), streptomycetes (Lehr et al. 2008) and
Trichoderma spp. (Harman et al. 2004). The soil isolate Streptomyces GB 4-24
prevented the development of Heterobasidion root rot in Norway spruce seedlings
(Schrey and Tarkka 2008). Treatment of plant roots with GB 44-2 led to a reduction
in the penetration ability of the pathogen. Whereas inner root cortex layers and
vascular tissues of plants were fully colonised in plants inoculated with
Heterobasidion only, pre-treatment with GB 4-2 resulted in inner root tissues
virtually free of the pathogen (Lehr et al. 2008). Reduced root colonisation was
accompanied by distinct changes in root anatomy, e.g., cell wall appositions,
lignified walls in a phenomenon termed ‘priming’ by Conrath et al. (2002).
The nature of systemic acquired resistance (SAR) was shown using split root
systems to demonstrate that treatment of one section of a root system with
Pseudomonas bacteria induced resistance to soil-borne pathogens in the untreated
roots (Chen et al. 1999). Pseudomonas may induce SAR directly by synthesizing
SAR signalling intermediates, such as salicylic acid, or by causing necrosis of host

cells through the action of toxins, enzymes or elicitation of the hypersensitive reac-
tion (HR). Preston (2004) showed that bacterization of carnation roots with P. fluore-
scens WCS417 reduced wilting caused by F. oxysporum f. sp. dianthii. Upon pathogen
infection, higher amounts of the anthranilate derived phytoalexins dianthalexin
(2-phenyl-7-hydroxy-1,3-benzoxazin-4H-one) and a group of diantramides accumu-
lated in plants that had received a root pretreatment with the biocontrol strain (Van
Peer and Schippers 1992).
Trichoderma spp. have the capacity to induce resistance to a range of diseases
caused by various classes of plant pathogen (fungi, bacteria, viruses) in a variety of
plants (Harman et al. 2004; Vinale et al. 2008). Trichoderma strains can produce
three different classes of compounds that can induce resistance in plants; proteins
with enzymatic functions e.g. xylanase (Anderson et al. 1993); homologues of
proteins encoded by the avirulence (Avr) genes (de Wit et al. 2002); oligosaccha-
rides/low molecular weight compounds released from cell walls by the activity of
Trichoderma enzymes (Kubicek et al. 2001). Pre-inoculation of cotton plants with
T. virens G6, G6-5, G-11 protected against attack by R. solani, as a result of the
induction of fungitoxic terpenoid phytoalexins (Howell et al. 2000). Induction of
phytoalexin synthesis in roots of cotton also protected the plants from vascular wilt
pathogens that enter through the root. Similarly, seed treatment of cotton with
T. virens resulted in colonisation of the developing root system, which led to sup-
pression of symptom development when the plants were inoculated with Verticillium
and Fusarium wilt pathogens (Hanson 2000; Zhang et al. 1996).
Constraints to Successful Biological Control
Abiotic Factors
Superficially, the soil appears an attractive environment in which to target microbial

control efforts, protected from environmental extremes of temperature and mois-
ture. Yet soil is a complex, heterogeneous environment, compartmentalised into
nutrient-rich competitive (e.g., rhizosphere) and harsh environs, and microbial
persistence and activity is influenced by a range of interacting environmental
factors. Tolerance to abiotic fluctuations is, therefore, a prerequisite for the
successful development of ecologically competent biocontrol agents for use in the
field. Yet in most cases these conditions are not adequately defined. Pertinent
examples of such interactions from the literature are discussed below.
Soil Type
Contrasting results have been obtained in studies investigating effects of soil

type on the natural incidence, persistence and efficacy of biological control agents.
For example, Queseda-Moraga et al. (2007) observed a correlation between high

soil organic matter and clay content and the incidence of entomopathogenic fungi,
particularly B. bassiana. This was attributed to enhanced adsorption of conidia to
soil particles which reduces potential biodegradation or leaching from the upper
layers of the soil profile, inhibition of germination by organic acids and other fungi-
static compounds produced in these soils, or greater incidence of potential arthro-
pod hosts in soils with high organic content. However, Vänninen et al. (2000) found
that persistence of M. anisopliae was poorer in peat than clay soils; Kessler et al.
(2004) showed that B. brongniartii declined more quickly in soils with a high
organic content when insect hosts were absent; while Milner et al. (2003) indicated
that soil type had negligible effects on persistence. The ability of a biocontrol agent
to perform in different soil types can differ significantly even between closely
related biocontrol agents. For example, Larkin and Fravel (2002) observed varia-
tions in the efficacy of non-pathogenic F. oxysporum (CS-20) and F. solani (CS-1)
used to control Fusarium wilt of tomato in different soil types. Isolate CS-20 effec-
tively reduced disease in four different field soils varying in texture (sandy to clay)
and organic matter content (0–3.2%), while isolate CS-1 reduced disease in sandy
and loamy soil (49–60%) but was as not effective in a heavy clay soil.
Soil Temperature
Temperature has a direct impact on persistence and efficacy. Although persistence is

generally extended in cooler soils, most soil-borne fungal biocontrol agent strains are
mesophilic so low temperatures in winter may constrain biocontrol activity. Also, these
effects may be compounded by soil moisture content (Jaronski 2007). Soil temperature
must be appropriate for insect infection to occur. While temperature optima for differ-
ent isolates will vary, fungal development will generally occur between 15°C and 30°C
(Jaronski 2007). At temperatures outside of this range, the infection process can be
slowed or totally inhibited. For example, Glare et al. (1994) identified low soil tem-
perature as a primary limiting factor to the effectiveness of M. anisopliae against New
Zealand grass grub larvae, advocating selection of isolates with activity at cooler
temperatures to overcome this limitation, similar to the approach taken in the develop-
ment of a fungal biocontrol agent for cockchafer in Tasmania (Rath et al. 1992).
Knudsen et al. (1991) reported that mycoparasitism of sclerotia of S. sclerotiorum by
T. harzianum was reduced in soil at 15°C compared to 25°C. Other studies on the
effects of temperature on spore germination and germtube growth, mycelial growth,
competitive saprophytic ability and antibiotic production support this. For example,
gliotoxin production by T. virens was low at 15°C increasing to a maximum at
25–30°C, thus identifying a potential adverse effect on biocontrol activity consid-
ering the low temperature of many soils at planting time (Lumsden et al. 1992).
However, some studies have contradicted this general trend. For example, degradation
of sclerotia of S. cepivorum by T. viride was optimal at 10°C with decreasing parasit-
ism as temperatures increased (Clarkson et al. 2004). Trichoderma viride is a species
group often associated with cold tolerance (Kredics et al. 2003), so there is clearly an
opportunity to select for low temperature tolerant strains. Isolates with higher or lower
thermal optima have been identified, and there does appear to be a link between the
geographic origin of an isolate and its performance at lower temperatures (Fernandes
et al. 2008); while more thermotolerant isolates have been recovered from agricultural
soils which are exposed to elevated temperatures (Meyling and Eilenberg 2007).
Osmotolerance
Many biocontrol fungi have a low osmotolerance level and do not proliferate in dry
soils with spore germination, conidial survival, germtube growth and mycelial
growth all inhibited at low water potential. However, there is also an inverse rela-
tionship between persistence and soil moisture; where longevity declines as mois-
ture levels increase, and the rate of decline increases as temperatures rise (Jaronski
2007). In trials examining effects of two irrigation schemes on conidial persistence
in turfgrass, Thompson et al. (2006) showed that survival was greater at higher
irrigation levels. Irrigation (and rainfall) only influence soil moisture levels in the
short term and enhanced survival in this study was thought to be due to movement
of the conidia into the soil profile where they were protected from higher tempera-
tures and damaging UV. Similar observations were made by Milner et al. (2003),
where rainfall appeared to have minor effects on persistence of M. anisopliae in
different sugarcane soils in Australia. Soil moisture also appears to have a signifi-
cant impact on efficacy. However, if a ‘standard’ soil treatment is applied, insect
infection is commonly higher at low rather than high soil moisture levels (Jaronski
2007). Water activity (aw) and pH were demonstrated to be the most important
environmental parameters affecting the biocontrol activity of mycoparasitic
Trichoderma spp. (Kredics et al. 2004). Optimal conditions for mycelial growth
were defined as aw 0.997 and pH 4. Kredics et al. (2004) also investigated optimal
(aw) and pH conditions for key extracellular enzymes known to play a role in myco-
parasitism identifying the potential for the development of xenotolerant mutants
with improved biocontrol performance in low aw soils. Further to this, Clarkson
et al. (2004) demonstrated a clear relationship between the efficacy of T. viride in
degrading S. cepivorum sclerotia and soil water potential. Sclerotia were degraded
at water potential levels as low as −4.03 MPa, however, degradation was most
efficient at water potential levels over −0.022 MPa.
Carbon and Nitrogen
Mineral nutrition is essential for microbial growth and sporulation and can stimu-
late the production of secondary metabolites. Therefore, any nutrient limitation
may constrain a biocontrol agent’s competitiveness in the soil environment and
ultimately its biocontrol activity. A soil sandwich assay was used to determine the
influence of nitrogen on the saprophytic growth through soil of an isolate of T. kon-
ingii. A key result showed that nitrogen added as ammonium sulphate (NH4+ N)
increased the saprophytic growth of T. koningii whereas nitrate (NO3− N) suppressed

growth (Wakelin et al. 1999). This supports the findings of Danielson and Davey
(1973) who reported that Trichoderma spp. grow best when supplied with
ammonium-nitrate compared with nitrite-nitrate on artificial culture medium. Brian
et al. (1946) showed that gliotoxin-levels produced by T. virens was optimum on
medium containing glucose and phenylalamine at C-to-N ratios of 18:1, 31:1 or
42:1. Similarly, Howell and Stipanovic (1984) showed that viridiol production by
T. virens was better on a rice substrate than on other substrates with lower C-to-N
ratio, while Jones and Hancock (1987) showed that high C-to-N ratios enhanced
and low C-to-N ratios suppressed viridiol production.
Soil pH
Soil pH may be influenced by a variety of agricultural practices, e.g., application

of fertilizers, organic content, etc., which will affect the diversity and make-up of
microbial populations. These, in turn, may exert a fungistatic effect on biocontrol
agents, which will impact on their incidence and efficacy. Thus, while pH may not
directly influence efficacy per se, its indirect effects via the soil biota may be more
significant. The occurrence of B. bassiana in cultivated and natural soils in Spain
was correlated directly with soil pH (Quesada-Moraga et al. 2007). The fungus was
recovered with greater frequency from soils at pH 8–8.5, and both B. bassiana and
M. anisopliae were infrequently recovered from soils with pH values >8.5.
Metarhizium anisopliae was isolated over a greater pH range and was the predomi-
nant species isolated from soils with pH <7; M. anisopliae appears to be more toler-
ant of acidic conditions, which could explain these findings (Padmavathi et al.
2003). Low soil pH (<7) has long been known to favour Trichoderma-mediated
suppression of a range of soil-borne pathogens. Harman and Taylor (1990) showed
that acidification of the soil and seed environment can improve the competitiveness
and biocontrol activity of introduced Trichoderma strains. Acidic conditions are
known to enhance conidia production and germination, mycelial growth, and
production and activation of antimicrobial compounds such as antibiotics and lytic
enzymes. For example, Weindling (1941) demonstrated that the production of glio-
toxin was higher at pH 3.5 than at 6.0, with this loss of activity at higher pH also
demonstrated for viridin (Brian and McGowan 1945). Alkaline soils tend to evolve
more ammonia than acidic soils, which reduces germination of Trichoderma
conidia and has been suggested to reduce the biocontrol activity of T. hamatum
(Papavizas 1985). Cellulase production by T. koningii, which is associated with
competitive saprophytic ability and rhizosphere competence (Cotes et al. 1996),
was negatively correlated with soil pH (Widden et al. 1988). Thus, soil pH will be
a major constraint to effective biocontrol by Trichoderma spp., particularly in more
alkaline soils and there is a need to screen for Trichoderma strains with tolerance
to more alkaline conditions.
The effect of soil pH on biocontrol efficacy of Pseudomonas spp. has also been
described (Ownley et al. 1992). Pseudomonas chlororaphis PCL1391 produces the
secondary metabolite phenazine-1-carboxamide, which is an antifungal metabolite

required for biocontrol activity of the strain. Identification of environmental/nutri-
tional conditions that influence production of the antibiotic showed that pH
decreasing from 7 to 6 and temperature from 21°C to 16°C could reduce production
significantly (Tjeerd van Rij et al. 2004). Similarly, biocontrol by P. fluorescens
2-79 against take-all of wheat caused by G. graminis var. tritici was shown to
increase with increasing pH (Ownley et al. 2003). Slininger and Shea-Wilbur
(1995) reported a direct effect of pH on phenazine-1-carboxylic acid (PCA) accu-
mulation by P. fluorescens strain 2-79. PCA production was optimal at pH 6–7 and
declined at lower and higher pHs.
Interactions Between Soil Abiotic Factors
Interactions between soil factors may be as important in determining the outcome

of biocontrol than any direct effect of one soil factor on the biocontrol agent. For
example, P. fluorescens 2-79 RN10 protects wheat against take-all disease caused
by G. graminis var. tritici, however, the level of protection in the field varies from
site to site. Ownley et al. (2003) evaluated the relative importance of 28 soil
properties on take-all suppression. Bacterized seed were planted in ten soils
representative of the wheat-growing region in the Pacific Northwest. Biocontrol was
positively correlated with ammonium-nitrogen, % sand, soil pH, sodium, sulphate-
sulphur and zinc. In contrast, biocontrol was negatively correlated with cation
exchange capacity (CEC), exchangeable acidity, iron, manganese,% clay,% organic
matter, % silt, total carbon and total nitrogen. Six key soil properties that accounted
for the variance in biocontrol activity were identified – ammonium-nitrogen, cation
exchange capacity, iron,% silt, soil pH and zinc. Using this information, the
researchers were able to identify specific crop management practices that could
enhance biocontrol, for example, the performance of 2-79 RN10 was improved by
amending a soil low in zinc with 50 mg of zinc-EDTA g-1 soil. A similar study was
conducted by Duffy et al. (1997) on T. koningii, used to control take-all of wheat
in Australia. Previous work had shown that soil treatment with T. koningii
reduces the saprophytic growth of the pathogen and increases survival of wheat
seedlings by ~50% and grain yield by 10% (Rovira et al. 1992). However, control
was inconsistent between sites and seasons prompting the need to understand the
influence of soil properties on the biocontrol activity of the introduced organism.
Duffy et al. (1997) evaluated suppression of take-all by T. koningii in eight silt
loam soils from Pacific North West, USA and the influence of 21 abiotic soil
parameters on biocontrol activity. Biocontrol was positively correlated with iron,
nitrate-nitrogen, boron, copper, soluble magnesium and percent clay and negatively
correlated with soil pH and available phosphorus. Principal component factor
analysis using these eight variables resulted in a three-component solution that
accounted for 95% of the variation in disease rating. The authors proposed that
this information could be used to help select sites where T. koningii will perform
optimally.
Biotic Factors
There is a large body of literature on the influence of abiotic factors on biocontrol

microbes in the soil environment, but surprisingly little is reported on the influence
of biotic factors. This is probably due to the difficulty in identifying specific biotic
influences. Soil microbial populations are relatively stable, so it would be naive to
think that introducing a single organism into the soil as an augmentative treatment
would always necessarily result in successful establishment. Clearly, there will be
situations where a member or members of the soil microbe population will act in
an inhibitory manner towards the biocontrol agent and constrain its ability to
establish.
We can identify three principle biotic components that have a major influence
on microbe persistence and efficacy. These are soil microorganisms, plants and
invertebrates and examples of their effect on several biocontrol agents are discussed
below. Generally speaking, entomopathogens are considered weak saprophytes in
the competitive soil environment and inoculum levels will decline in the absence of
an arthropod host. Metabolites produced by other soil microbes can adversely affect
germination and growth, or be directly toxic, leading to reduced infectivity or
multiplication; consequently, survival and efficacy of biocontrol agents is com-
monly observed to be greater in sterilized versus non-sterilized soils (Jaronski
2007). Even so, in natural soils conidia from entomopathogenic fungi will infect a
susceptible host when they contact the insect cuticle; Metarhizium and Beauveria
will germinate, grow and conidiate when applied to soil on nutrient granules or as
mycelial aggregates; and amendment of soil with nutrients can overcome (appar-
ent) fungistasis (e.g., Jaronski 2007; Brownbridge 2006). Hubbard et al. (1983)
reported that Trichoderma biocontrol agents may decline in the soil or fail to pro-
liferate in the plant rhizosphere due to the presence of antagonistic or competing
microorganisms at the rhizosphere or rhizoplane level. Bae and Knudsen (2005)
showed that higher levels of microbial soil biomass reduced the biocontrol activity
of T. harzianum and postulated that this was due to a soil fungistatic effect brought
about by the presence of Pseudomonas spp. and the production of antibiotic com-
pounds. Indeed, Pseudomonas spp. were shown to compete with the biocontrol
agents for iron in the rhizosphere and produce toxic metabolites that were inhibi-
tory to the Trichoderma. Multiple logistic regression analysis used to determine the
significance of associations between nine abiotic factors, total populations of fungi,
bacteria and actinomycetes and the presence of 42 individual fungi over a 12-month
period revealed that T. harzarium was positively associated with the total bacterial
population and the occurrences of Aspergillus ustus, A. ramarii, Penicillium
citrinum, P. chrysogenum and P. griseoroseum, but negatively correlated with the
occurrences of A. flavipes and P. miczynskii (Eastburn and Butler 1988a, b). This
suggests that biotic interactions in the soil occur at both the species and subspecies
level. Inhibition of Trichoderma spp. by certain mycorrhizal fungi has also been
reported. For example, the ectomycorrhizal fungus Laccaria laccata inhibited
the germination of T. virens and T. harzarium conidia. Mantle hyphae of the
e ctomycorrhizal fungus coiled around the spores and occasionally caused breaks in
their walls (Zadworny et al. 2004). Reduced growth of T. harzarium and T. virens
as well as cytoplasm aggregation of their hyphae was observed in dual culture with
L. laccata (Zadworny et al. 2004). These observations have not been validated in
the soil environment so limited conclusions can be drawn on the significance of this
inhibition to biocontrol activity.
Crop plant species and tillage practices affect the incidence and persistence of
fungi. Some entomopathogens (M. anisopliae) are more commonly associated with
agricultural (tilled) soils than natural habitats, although fungal prevalence and
diversity is normally greater in undisturbed soils. Plant root exudates contain many
nutrients that will support the development of microbial populations in the rhizo-
sphere; in vitro tests demonstrated that carbohydrates and nitrogen compounds
stimulate germination and growth of M. anisopliae conidia, while organic acids
may inhibit germination (Li and Holdom 1995). This observation pre-empted later
work showing that some M. anisopliae isolates are in fact rhizosphere-competent,
a trait that enhances persistence in the root zone. Beauveria bassiana also shows
specific plant adaptations in its existence as an endophyte; not all isolates can func-
tion as endophytes, but this capacity has been demonstrated in a diverse range of
plant species (Vega et al. 2008).
Invertebrates have many effects on biocontrol agent levels in soil. Some, such
as Collembola and earthworms, ingest conidia and play a role in their distribution
within, and removal from, the soil profile. Insect hosts are critical to the long-term
survival of entomopathogenic fungi. Access to and successful infection of a host is
the only way in which fungi can significantly multiply within the soil; fungal load-
ings over time may thus be closely correlated with the presence of susceptible
insect populations in both natural and agricultural soils (Meyling and Eilenberg
2007). Use of insecticides may contribute to the decline of fungal populations by
reducing the availability of suitable hosts, as opposed to their having direct nega-
tive effects on fungal viability (Mietkiewski et al. 1997).
Fungi and arthropods have evolved complex relationships, and some soil-dwelling
species show adaptive behavioural responses that prevent their coming in to contact
with fungal inoculum. An avoidance response has been observed in mole crick-
ets to conidia of both M. anisopliae and B. bassiana, which may lead to inconsis-
tent performance of these fungi in the field (Thompson and Brandenburg 2005).
However, there appears to be variation in the level of response to different isolates
(Thompson and Brandenburg 2005). Insects may also be attracted to fungi. Engler
and Gold (2004) showed that termites were attracted to mycelial preparations
and volatile extracts of M. anisopliae, while Japanese beetle females preferen-
tially oviposited in soils treated with mycelia (Villani et al. 1994). This recruitment
effect was also seen with black vine weevil larvae that responded positively to
M. anisopliae-treated media (Kepler and Bruck 2006). Such behavioural responses
should be taken into consideration when selecting appropriate strains for insect pest
management and may be useful in the development of more effective biocontrol
strategies. Understanding the influence of soil properties on the pest and disease
suppressive activities of introduced microbes should facilitate tailoring biocontrol
agents for use in fields where biocontrol activity is maximised (Cook 1993).
Further to this, the identification of soil factors that influence biocontrol will pro-
vide a biological basis for improved integration of biologicals with cultural prac-
tices that manipulate soil properties with an aim towards improved pest and disease
control (Pierson and Weller 1994).
Production and Formulation
Fungal Inoculum
Product formulation is critical to the successful delivery and establishment of an

effective population of a biocontrol agent. For soil pest control, fungi have gener-
ally been applied by direct incorporation of conidia, mycelial pellets, or inert or
nutrient-based granules containing fungal propagules (conidia or mycelia). Suitable
in vitro mass production systems are needed to provide sufficient inoculum for
large-scale applications at a competitive price. The inoculum produced must be
virulent, stable in storage, and environmentally competent after application. In the
majority of cases, though, research emphasis has been placed on optimizing bio-
mass production, with the assumption that control could be achieved if sufficient
inoculum could be produced cheaply enough and applied at a high enough rate to
the soil. However, soil is a complex environment and fungal activity against soil-
inhabiting pests and pathogens is affected by many biotic and abiotic factors; envi-
ronmental integrity is critical to performance, and maintenance of bioactivity must
be a primary consideration when developing production media (Jaronski 2007;
Brownbridge 2006). Formulation can enhance characteristics or render fungal
preparations easier to apply, but their performance is ultimately reliant upon inclu-
sion of robust biological material that is ‘fit for purpose’. The production method
selected will depend upon the nature of the inoculum required and isolates may
have different growth characteristics on different production media.
Solid substrates have been widely used to produce aerial conidia of beneficial
fungi. Temperature, pH, aeration and substrate components all influence conidial
yield, viability, stability and virulence. Although these parameters are harder to
regulate in a solid-substrate system, this remains the predominant method used for
commercial products. This is due, in part, to the flexibility of a system that lends
itself to the ‘cottage-industry’ scale used in many parts of the world, while solid-
state fermentation bioreactors yielding 3 × 1013 conidia/kg of substrate have also
been developed (Wraight et al. 2001). Small cereal grains are commonly used to
produce fungi owing to the relatively high surface area-to-volume ratio of the
substrate (which allows better aeration and access to nutrients) and their ready
availability. Production varies on different grains, however, and virulence may be
similarly affected (Wraight et al. 2001). Grains have been used to mass-produce
conidia, chlamydospores or mycelial granules for soil incorporation (Jaronski
2007; Pérez-Rodriguez et al. 2007; McLean et al. 2005).
Large-scale liquid fermentation systems are successfully used for agriculturally

important bacteria (e.g., B. thuringiensis, S. entomophila). In submerged culture,
fungi generally produce vegetative propagules such as mycelia or yeast-like blas-
tospores and culture conditions and media composition have a primary influence on
the type and amount of inoculum produced. Production systems have largely been
designed with high yield as a primary goal, but again the relative infectivity of the
resulting biomass, its ecological competence and stability are key factors that must
be considered during process development. Conditions and media can be manipu-
lated to impart specific traits on the resulting biomass, including enhanced stability
during drying and in storage, and infectivity. For example, conidia of M. anisopliae
generated under nutritive stress (MM + 3 g L–1 lactose; MML) had increased viru-
lence and increased germination speed than conidia produced under optimal nutri-
tive conditions (e.g., potato dextrose agar + 1 g L–1 yeast extract; PDYA). MML
medium produced conidia with greater adhesion capacity and that were more
hydrophobic. There was also evidence that conidia production on MML were more
reactive than PDYA produced conidia and enhanced binding efficiency to the sur-
face of insect cuticles (Rangel et al. 2008). Similarly, studies by Jin et al. (1991)
with T. harzarium demonstrated that by manipulating the growth conditions with
polyethylene glycol (PEG), the trehalose content of the conidia could be signifi-
cantly increased and this enabled the inocula to survive dessication better than
unmodified, control conidia. Jaronski and Jackson (2008) recently described
methods to induce production of microsclerotia by M. anisopliae in liquid media.
The aggregates were readily air-dried, stable at room temperature, showed superior
efficacy against sugarbeet root maggot in soil assays compared with conventional
corn-grit granules, sporulated profusely in non-sterile soils and were active at low
soil moisture levels. Such production/formulation techniques may overcome some
of the biotic and abiotic constraints to fungal efficacy and result in increased oppor-
tunities to utilize these biocontrol agents against soil pests and pathogens.
Bacterial Inoculum
Bacterial agents are usually produced by liquid or solid state fermentation on low-
cost carbohydrate or protein substrates. The method of production is determined by
the characteristics of the specific organism with its economic viability dependent
on the cost per unit of active ingredient applied in the field. The cost of production
per unit can be reduced if the quality of the cells harvested is maximised during the
fermentation. This can be achieved by optimising the harvest time for high yields
of competent cells, maximising the cell density within the fermenter and by raising
the scale of production. Visnovsky et al. (2008) showed that the most robust
S. entomophila cells were produced after the cell culture entered the stationary
phase of fermentation and that bacterial cells comprised 30–50% of the fermenter
volume by the end of fermentation.
Media composition is also critical to the production of high yields of competent
cells at harvest. Media can be optimised in the laboratory at a small scale then tested
through scale up to high volumes. Liquid fermentation lends itself well to up-scaling
to very large volumes. For production of B. thuringiensis, Valent Biosciences utilise
fermenters of 50 m3 volume utilising starch-based carbohydrates from the maize belt
of the United States as a cheap and successful substrate (Georgis and Gaugler 1991).
Where efficacy is determined by a toxin, increasing the production of the toxin can
raise efficacy and modified media compositions have been used to develop high
toxin cultures of B. thuringiensis (Marrone 1994).
Longevity and the maintenance of competency during storage are also critical to
the development of a commercial product. Unfortunately, large-scale production
creates a specific problem in that the large volume of cells may be vulnerable to
factors in their surrounding environment. For non-spore forming bacteria, main-
taining viability in the period between production and application poses even
greater problems. These microbes lack a resistant stage and are susceptible to envi-
ronmental factors such as temperature, toxins and desiccation, which usually lead
to a short shelf life. Microbe producers have addressed this issue through mail order
of sensitive microorganisms to reduce storage time, but the handling conditions
after arrival cannot be guaranteed. Alternatively, the cell mass can be stabilised by
cooling. However, low temperature storage is expensive and organisms can be
sensitive to temperature fluctuations during distribution and application. Thus,
other methods are usually required for long-term storage. For production of Bt
toxins, the cell paste is concentrated by centrifugation and toxins subsequently
stabilised by spray-drying or freeze-drying. The resultant dry powder containing
spores and toxins can be stored for several years without loss in activity. An unpro-
cessed fermenter broth containing a high-density, cell culture of S. entomophila was
originally marketed as Invade®. This product could be stored for several months
under refrigeration without deterioration (Johnson et al. 2001). Since then, a dry
substrate coating system that allows cell survival at ambient storage temperatures
has been developed for bacterial formulation and has been applied to a wide range
of Enterobacteriaceae and other non-spore forming bacteria (Swaminathan et al.
2008). A second-generation S. entomophila product has been developed using
Bioshield™ cell broth, which is incorporated into a biopolymer gel and coated onto
a zeolite granule (Townsend et al. 2004). The resultant product can be stored at
ambient temperatures without cell deterioration for 6–9 months at 25°C prior to
application. Furthermore, the granules can be applied using standard farm equip-
ment rather than the modified machinery used for application of Invade™. Similar
products have been developed with Gram-positive bacteria, Lactobacillus,
Bifidobacterium spp., Pantoea and Pseudomonas and other species, which can also
be stabilised using these technologies, but with a lower water activity of the final
product (Swaminathan et al. 2008).
Advances in formulation technologies now permit stabilization of environmen-
tally sensitive microbes, and have applications to a diverse variety of beneficial
organisms. Formulations can improve the handling characteristics and safety of a
microorganism (e.g., by eliminating spore dust during preparation of a spray mix-
ture), enhance stability pre- and post-application, improve persistence, promote
efficacy, and facilitate easy delivery to the target pest/pathogen. Effective formulation
is integral to the wider utility of biopesticides in agricultural production systems,

and microbes can fail if formulated poorly (Jaffee et al. 1996). Formulations may
be tailored to suit the environment in which the microbe will be used, the delivery
system envisaged, and the nature of the inoculum being used. Like production
systems, they must be rationally developed to ensure retention of key characteris-
tics that are critical to microbial efficacy.
The Way Forward
Characteristics of Successful Biocontrol Agents
To be successful, a microbial control agent must provide the desired level of control
of the target pest or disease. Some microbes that have been commercialised after
providing consistent results in large-scale trials over a number of years or have been
widely used in commercial operations are listed in Table 1. It is useful to examine
this list to identify common biological factors that could assist in selection and
accelerated development of new biocontrol agents.
For soil-dwelling insect pest species, a wide range of microbial agents (virus,
bacteria, fungi, protozoa and nematodes) have been developed (Table 1).
Interestingly no one agent has been developed as a general control for several soil
dwelling species. This single product/single target effect appears to result from the
intrinsic specificity of the relationship between microbial pathogens and their soil
dwelling pest hosts. This contrasts with arboreal habitats where generalist strains
of pathogen, such as B. thuringiensis var. kurstaki, have been used to control a
very wide range of species (Navon 1993). Intrinsic specificity is demonstrated by
plasmid bearing strains of the bacteria Serratia spp. which are only known to
infect one species, the New Zealand grass grub (Jackson 2003). Similarly,
B. popilliae, the cause of milky disease, is only known as a pathogen of the
Scarabaeidae with a high degree of host species/bacterial strain specificity. While
fungal products, based on Beauveria and Metarhizium, are listed several times in
Table 1, the strains used are highly specific. For example, B. brongniartii strain 96
used against the scarab pest Hoplochelus marginalis in Reunion was isolated from
that host in its original habitat of Madagascar and has little effect on other insects
(Couteaudier et al. 1996). Other strains of fungi used for soil insect control,
such as M. anisopliae DATF001 for pasture cockchafer control in Australia or
B. brongniartii for May beetle control in Europe, also appear to be highly specific
to the target pest as they provide effective control with little impact on non-target
species (Traugott et al. 2005; Rath et al. 1995). While entomopathogenic nema-
todes appear to be less specific, the use of the mole cricket specific S. scapterisci
(Parkman and Smart 1996) has proven more successful and consistent than the
generalist nematode Steinernema carpocapsae in control of mole cricket and other
turf pests (Georgis and Gaugler 1991).
Table 1 A selection of microbial control agents and products used for control of soil dwelling pests and diseases
348
Pest control agent Pest targets Product name Reference

Oryctes virus Oryctes rhinoceros – (Bedford 1981; Ramle et al. 2005)
Bacillus popilliae Popillia japonica Doom (Klein 1992)
Serratia entomophila Costelytra zealandica Invade/Bioshield (Jackson et al. 1992; Jackson 2007)
Bacillus thuringiensis var. Popillia japonica, Anomala spp. – (Alm et al. 1997)
buibui
Beauveria bassiana Cornitermes cumulans Boverial (Alves et al. 1995)
Beauveria bassiana Heterotermes tenuis Termitrap (Almeida et al. 1997; Almeida and Alves
1996)
Beauveria bassiana Cosmopolites sordidus – (Badilla and Alves 1991)
Beauveria brongniartii Melolontha melolontha Engerlingspilz (Keller 1992)
Beauveria brongniartii Hoplochelus marginalis Betel (Vercambre et al. 1994)
Metarhizium anisopliae Adoryphorus couloni BiogreenChaferGuard ®; (Rath et al. 1995; Rath 2002)
Metarhizium anisopliae Cornitermes cumulans Metarril (Alves et al. 1995)
Steinernema scapterisci Scapteriscus spp. – (Parkman and Smart 1996)
Heterorhabditis Phyllopertha horticola Nema-green (Sulistyanto and Ehlers 1996)
bacteriophora www.e-nema.de
Heterorhabditis spp. Otiorhynchus spp. Dickmaulrüssler nematoden Product literature, Andermatt
Biocontrol AG
Heterorhabditis megidis Otiorhynchus spp. Larvanem www.koppert.com
Thelohania solenopsae Solenopsis invicta – (Oi and Williams 2002)
Nematode control agent Nematode targets Product name Reference
Paecilomyces lilicanus Meloidogyne, Globodera spp Bionemat www.biotech-int.com
Bacillus firmus Meloidogyne spp Bionem WP (Blanchinsky et al. 2007)
Microbial control agent Microbial targets Product name Reference
Agrobacterium radiobacter Agrobacterium tumefaciens NoGall™ (Ryder and Jones 1990), www.
beckerunderwood.com
Bacillus subtilis Rhizoctonia, Fusarium spp. Kodiak ® (Brannen and Kenney 1998)
A. Stewart et al.
Pest control agent Pest targets Product name Reference
9
Bacillus subtilis Fusarium, Verticillium, Pythium spp Biosubtilin www.biotech-int.com

Pseudomonas fluorescens Sclerotinia, Rhizoctonia, Pythium spp. Biomonas www.biotech-int.com
Streptomyces griseoviridis Fusarium, Phytophtora, Pythium, Mycostop www.verdera.fi
Rhizoctonia spp.
Coniothyrium minitans Sclerotinia spp. Contans ® www.prophyta.de
Pythium oligandrum Sclerotinia, Pythium, Alternaria spp. Polyversum www.polyversum.cz
Trichoderma atroviride Sclerotium cepivorum Tenet® (Stewart and McLean 2007), www.agrimm.
co.nz
T. harzianum T-22 Pythium, Rhizoctonia, Fusarium spp. Rootshield®, PlantShield® (Harman and Bjorkman 1998), www.
bioworksinc.com
T. virens Pythium, Rhizoctonia spp. SoilGard® (Lumsden et al. 1996; Lumsden and
Knauss 2007)
Utilizing Soil Microbes for Biocontrol
349
In contrast to the soil pest situation, there is a much broader range of specificities
exhibited by microbes targeted for plant disease control. This is likely to be a reflec-
tion of the broader mechanisms of action exhibited by these agents (e.g., parasitism,
competition, antibiosis, induced resistance). For example, there is an extremely high
degree of specificity and a single target focus exhibited by A. radiobacter due to the
specific activity of the antibiotic produced and there is a relatively high degree of
specificity where sclerotial parasitism occurs in Sporidesmium and Coniothyrium.
In contrast, there are several biocontrol fungal and bacterial species that have a
wider range of host targets covering several different taxonomic groups of soil-borne
plant pathogens. This can be evident at the individual strain level, for example,
T. harzianum T-22 is widely utilised for control of a range of soil-borne pathogens
(e.g., Rhizoctonia, Pythium and Fusarium spp.) in numerous glasshouse and broad-
acre cropping situations (Harman 2000).
The primary factors in the selection of biocontrol microbes are intrinsic pathogenic-
ity or suppressive ability, but environmental competence – the ability of an applied
microbe to persist and multiply in the pest’s environment – is also essential (Jackson
and O’Callaghan 1997). The soil comprises a microbial reservoir, where it is estimated
that a Gram of fertile soil may contain as many as 105–108 bacteria, 106–107 actinomy-
cetes and 105–106 fungal colony forming units (Metting 1993), and with as many as
3,000 species represented in a fertile pasture soil (Ovreas and Torsvik 1998). Therefore,
applied microbes must be able to establish and survive in this competitive environment
to be successful in pest or disease control. Some species, such as the spore-forming
B. popilliae, are highly robust and can survive for many years in the soil before inges-
tion and germination in the specific conditions provided by the insect gut (Klein and
Jackson 1992). Another spore former, M. anisopliae DAT F001 can survive in the soil
for several years after application with persistence aided through recycling in the host
population (Rath and Bullard 1997). Non-spore forming microbes, such as the bacte-
rium S. entomophila, primarily persist by recycling through the host population. The
nests of social insect are particularly challenging environments, where applied
microbes must be able to survive both the stringent conditions within the nest and
deterrent chemicals produced by the target insects. Testing from a wide variety of
isolates has proven essential for selection of isolates that can survive these conditions
and provide effective control of ants and termites (Almeida et al. 1997; Alves and
Pereria 1998). Furthermore, lack of a repellent effect is a key trait when introducing a
pathogen into the nest of social insects; this is important for acceptance of contami-
nated bait if this method of delivery is used, but also to promote development of an
epizootic within a nest. Differences in repellency of fungal conidia have been previ-
ously observed and may be a critical factor in the selection of appropriate isolates for
control of social insects (Wright et al. 2005). Persistence in soil of microbial agents
used for plant disease control is highly variable depending on the system under inves-
tigation. Those microbes effective as sclerotial parasites often have good persistence
because the host pathogen target acts as the survival conduit. Fravel et al. (1986)
showed that Talaromyces flavus, a mycoparasite of Rhizoctonia solani and Sclerotinia
sclerotiorum was able to persist in the soil on parasitized sclerotia from one season to
the next. Similarly, a single application of Sporidesmium sclerotivorum to a field at
1,000 macroconidia g–1 soil provided significant control of lettuce drop for three
consecutive crops over a 2 year period, indicating that the mycoparasite could be added
to the field, establish, proliferate and persist (Adams and Ayers 1982). Although, bio-
control strains of Trichoderma and Gliocladium are common soil saprophytes able to
survive on organic debris, they are not highly competitive in the soil environment with
persistence often declining over a 3–6 month period. To utilize these agents effectively
in biocontrol requires the addition of a nutrient base to sustain the population levels.
Identification and Selection of Desired Traits
Insect Pests
As outlined above, specificity and persistence are key traits of a successful biocon-
trol agent for soil pests. The importance of specificity in pest biocontrol suggests
that targeted discovery will be more useful than high throughput screening from
random sources in the selection of microbes. A review of the organisms developed
into successful pest control products (Table 1) confirms that most isolates used in
commercial products were taken from diseased insects in declining populations.
Once a genetic mechanism is identified determining a desired trait, the strain selec-
tion process and quality control of developed products can be progressed to a higher
level. Discovery that genes encoding pathogenicity were located on a specific plas-
mid (Glare et al. 1993) explained differences in pathogenicity between phenotypi-
cally similar strains and also loss of pathogenicity in extreme conditions.
Identification of the gene sequence controlling pathogenicity allows pathogenic
strain differences to be determined (Hurst et al. 2000). Knowledge of the genetic
determinants allows quality control systems to be developed for monitoring stabil-
ity in production and persistence in the field (O’Callaghan and Jackson 1993).
Insect-pathogenic fungi have generally been derived from the target pest directly,
or from its environment. However, geographically exotic isolates or those from
other hosts may be equally effective (Charnley and Collins 2007), and selection
may simply be based on the prevailing regulations in force in the country where the
pathogen will be used. The pathway to registration may be easier if a product con-
tains a ‘local’ isolate (Chandler et al. 2008). Virulence, environmental competence
and biological fitness all need to be considered in the selection of strains for further
evaluation. Selection of strains, which can germinate, grow and infect the target
pest under defined environmental parameters, will provide strains that have the
desired pre-requisite characteristics for field use. Their suitability for mass-
production and their genetic stability are additional important selection criteria.
Searches for fungi with specific traits have also been carried out. For example, sur-
veys carried out to recover cold-tolerant isolates led to the discovery of M. anisopliae
DATF001 and its development as a biocontrol agent for A. couloni (Rath et al. 1995).
Differences in germination and growth of M. anisopliae and B. bassiana isolates at both
high and low temperatures have been demonstrated with implications for strain selection
and application in insect control strategies (Fernandes et al. 2008). Baiting of soil sam-
ples taken underneath pecan trees that had previously been sprayed with a regime of
fungicides yielded fungal isolates with enhanced resistance to these chemicals (Shapiro-
Ilan et al. 2002). Thus, selection pressures exerted on the natural population of entomo-
pathogens probably results in the evolution of resistant strains and targeted sampling
could yield strains with superior chemical compatibility (Mietkiewski et al. 1997).
Microbial Pathogens
For many years the standard laboratory dual culture test was used to identify and select
microbes with antagonism towards plant pathogens. However, it has become apparent
that isolate selections made using this method do not correlate well with results and
efficacy in the field. The main exception to this is with sclerotial parasites where Henis
et al. (1984) found a good correlation between sclerotial parasitism in agar plates and
biocontrol in glasshouse and field tests. Thus, there is no general reliable laboratory
test for biocontrol activity due to variability in plant pathogens, cropping systems, soils
and temperatures. Selection assays are usually developed for specific mechanisms of
bioactivity that are deemed likely to bring about biocontrol in any particular system.
For example, many researchers have identified rhizosphere competency as a key
biological attribute for microbes targeted to control root pathogens and so selection
assays have been developed to identify this trait. Other researchers have targeted the
selection of plant endophytic microbes on the basis that they would have greater
induced resistance capability (Coombs et al. 2004) and assays comparing the carbon
utilisation profiles of test strains relative to the target pathogen have been developed to
select for microbes with high soil competitiveness (Marin et al. 1998).
Ecological Data
There has been considerable investment in the development of microbial biological

control agents for inundative use, yet ways of preserving and enhancing the
ecosystem services provided by these organisms, and developing a greater under-
standing of their fundamental ecology in soil have received scant attention.
Research on factors affecting the performance of microbial biocontrol agents in soil
is sparse. This is, in part, due to the complexity of the soil environment and the
intricate interactions between different environmental and biological factors that
can confound observations around cause and effect. While in vitro testing can pro-
vide valuable insights into microbial responses to specific inputs, they rarely yield
data that can be directly extrapolated to predict field responses. More effort must
be invested to evaluate effects of agricultural practices (e.g., Mietkiewski et al.
1997) on persistence and (particularly) efficacy under field conditions.
Production of good ecological data has also been impeded by a historic lack
of tools to examine and quantify microbial populations in soil. Traditionally,
studies investigating interactions with other soil microorganisms have relied on
time-consuming isolation and plating techniques. Similarly, risk assessments have

tended to focus on macroorganisms, while monitoring of interactions with other
microbes has been limited and biased by our inability to culture all soil
microorganisms. However, new tools and increasingly powerful molecular methods
are becoming available to examine microbial communities in soil, and may be
applied to the study of microbial biocontrol agents. Use of, for example, nuclear
ITS and EF1-alpha sequences have enabled isolates of entomopathogenic fungi to
be differentiated and phylogenetic relationships within species to be determined,
enabling links to geographic and host origins to be defined. The ability to transform
microbes to express beta-glucuronidase (GUS) and the green fluorescent protein
(GFP) allows transformants to be observed in situ, and key ecological questions
related to growth, sporulation, parasitism, and root colonisation etc. to be addressed
(Bae and Knudsen 2005; Lorang et al. 2001). A variety of other molecular
techniques such as RFLP, T-RFLP, AFLP and strain-specific microsatellite markers
have been used as diagnostic tools allowing microbes to be tracked in the
environment (Dodd et al. 2004; Enkerli et al. 2005; Inglis et al. 2008). Advances
in the use of PCR techniques provide highly specific methods of monitoring micro
bial populations in ‘real time’ and in a quantitative manner, in soils, insects and
in planta (Ownley et al. 2004; Wang et al. 2004; Entz et al. 2005; Schwarzenbach
et al. 2007a; Meyling et al. 2009). Use of quantitative PCR with automated
ribosomal intergenic spacer analysis (ARISA) allow soil microbial communities to
be profiled and responses to specific events to be monitored; these techniques are
set to be increasingly applied to the study of microbial biocontrol agents to assess
their fate and impacts on microbial community structure and ultimately plant health
(Schwarzenbach et al. 2007a, b; Enkerli et al. 2008).
Our ability to capitalize on the potential shown by microbes to regulate plant
pest and pathogen populations will be greatly increased by improved insights into
their ecology and behaviour in soil, their response to different environmental fac-
tors and farming practices, and interactions with other members of the soil biota
(Jaronski 2007; Meyling and Eilenberg 2007). This knowledge can then be applied
to the selection of robust, environmentally-competent strains; development of pro-
duction systems that provide inoculum that is ‘fit for purpose’; and to improve
formulation and delivery techniques, etc., such that constraints to the effective
utilization of these microbial control agents can be overcome.
Integrated Control
For the most part, this review has focused on the utilization of microbial biocontrol
agents to control either soil-borne pests or diseases. In any given system, though,
crops are likely to be affected by a suite of pests, requiring the concurrent use of
more than one control agent. It is important, therefore, to ensure compatibility
among the different control strategies so that their efficacy is not impaired in an
integrated crop management programme.
The benefit of combining different biocontrol agents has been reported, for example,
Alabouvette et al. (1996) demonstrated a synergistic effect in controlling Fusarium
oxysporum f.sp. radicis-lycopersici by combining a fluorescent Pseudomonas sp. with
a non-pathogenic F. oxysporum. The non-pathogenic F. oxysporum competes for
carbon sources while the bacterial antagonist produces a siderophore competing
for iron (Lemenceau et al. 1993). Effective disease control was also demonstrated by
Domenech et al. (2006) for a combination of several plant growth promoting
rhizobacteria. They reported enhanced biocontrol of Fusarium wilt and Rhizoctonia
damping-off on tomato and pepper by the product LS213 that contained mixed
inocula of Bacillus subtilis GB03 and B. amyloliquefaciens IN937a. A combined
application of M. anisopliae with S. entomophila increased grass grub mortality in pot
trials, suggesting synergies between biocontrol agents for pest and disease control
could be explored (Jackson and Chinn 1993). However, a range of mortality responses
were observed when entomopathogenic nematodes were combined with various
beneficial fungi or bacteria against pecan weevil, thus, demonstrating the need to
consider these interactions on a case-by-case basis rather than simply assuming that
all biocontrol agents are compatible or interact positively (Shapiro-Ilan et al. 2004).
More usually, microbial biocontrol agents have been combined with a range of
other crop management and pest management practices. Incorporation of antagonists
following disinfestation of the soil with fumigants has been shown to be effective in
controlling R. solani on carrots (Strashnow et al. 1985), and soil solarization proved
a good control strategy when combined with Gliocladium virens against southern
blight of tomatoes (Ristaino et al. 1991). Minuto et al. (2004) demonstrated that the
combination of soil solarisation and Streptomyces griseoviridis was effective against
Fusarium and Verticillium wilts and corky root rot, increasing the range of pathogens
controlled with respect to the single treatments. Similarly, Stevens et al. (2003)
showed that integration of soil solarisation with the biological control agent T. virens
and the chemical fungicide PCNB significantly reduced southern blight and root knot
disease on tomato compared to individual applications. The use of biocontrol agents
with fertilization has also been shown to provide enhanced disease control. Hoynes
et al. (1999) reported that soil treatment with ammonium sulphate followed by
Gliocladium virens G1-3 resulted in greater reduction in viability of sclerotia of
S. rolfsii and a higher bean seed germination than that achieved with each individual
component. Howell (2007) reported optimum control of pre- and post-emergence
damping-off of cotton seeds with a seed treatment of chloroneb plus Trichoderma.
Trichoderma controlled the pre-emergence damping-off caused by Pythium spp. and
the fungicide controlled post-emergence damping-off caused by Rhizoctonia solani.
Similarly, T. virens in combination with metalaxyl as a seed treatment on cotton was
shown to provide increased control of seedling diseases (Howell et al. 1997).
Similar synergistic interactions have been reported with insect pathogens, for
example, M. anisopliae was applied to horticultural growing media or soil with sub-
lethal doses of insecticides (imidacloprid or fipronil) to control black vine weevil
(Shah et al. 2007) and citrus root weevil (Quintela and McCoy 1998) and co-application
of M. anisopliae with neem seed cake enhanced fungal efficacy against black vine
weevil (Shah et al. 2008). Improved efficacy of the biocontrol agents was attributed to
effects of the insecticides on insect behaviour, which promoted acquisition of infec-

tious material or reduced mechanical removal of conidia from the cuticle. Such an
approach for insect pest management in soil serves to reduce pesticide use while
improving opportunities to utilize fungi cost-effectively in this environment.
Conclusions
Microbial biocontrol agents have a demonstrated capacity to regulate pests and

diseases, and possess numerous beneficial traits that favour their use in integrated
crop management systems. The challenge lies in capitalizing on this potential, and
developing products and strategies that exploit their unique characteristics. Presently,
products containing insecticidal bacteria, primarily Bacillus thuringiensis (for foliar-
feeding insects), dominate the biopesticides market. The bacterium has lent itself well
to commercialization; industrial-scale liquid fermenters provide a raw material that is
stable, readily-formulated and efficacious, for distribution and sale into to a global
agricultural market. Few other microbial biocontrol agents (bacteria and fungi) have
similar characteristics, which has been a limiting factor to their wider use. Products
based on these microorganisms largely occupy niche markets, often within individual
countries or geographically-linked regions. To increase utilization of these microbial
biocontrol agents, several technical and regulatory challenges need to be overcome
which, by-and-large, are common to all of these organisms. Fundamentally, we must
recognize the strengths and weaknesses of microbial controls, and not simply try to
replace chemical insecticides with biologically-derived materials.
In general, there is no shortage of excellent candidate organisms, some of
which are already commercialized. To ensure their broader uptake in agriculture,
more efficient mass production, formulation and delivery systems must be
devised simply to supply a larger market; more testing under field conditions is
required to identify effects of biotic and abiotic factors (and interactions between
them) on efficacy and persistence, and potential limitations to the use of these
biocontrol agents in certain crops or locations; and there has to be greater invest-
ment in the optimization of use practices. Advances in our understanding of
infection processes, combined with the availability of new molecular tools which
aid our ability to monitor the fate of these organisms in the environment and
quantify effects of environmental factors on efficacy and persistence, continue to
provide insights that will support the rational development of these technologies.
Outreach and demonstration programmes that promote understanding of what
growers can (or cannot) expect from microbial control agents, coupled with
appropriate training on their use according to best practices, will further enhance
their successful implementation.
The regulatory environment is creating conditions whereby biocontrol strategies
are becoming the only available option for pest management. An increased interface
between agricultural and urban environments and ‘Greenbelt Legislation’ is often
preventing or strictly limiting the use of conventional crop protection materials.
Also, consumer awareness and demand is driving the move to implement more sus-
tainable pest and disease management techniques. By ensuring that quality products
are available and that farmers are equipped to apply them, microbial biocontrol
agents can become important components of integrated crop production systems.
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Chapter 10
How Will Climate Change Impact Soil
Microbial Communities?
Teri C. Balser, Jessica L.M. Gutknecht, and Chao Liang
Introduction
More than a century ago Svante Arrhenius predicted that continued combustion
of fossil fuels would lead to a doubling of carbon dioxide in the atmosphere and
associated climate warming (Arrhenius 1896). Despite this warning, we are now
faced with the predicted doubling of atmospheric carbon dioxide and global
temperature increase of 1.3°C by the end of this century if no policy changes are
made (Cubasch et al. 2001). Furthermore, not only are we faced with rising global
temperature but also shifting weather patterns, ocean acidification, and the potential
loss of many species on earth (Intergovernmental Panel on Climate Change (IPCC)
2001). These factors will all have a marked impact on land use, land cover, soil
quality, and productivity.
Climate change will have direct and indirect impacts on terrestrial ecosystems,
both above- and belowground (Fig. 1). Aboveground, the effects of global change
will be largely direct: elevated atmospheric carbon dioxide as well as changes
in temperature, precipitation, and nitrogen availability will all result in changes to
the abundance of plant species and altered land cover in unmanaged ecosystems
(Tylianakis et al. 2008). In managed systems, changes in seasonal climate and
precipitation will influence our choice of crop species, and the production of and
stresses on those chosen species, which will in turn influence management decisions
for irrigation, fertilization, and pathogen dynamics (Dixon 2009). Indirectly, land
use, plant species composition, and plant productivity all feedback to further alter
plant communities via changes in the belowground microbial community.
T.C. Balser (*) and C. Liang

Department of Soil Science, University of Wisconsin-Madison, 1525 Observatory Drive,
J.L.M. Gutknecht
Department of Soil Ecology, Helmholtz-Centre for Environmental Research-UFZ,
Theodor-Lieser-Str, 4, D06120 Halle (Saale), Germany
+CO2 changing
Shift in plant temperature
+N species range
or moisture
trace gas
Short term Changes what we
increase in release to
can grow where atmosphere
productivity CO2 release to
atmosphere
pathogens, plant
nutrient availability
Soil
Surface
increase in differential plant
belowground microbial nutrient
C allocation community and water use
carbon structure, +/− soil O2
availability function
+ −
nutrient
imbalance, +/− decomposition substrate
limitation availability
(e.g. Fe, S,
NO3−)
Fig. 1 Schematic of direct and indirect effects of global change drivers an below grand communi-
ties, including potential Feedbacks. Direct effects: elevated CO2 or N deposition;
Indirect effects: elevated CO2 or N deposition; Direct effects: altered temperature or pre-
cipitation; Indirect effects: altered temperature or precipitation; Feedbacks to
atmosphere or plant growth
Belowground, the response to climate change is more complex. Plant responses

affect the type and amount of carbon entering the soil system as well as the physical
architecture of the plant root zone. This has an indirect impact on microbial com-
munity composition and biomass. Water, temperature, and nitrogen all have direct
effects on the microbial community as the organisms respond to temperature or
drought stress along with altered resource availability. Microbial responses to cli-
matic changes, whether direct or indirect, will feedback to influence plants via nutri-
ent availability or pathogen productivity (Fig. 1). Thus a critical step in our
understanding of ecosystem response to climate change must be to increase our
understanding of the microbial community response.
However, too often researchers study ecosystem response to climate change with-
out explicitly including the microbial communities. Nevertheless, through their diverse
metabolic activities soil microbial communities are the major drivers of soil nutrient
cycling and their importance in mediating climate change and ecosystem functioning
should not be underestimated (Balser et al. 2001). Microbial metabolic activities can
be altered by many ecosystem-scale factors such as climate change, nitrogen
deposition, elevated carbon dioxide, or disturbance (Dhillion et al. 1996; Ajwa et al.
1999; Mayr et al. 1999). Shifts in microbial activity can in turn lead to changes in
decomposition, nitrogen mineralization, organic carbon storage, and other ecosystem
10 How Will Climate Change Impact Soil Microbial Communities? 375
processes (Carreiro et al. 2000; Sinsabaugh et al. 2002; Henry et al. 2005a; Sowerby
et al. 2005). Predicting microbial metabolic responses to these global changes and
disturbance factors is thus essential for an understanding about how global change will
affect soil and ecosystem function. For example, nitrogen deposition is likely to
decrease mycorrhizal fungal biomass while increasing bacterial and saprotrophic fun-
gal biomass (Treseder 2004; Rinnan et al. 2007) and has the potential to increase
carbon cycling by increasing the activity of microbial enzymes related to carbon
cycling (Henry et al. 2005a; Gutknecht et al. 2010). Elevated carbon dioxide may miti-
gate the effect of nitrogen deposition, but results have been highly varied (Treseder
2004, 2008). The response of microbial communities to climate change, and interac-
tions between climate change and other global change factors, may be highly depen-
dent on specific ecosystems and historical adaptation of the community (Rinnan et al.
2007; Balser et al. 2005; Fraterrigo et al. 2005, 2006).
In this chapter we will discuss the impact of climate change on belowground
microbial community structure and function.
Microbial Response to Climate Change
As a preface to a more detailed discussion of microbial response to specific global

change drivers later in the text, here we give an overview of microbial community
climate change research. Until recently, the focus of most climate change research
has been on how ecosystems respond to elevated atmospheric carbon dioxide.
While it is relatively well established that elevated carbon dioxide changes
belowground carbon allocation by plants (root biomass and root respiration), there
is less consensus on the response of microbial communities (Zak et al. 2000a). Root
symbionts such as mycorrhizal fungi and nitrogen fixing bacteria may (O’Neill
1994; Treseder and Allen 2000) or may not (Treseder and Allen 2000) increase in
biomass under elevated carbon dioxide. Microbial total biomass and community
structure also have shown highly varied responses to elevated carbon dioxide (Zak
et al. 1993, 2000b; Diaz et al. 1993; Hungate et al. 1996; Lussenhop et al. 1998;
Bruce et al. 2000; Montealegre et al. 2002), even between similar soils or between
plant species (Hungate et al. 1996; Niklaus 1998). Over longer time spans
especially, elevated carbon dioxide may have little direct effect on soil microbial
communities (Niklaus et al. 2003). For instance, after 6 years of elevated carbon
dioxide in a temperate grassland ecosystem, Niklaus et al. (2003) reported no major
effect on microbial biomass, microbial community composition, or microbial
carbon and nitrogen despite an increase in plant productivity and shift in soil
aggregation (although there were small effects on microbial variables that changed
each year of the study).
In addition to the effort to understand the microbial response to elevated carbon
dioxide, other major drivers of global change, namely nitrogen deposition, elevated
temperature, and altered precipitation have been the focus of much research.
Nitrogen addition to unmanaged as well as managed ecosystems is consistently
associated with a decrease in mycorrhizal fungal biomass (Balser et al. 2001;
Treseder 2004; Klironomos et al. 1997) as well as shifts in microbial community

composition and decomposition rates (Balser et al. 2001; Carreiro et al. 2000;
Henriksen and Breland 1999). Increased precipitation and hence increased soil
moisture may result in increased microbial predation by mesofauna (Clarholm 1985;
Kuikman et al. 1991; Taylor et al. 2004). It is also thought that the osmotic potential
of the soil solution (soil salinity effects on water movement and availability) may
play a more important role than absolute water content (Stark and Firestone 1995;
Zak et al. 1999). Finally, increased temperature may increase microbial metabolic
activity in temperate ecosystems (Zak et al. 1999; Contin et al. 2000; Vinolas et al.
2001), but not as much in northern ecosystems (Allison and Treseder 2008).
Temperature increase is also predicted to shift carbon use toward old carbon or
toward new carbon pools (MacDonald et al. 1995; Zogg et al. 1997; Andrews et al. 2000)
or just generally increase soil respiration (Lin et al. 1999; Niinisto et al. 2004).
Experiments focused on manipulating single factors alone (such as those with
elevated carbon dioxide, or altered moisture or temperature) offer valuable insight
on fundamental responses, but there is a need for studies which examine the realis-
tic future of global change: that of multiple, simultaneously interacting factors
(Intergovernmental Panel on Climate Change (IPCC) 2007). Multiple factor, long-
term studies may allow for a more mechanistic, predictive understanding of how
microbial communities will respond to future global changes.
Those studies that are focused on interacting factors tend to focus on two general
groups; (1) interactions between microbial resources (such as carbon and nitrogen),
and (2) interactions between climate factors and resources (for instance, tempera-
ture with nitrogen addition). Many investigations have focused on the possible
interaction between elevated carbon dioxide and nitrogen deposition, with less
attention on how elevated carbon dioxide or nitrogen deposition may interact with
climate change factors (temperature or precipitation).
Elevated carbon dioxide or nitrogen deposition may push a system toward nitro-
gen or carbon limitation (by increasing available soil carbon or nitrogen, respec-
tively), so when both are elevated, the responses may mitigate each other (Hu et al.
1999; Lee et al. 2003; Schaeffer et al. 2003). For instance, elevated carbon dioxide
lessens the positive effect of nitrogen addition on soil respiration and decomposi-
tion of litter (Lutze et al. 2000). Elevated carbon dioxide may also slow a decline
in the abundance of saprotrophic fungi seen with nitrogen addition alone (Řezáčová
et al. 2005). At the same time that effects of nitrogen deposition may be altered by
elevated carbon dioxide, the effects of elevated carbon dioxide on plants and micro-
organisms may be dependent on soil nitrogen and fertility levels (thus, creating a
cycle of nutrient limitation; Zak et al. 2000a; Lee et al. 2003; Tate and Ross 1997;
Martin-Olmedo et al. 2002). Elevated nitrogen has been shown to slow the increase
in carbon storage seen with elevated carbon dioxide alone (Tate and Ross 1997; van
Groenigen et al. 2002), perhaps by increasing carbon cycling rates. The response of
arbuscular mycorrhizal fungi (AMF) to elevated carbon dioxide may also depend
on soil nutrient levels (Rillig and Field 2003) and determine whether AMF biomass
becomes a carbon sink or decomposes organic matter and contributes to carbon
cycling (Treseder and Allen 2000).
Climate change factors (e.g. elevated moisture or temperature) also interact with
elevated carbon dioxide and nitrogen addition. For instance, increased moisture can
increase the apparent effect of nitrogen addition by increasing the movement of
nitrogen to the plant (Henry et al. 2005a). Or water may act in concert with nitrogen
addition to increase decomposition of plant tissues (Henry et al. 2005b). Increased
moisture, or alleviation of water stress, can also alter the lignification of plant cell
walls (Henry et al. 2005b), increase grassland productivity, or impact soil carbon
(Tate and Ross 1997). Soil moisture coupled with elevated carbon dioxide also
decreases abundance of ammonium oxidizing bacteria, potentially altering the soil
nitrogen cycle (Horz et al. 2004). Warming with elevated carbon dioxide may act
additively to increase soil respiration (Niinisto et al. 2004; van Veen et al. 1991;
Körner and Arnone 1992; Peterjohn et al. 1993; Johnson et al. 1994; Nakayama
et al. 1994; Pajari 1995; Vose et al. 1995; Hungate et al. 1997). Although there have
been few reports on the interactions between elevated temperature and moisture,
there is evidence to suggest that together they may lead to shifts in the structure of
methane oxidizing bacterial communities (Horz et al. 2005). In sum, while there are
reported differences, it is difficult to synthesize the varied patterns seen among
temperature or soil moisture and elevated carbon dioxide or nitrogen. There is also
little information about how climate factors will interact with elevated carbon diox-
ide and/or nitrogen to affect microbial biomass or community structure (Pendall
et al. 2004). More research is needed to generate a synthetic explanation and build
usable conceptual models.
It is apparent from the research outlined above that there is no straightforward
or clear model or prediction of how microbial communities will respond to future
global climatic changes. In fact, several recent reviews have commented on our
need for better general understanding of soil communities in order to understand
soil feedbacks to future global change (Zak et al. 2000a; Pendall et al. 2004; Foley
and Ramankutty 2004). In the next section of this chapter we will review microbial
response to specific climate perturbations in more detail.
Response to Specific Changes in Climate
Impact of Increasing Temperature
As climate warms, microbial populations must acclimate or die. Many studies have
shown an increase in microbial biomass in short-term experiments but over the
long-term under elevated temperature biomass is more likely to decrease. This is
because the efficiency of microbial growth changes at higher temperatures (Schimel
et al. 2007; Hyvonen et al. 2005). Rather than biomass being directly correlated
with decomposition, the reverse becomes true as organisms utilize labile carbon for
energy production rather than biomass production (Contin et al. 2000; Zogg et al.
1997; Schimel et al. 2007). A specific instance is that higher temperatures alter cell
membrane fluidity and permeability, requiring membrane lipid re-synthesis
(Petersen and Klug 1994). The high energetic cost of this stress response is one
mechanism by which carbon can be utilized for energy instead of biomass. When
the microbial energy demand exceeds the limit of labile carbon pools, biomass
cannot be maintained and may decline at higher temperatures (Balser 2000; Balser
and Firestone 2005). If however, the microbial community can access necessary
labile carbon, then increased temperature will result in a shift in carbon allocation
from growth to acclimation with a concomitant decrease in growth efficiency (i.e.
increase in respiration per unit biomass; Schimel et al. 2007). In this case microbial
biomass may be maintained rather than decline.
In one of the few long-term studies including elevated temperature that explicitly
focused on the microbial community, Gutknecht (2007) considered 8 years of field-
based data. They found that ambient inter-annual and seasonal variations in the
microbial community were greater than effects of elevated temperature or moisture
treatments. Overall there were few significant responses of microbial biomass or
community structure to climatic treatments (elevated precipitation or temperature),
with responses to elevated temperature being stronger than elevated precipitation.
Importantly, however, these treatments did impact community process responses.
Exposure to 8 years of elevated temperature or elevated precipitation in the field
affected community responses to a short-term increase in substrate availability. In
detail, Gutknecht (2007) reported a microbial response to elevated temperature that
was similar to that obtained with nitrogen addition. Mycorrhizal abundance
decreased, but other bacterial and general fungal microbial indicators increased in
relative abundance. Elevated precipitation was related to lower relative abundance
of a mycorrhizal indicator, but this trend was only statistically significant in one of
6 years of this study. While there were few significant effects from climate manipu-
lation alone, temperature interacted to modify the effects of elevated carbon diox-
ide or nitrogen addition alone. Increased temperature may directly enhance
arbuscular mycorrhizal (AM) colonization and development (Pendall et al. 2004;
Fitter et al. 2000; Gavito et al. 2003), or may have the opposite effect. These
changes in mycorrhizal fungal biomass may be important as mycorrhizas play such
an important role in plant nutrition and plant, community, and ecosystem responses
to global change.
Model systems can also be valuable for looking in detail at microbial responses to
elevated temperature. Bardgett and Shine (1999), in a model ecosystem study, explored
how a microbial community responded to elevated temperature over three plant gen-
erations. Microbial analyses revealed that biomass increased significantly, but only
during the first plant generation. By the last plant generation, total microbial biomass
was in decline. The initial increase in microbial biomass was likely due to fast-growing
bacteria that initially responded to elevated temperature, while slower growing
microbes such as fungi and actinomycetes were unaffected. Similarly, the relative
abundance of Gram-positive and Gram-negative bacteria may increase with tempera-
ture, perhaps due to a shift in available substrates (Zogg et al. 1997), while fungal and
actinomycete biomarkers may decline at higher temperatures (Waldrop and Firestone
2004). These studies together illustrate the importance of understanding how different
microorganisms respond to elevated temperature, and how these varied responses
impact on the timing and duration of the overall community response to elevated
temperature or other potentially to other global changes.
Finally, there is a growing body of work indicating that the size and quality of
the carbon pool accessed by microorganisms changes with varying temperature
(MacDonald et al. 1995; Zogg et al. 1997; Andrews et al. 2000; Tison and Pope
1980; Linkins et al. 1984; Ellert and Bettany 1992; Nicolardot et al. 1994). Models
that define decomposition or respiration rate coefficients (k) as a function of tem-
perature assume constant substrate pool size and uniform substrate preference
(Ellert and Bettany 1992). However, MacDonald et al. (1995) found that the pool
size of carbon substrate available to the microbial community can vary substantially
with temperature. In addition, several researchers have found that not only does the
size of the carbon pool accessed change with temperature, but microbial use of
specific substrates also changes (Zogg et al. 1997; Andrews et al. 2000; Balser
2000; Waldrop and Firestone 2004; Nicolardot et al. 1994). Further, some data sug-
gest that the response is not always consistent with simple kinetics. In the microbial
community of a Californian annual grassland for instance, substrate-utilization
varied substantially with incubation temperature (Balser and Wixon 2009).
Strikingly, despite predictions that polymeric carbon will be more readily degraded
at higher temperatures, we have seen the opposite effect. At cooler incubation tem-
peratures, polymers were preferentially degraded (Balser and Wixon 2009). The
significance of this for soil carbon dynamics remains to be explored.
However, the issue of whether soil will act as a net carbon source or sink in
response to climate warming remains a matter of intense interest in global change
policy and research communities (Cox et al. 2000; Shaver et al. 2000; Rustad et al.
2001; Knorr et al. 2005). Because global soil organic carbon concentration is greater
than twice that of the atmosphere (Post et al. 1982; Schimel 1995; Schlesinger 1996),
even small changes in flux can have a significant impact on atmospheric carbon diox-
ide (Kirschbaum 2000; Rustad et al. 2000; Schlesinger and Andrews 2000).
In particular, the sensitivity of recalcitrant (‘older’) carbon to temperature is a critical
parameter for predicting the role of soil as a feedback agent in climate warming (Knorr
et al. 2005; Giardina and Ryan 2000; Luo et al. 2001; Fang et al. 2005). While there
is a reasonable level of agreement that younger (labile) carbon will generally display
a predictable pattern of response to temperature (e.g. it has a Q10 of approximately 2.4
and increasing rate of mass loss as temperature rises), the dynamics of older carbon
largely remain a mystery (Kirschbaum 2000; Agren and Bosatta 2002). The sensitivity
of recalcitrant carbon to rising temperature has been predicted to increase (Knorr et al.
2005; Fierer et al. 2005), decrease (Agren and Wetterstedt 2007; Wagai et al. 2008), or
remain invariant (Giardina and Ryan 2000; Luo et al. 2001). This variability is likely
due to the web of interacting factors that influence carbon stability in soil (Davidson
et al. 2000). As litter transforms to ‘soil organic matter’, and then ages to stable
(humic) forms it becomes increasingly chemically altered (Balser 2005) and associ-
ated with soil minerals (Sollins et al. 1996). Further, litter of differing chemical quality
will vary in its transformation, as will the availability of organisms to degrade it (Balser
2005). As a result, temperature sensitivity of older carbon is not a simple function of
enzyme response, but instead is the product of a complex suite of interactions among
the varying temperature responses of competing processes such as activation energy

(Bosatta and Agren 1999), altered substrate diffusion (Mikan et al. 2002), mineral
adsorption or occlusion (Wagai et al. 2008; Thornley and Cannell 2001), historical
carbon input and land use (Davidson et al. 2000), and acclimation of the decomposer
community (Balser and Firestone 2005; Waldrop and Firestone 2004; Agren and
Bosatta 2002). To date, studies including or focusing on more than one of these factors
at a time are rare, and consequently, results of existing studies often appear idiosyn-
cratic or surprising (Davidson et al. 2000; Mack et al. 2004).
Impact of Elevated Carbon Dioxide
To date, the majority of studies investigating ecosystem carbon storage response to

increasing atmospheric carbon dioxide have been focused on the role of above-
ground tissue chemistry and biomass production (Pan et al. 1998; Mooney et al.
1999). However, initial predictions that increases in plant biomass and decreases in
above-ground litter quality in response to elevated carbon dioxide concentrations
would lead to increased soil carbon storage have not been borne out (Norby and
Cotrufo 1998; Schlesinger and Lichter 2001). In fact, it has become clear in recent
years that more often than not, aboveground biomass remains unchanged, and plant
tissue chemistry at senescence is indistinguishable from that grown at ambient carbon
dioxide concentrations (O’Neill 1994; Curtis et al. 1989; Franck et al. 1997).
However, there is a growing body of evidence indicating that carbon dioxide enrich-
ment coupled with increased plant nitrogen requirements leads consistently to
enhanced carbon allocated belowground (O’Neill 1994; Niinisto et al. 2004; Cardon
et al. 2001). Although responses vary across systems, increased carbon flow to below-
ground pools appears to be driven by mechanisms for nitrogen acquisition such as:
1. Increased allocation to root structural tissues (Zak et al. 2000a; Owensby 1993;
Kampichler et al. 1998)
2. Accelerated root turnover (Norby 1994; Pregitzer et al. 2000)
3. Rhizodeposition (Cardon et al. 2001; Hungate et al. 1999)
4. Mycorrhizal development (O’Neill 1994; Treseder and Allen 2000)
5. Nitrogen-fixation (Hungate et al. 1999; Montealegre et al. 2000)
Few studies to date have addressed, however, the relative importance and long-term
consequences of different mechanisms by which increases in belowground carbon
impact microbial communities, and thus carbon dynamics in soil.
Increasing atmospheric carbon dioxide can affect plant responses in several ways:
plant growth can increase or decrease (Poorter 1993; Joel et al. 2001), nutrient usage
and allocation can change (Curtis et al. 1989; Cotrufo et al. 1998), and above- and
belowground patterns of biomass can be altered (Zak et al. 2000a; Hungate et al.
1997). These responses to elevated carbon dioxide affect the competitive abilities of
plant species, and in turn, can alter plant community composition (Zangerl and
Bazzaz 1984). Within plant communities containing exotic, invasive species, the
growth and competitive dynamics of plants may change with rising carbon dioxide to
favour the exotic species or the native species. Fast-growing C3 species, such as many
exotic, invasive species, tend to respond most strongly to elevated carbon dioxide
(Poorter 1993). Therefore, there is concern whether or not invasive species will
become more aggressive in their growth and establishment under enriched atmo-
spheric carbon dioxide. Hungate et al. (1996) showed that the impact of invasive
grasses on the cycling of nitrogen in Californian grassland was greater under elevated
carbon dioxide than under ambient levels. The invasive grasses showed increased
plant nitrogen pools and 15NH4+ (ammonium) uptake under elevated carbon dioxide
while the native species exhibited smaller increases or decreases. A study by Dukes
(2002) showed that growth and competition of Centaurea was enhanced under ele-
vated carbon dioxide. Similarly, Smith et al. (2000) found that both the native desert
annuals and the exotic, invasive grass (Bromus tectorum) showed greater total above-
ground biomass and individual plant biomass under elevated carbon dioxide, but the
total density of the native annuals decreased while the density of Bromus increased.
Additionally, Bromus exhibited a three-fold increase in seed rain, while the whole-
plot seed rain of the native annuals did not increase significantly. These studies illus-
trate that changes in the growth dynamics of invasive and native species under
elevated carbon dioxide may lead to ecosystem-level changes in primary productivity,
which will in turn impact on soil microbial and carbon dynamics as plants senesce.
Impact of Changing Soil Moisture
The soil moisture relationship to microbial community is more highly variable and
complicated than that of temperature, and less studied (Lavigne et al. 2004; Saiz et al.
2007). It is easy to understand intuitively that soil water content will vary negatively
with temperature, and has been shown to be the case (Luo et al. 2001; Davidson et al.
1998). However, the relationship is challenging to describe empirically. There is no
consensus as to an equation describing soil respiration and moisture (Emmett et al.
2004), or moisture and temperature (Lavigne et al. 2004). As with other factors,
interactions may be seen as a key reason that temperature relationships are not clear.
The time scales and spatial scales of water change are different from those of tem-
perature change. Moisture changes may come in the form of wet-dry cycles, drought,
flooding, or smaller shifts. These different changes have different community struc-
tural and functional impacts, and are conditioned by a community’s native regime.
There are several mechanisms or physical processes affecting microbial com-
munities that vary with moisture content (Rodrigo et al. 1997). Precipitation is
generally agreed to constrain decomposition at its extremes of dry (water stress)
and wet (anoxia). Although a general interaction between oxygen concentrations
and soil moisture is intuitively obvious, soil moisture effects are not limited to
anoxia. For example, considering aerobic respiration at low moisture contents,
substrate diffusion has been shown to be the main regulating process at low water
contents in clay soils (Schjonning et al. 2003).
Despite logical mechanisms by which microbial communities may be altered by

changes in soil moisture, these effects have so far only been observed in some studies,
in dry summers (Hui and Luo 2004; Rey and Jarvis 2006). The impact of experimental
drought has been likewise inconsistent (Emmett et al. 2004). Water is a critical factor
in global change, nevertheless. Wetter soils such as peats and wetlands constitute large
carbon sinks. Heterotrophic respiration overall is commonly seen as negatively related
to water content above the soil moisture optima, typically considered 60–80% of water
holding capacity. Waterlogged soils are so successful at creating physical and chemical
barriers to aerobic respiration that a future carbon sequestering management strategy
might involve saturating soils (Sylvia et al. 2005). Other evidence considering the
interacting factor of elevated carbon dioxide suggests that anaerobic wetland condi-
tions may be less of a carbon sink with global climate change (Wolf et al. 2007).
The adaptation of a microbial community to a local precipitation regime can
make generalizing moisture response figures as dangerous as stating a universal
temperature optimum. Response to conditions such as flooding depends on the life
history of the community. Communities more tolerant of flooding, or drying and
rewetting, have different responses to these disturbances (Fierer et al. 2003; Mentzer
et al. 2006). Fierer et al. (2003) provide an example of community adaptation and
process response to moisture levels. Drying-wetting regimes were found to signifi-
cantly impact on bacterial community composition in oak woodland soils, which are
less frequently exposed to moisture stress, but not in grassland soils. Size and func-
tion of litter decomposers can also be strongly affected by their moisture stress his-
tory (Schimel et al. 1999). Thus, the historical adaptation of the microbial community
may determine how an ecosystem responds to changing moisture regimes.
Drought, irrigation, flooding, and re-wetting or other pulse events all can be
expected to have different responses compared with modest water stress or modest
increases in soil moisture (Schimel et al. 2007). Microbial activity or biomass may
increase during wet seasons or after ‘wet-up’ (the period when soils regain moisture
after the dry season; Fierer et al. 2003; Waldrop and Firestone 2006). Rewetting
after drying also has unique process implications, and several studies address it
(Reichstein et al. 2005; Wu and Brookes 2005). For example, dissolved organic
carbon release is enhanced when dry soils are rewetted (Marschner and Bredow
2002), but these effects may be community-specific (Reichstein et al. 2005).
Increasing soil moisture may also alter microbial activity but not biomass. For
instance one study (Waldrop and Firestone 2004) reported that increased soil water
did not affect the size of the available substrate pool, the utilization of differing ages
of soil carbon, or microbial community composition. However, increased soil mois-
ture did decrease hydrolytic enzymatic activity and reduced peroxidase enzyme
activity, associated with complex carbon degradation, by 42%.
It is likely in at least some cases, however, that soil moisture has an influence
even at levels other than the extremes. Considering the importance of soil micro-
sites, water films, osmotic stress tolerances, ion concentrations, and the differential
water retention of different pore size, it is clear that the effect of moisture is structurally
and biologically complex. Any environmental conditions, including smaller moisture
changes that limit or accelerate the diffusion of carbon dioxide from soils or the
surface layer, can create non-equilibrium conditions that impact carbon dioxide
efflux rates (Hanson et al. 2000). Studies have shown that moderate drying can
significantly affect decomposition (Reichstein et al. 2005). Even modest water
stress can cause a 25–50% decline in soil respiration (Lavigne et al. 2004).
A last consideration is that certain factors may interact with soil moisture regimes
to either affect microbial community structure, or activity, or both simultaneously.
A study by Chena et al. (2008) considered two near-optimal moisture levels (60%
and 80% of field capacity) and two different plant covers (with differing biomass).
The community structure (assessed via phospholipid fatty acid analysis) was more
affected by plant species, but community-level physiological profiles were affected
by both plant species and soil moisture. Both physiological and acclimation mecha-
nisms occurred, with differing factors contributing to each mechanism.
Changing Agricultural Management Regimes

and Soil Microbial Communities
Some of the most critical impacts of climate change on microbial communities will
not be through direct changes to the soil environment, but through those that occur
indirectly via changes in land management and vegetation cover as humans respond
to a changing climate.
It is well-established that conversion of native ecosystems to agricultural uses
can strongly affect microbial community structure, composition and diversity.
For example, conversions of tropical forest to plantations (Waldrop et al. 2000)
have been found to engender distinct soil microbial community structures, and
agricultural intensification has been reported to decrease microbial diversity
(Steenwerth et al. 2005). Additionally, the type of land management practices in
agroecosystems also affects microbial community structure and function through a
variety of different mechanisms. Numerous studies have documented changes in
microbial community structure as a result of physical disturbance, especially tillage
(Frey et al. 1999; Guggenberger et al. 1999). Tillage represents a severe disturbance
to fungi by severing hyphal connections and no-till systems favor fungi over bacte-
rial community components (Minoshima et al. 2007; Kennedy and Schillinger
2006). Conversions to agriculture and cultivation practices also alter microbial
communities through changes to temperature, soil moisture (through irrigation and
alteration of soil structure), and other physical parameters.
Land-use changes also alter soil microbial community structure through altera-
tions in carbon availability and quality, pH (Cookson et al. 2007), nutrient avail-
ability, or other chemical parameters. For example, fungal-to-bacterial ratios are
commonly measured as indicators of microbial community structure, and the rela-
tive proportions of fungi are increased by no-till practices, crop rotations, and use
of cover crops (Six et al. 2006). Studies in both agroecosystems and natural systems
report that nitrogen additions decrease the relative abundance of fungi to bacteria
(Bardgett and Shine 1999; Bradley et al. 2006). Seghers et al. (2004) found that
nitrogenous fertilizers decreased populations of methanotrophs in the bulk soil

microbial community, as well as members of the root endophytic community. They
also found differential effects of organic fertilizers versus inorganic, consistent with
other studies. For example, Wander et al. (1995) reported that manure-amended
plots contained a less diverse population of microorganisms than cover cropped
soil, but that the microbial biomass was more metabolically active (Wander et al.
1995). Ulrich et al. (2008) found that manure applications led to an increase in the
population densities of cellulytic bacteria within the soil microbial community.
In addition to physical disturbance effects, alterations in vegetation, including
alterations in plant diversity and species-specific plant traits, can in turn cause altera-
tions in aboveground litter quantity and quality, and belowground root dynamics.
However, alterations in vegetation tend to be idiosyncratic effects of particular plant
species or particular plant functional traits, and difficult to draw more generalized
patterns from (Porazinska et al. 2003). The effects of plant litter quality and quantity,
in particular, are limiting factors to microorganisms, and thus species-specific differ-
ences in plant litter can especially affect microbial community structure and function
(Wardle 2002).
Additionally, effects may persist for many years and decades after a given land-
use has stopped (Steenwerth et al. 2003). For example, Fraterrigo et al. (2006) found
long-term alterations to microbial community structure in forest stands that had been
cultivated but not logged. Fungal markers, especially, were lower in previously cul-
tivated sites, suggesting that fungi may need more time to recover from agriculture
(Fraterrigo et al. 2006). Likewise, Spiegelberger et al. (2006) found, 70 years after
agricultural abandonment, changes to the microbial community due to lasting
changes in pH due to former agricultural liming. Evidence is accumulating that the
history of land-use can leave contingency effects in the soil microbial community,
ultimately influencing successional dynamics of future plant communities (Kardol
et al. 2007) and hence providing a mechanism by which changes due to agricultural
management practices may persist far into the future.
Linking the Structure and Function of Microbial Communities

in Changing Landscapes
As conversion of native ecosystems to arable land is likely to continue into the

future, future agricultural management practices are likely to alter the essential
functions microbial communities provide to plant productivity, including providing
structural stability to soils, nutrient (especially limiting nutrients such as nitrogen
and phosphorus) mineralization and transformation, and disease suppression (Barea
et al. 2002; Peterson et al. 2002; Kennedy and Papendick 1995). In addition to
effects on plant productivity, land-use shifts are likely to affect numerous other
processes that the microbial community carries out in agroecosystems, including
decomposition, carbon cycling, soil aggregation and stability, plant productivity, and
greenhouse gas emissions. For example, Bossuyt et al. (2002) found that elimination
of soil fungal populations decreased macroaggregate formation in the soil. The

impact of climatic change on soil ecosystem services and products is likely to be
profound and demands immediate and detailed studies not least because of its
relevance to food security.
Studies that link community structure and functional measurements of land-use
change effects are relatively rare but increasing. For example, the study of Waldrop
et al. (2000) on conversion of tropical forest to plantation linked measurements to
community structure to enzyme activities, and found that land conversion to planta-
tions increased the activities of key enzymes involved in the carbon cycle, such as
phenol oxidase and peroxidase (lignin degradation) and cellobiohydrolase (cellulose
degradation). Thus changes to the overall physiology of the soil microbial commu-
nity provide a mechanism whereby land-use can affect ecosystem functions.
It is well established that land-use changes alter mineralization rates of key lim-
iting nutrients and thus affect nutrient availability to plants. Recent studies have
found that alterations to microbial community structure can often explain differ-
ences in mineralization rates in different altered ecosystems (Fraterrigo et al. 2006;
Hawkes et al. 2005). Shifts in nitrogen processes important to agricultural produc-
tivity, such as nitrogen mineralization rates (Balser et al. 2005; Fraterrigo et al.
2005) or phosphorus cycling rates (Cleveland et al. 2003), can thus be altered with
shifts in microbial community composition.
Land-use caused shifts in microbial mediated processes ultimately feed back to
plants themselves, impacting agricultural productivity, although the mechanisms
linking aboveground and belowground systems may be complex and context-
dependent (Wardle et al. 2004). Additionally, recent research has suggested that
particular land uses (including cultivation) may cause changes in microbial com-
munity structure and function that may persist many years after cultivation has
ceased (Fraterrigo et al. 2006; Dupouey et al. 2002). These biotic legacies in the
soil may influence future plant communities and environmental conditions at the
site, and so microbial functions may reflect past land uses as much as contemporary
ones. Through biotic legacies left by previous vegetation that has altered the soil
microbial community, changes to soil microbial physiology and function induced
by land-use changes can ultimately feed back to subsequent vegetation that is
planted, succeeds, or is restored. More work is needed to elucidate how interacting
biotic and abiotic factors can influence microbial community structure and func-
tion, over short and long time periods of land-use changes.
Caveats and Conclusions
Climate Modulators and Native Regimes
Temperature and water are both environmental factors that are important for micro-
bial growth. They are considered to be environmental “modulators” (that influence
organism activity) as opposed to “resources” that are used by organisms to grow
and reproduce (Chapin 2003). Modulator changes affect the whole community, but
often ultimately cause change by their impacts on resources. For example, a reduc-
tion in water potential (modulator) may act to limit substrate availability (resource)
via diffusion, improving the success of hyphal strategists. Energy must be used to
adjust processes when a modulator is not optimal (Balser et al. 2002). Microbial
responses to temperature and moisture stress can occur via several mechanisms,
including physiological changes such as lipid membrane changes, substrate prefer-
ence changes, or the formation of dormant structures. Regarding such stresses,
Schimel et al. (2007) conclude that even when microbial community response to
stress is limited, the physiological costs imposed on soil microbes are large enough
that they may cause large shifts in the allocation and fate of carbon and nitrogen.
Response to temperature and moisture change is dependent on native regimes.
This importance of native regimes makes theoretical sense. From a microbial physi-
ology perspective, a stressed microbial community is more likely to show changes
than one operating within accustomed conditions. Prevailing conditions at a given
location select for communities with particular adaptations to temperature (Dalias
et al. 2001), and to its variability. If an environment rarely changes, there is a dis-
advantage to maintaining sufficient genetic material to have a broad functional
range (Balser et al. 2002).
A community may be more sensitive to temperature and moisture changes if the
new condition is outside of its normal climate range. This has been demonstrated
experimentally (Balser and Firestone 2005; Wolf et al. 2007). For example, Waldrop
and Firestone (2006) demonstrated that a forest microbial community perturbed
beyond its environmental range due to transplant was more sensitive to climate
changes than a transplanted grassland microbial community with a broader native
range. In this case, both community structure and biomass changed. This is consis-
tent with a study by Balser and Firestone (2005) showing that response to soil trans-
plant depended on the deviation from the community’s ambient regime.
Temperature optima do not necessarily relate to average local temperature
(Lipson 2007), but the community may instead be driven by substrate availability
resulting from plant phenologies (Yuste et al. 2004). Optima changes with high
temperature incubations may also be more related to substrate limitation, and may
not increase as might be expected (Dalias et al. 2001).
Importance of Study Context and Length
Even among studies that examine multiple interacting global change factors, few have
studied microbial responses for more than one growing season. Variation caused by
seasonal oscillations in weather over time significantly affects microbial community
structure and function (Wolf et al. 2007; Ebersberger et al. 2003; Jin and Evans 2007),
as well as changing responses to global change manipulations (Ebersberger et al.
2003). Year to year climate variation may also change the intensity of treatment effects
(Saiya-Cork et al. 2002). Neglect of seasonal trends can lead to misinterpretation
of results (Boerner et al. 2005), and in much the same way neglect of inter-annual
variation could lead to misinterpretation or over-generalization of results that may
have been particular to one growing season. Multiple year studies are more able to
take into consideration seasonal changes in weather, perhaps giving a broader or more
realistic perspective of how multiple, interacting factors alter soil communities.
A critical reason to study microbial communities across long-term global change
manipulation is to understand how chronic, multiple long-term stresses, such as the
stress of elevated temperature or shifting nutrient dynamics, will alter the micro-
bial community. It’s possible, for instance, that cumulative stress could result in
large community shifts depending on external ‘tipping points’, such as the
extreme wet or dry years that may occur more often in a globally changing climate
(Intergovernmental Panel on Climate Change (IPCC) 2007). Multiple stresses
simulated in global change experiments may interact to impact above and below-
ground communities in ways distinct from single factor responses (Henry et al.
2005a, b; Flannigan et al. 2006; Kandeler et al. 1998). For example, nitrogen depo-
sition from urban and agricultural areas may result in a shift from mycorrhizal to
bacterially dominated communities (Aber et al. 1998) that may be altered further
by elevated carbon dioxide or climate factors (Treseder and Allen 2000). Despite
the abundance of work on single factors there is still relatively little known about
how microbial communities will respond to multiple simultaneous stresses repre-
sented by global change treatments (Pendall et al. 2004; Kandeler et al. 1998).
This added interpretive strength and understanding of temporal aspects of soil
community response to global change is also important from a standpoint of its con-
sequences. What does it mean that the community acts in one way or another under
global change treatments? How will ecosystem function change as a result of changes
in small scale, microbial processes? Should soil management decisions change in wet
versus dry years? These questions can only be answered from understanding the
microbial community on a longer time-scale than has traditionally been studied. For
instance, several studies have shown that it takes from 2 to several years for microbial
activity and nutrient cycling to recover after burning (Smithwick et al. 2005; Zhang
et al. 2005; Yong-Mei et al. 2005; Boerner et al. 2006; Turner et al. 2007). Over this
time frame, or until final recovery, any one time-point may give a very different pic-
ture of response to disturbance that may not accurately reflect long-term patterns of
recovery and thus, poor interpretations for proper landscape management.
Conclusions
Climate change will impact microbial community structure and activities both directly,
through alteration of the soil chemical and physical environment, and indirectly
through changes in land use and cover. We have most often studied climate change
impacts by isolating the various change drivers and manipulating them independently.
However, in reality changes in the soil habitat will occur in concert and may produce
unexpected results as factors like temperature interact with changes in water or
nitrogen availability and plant species cover. We need a better understanding of the
nature of climate change interactions from studies that include multiple factors. It is
especially critical to note that the specific response of a community to a given environ-
mental change will not be dependent only on the severity of the disturbance, but also
on the history of the affected soil and soil community. Evidence is accumulating that
rather than being infinitely plastic in their response to and recovery from environmen-
tal perturbations, microbial communities are uniquely adapted to their native climatic
regime and vegetation cover. They will thus respond uniquely to climate and land use
change across ecosystem types or climate regimes. Further, it is increasingly being
shown that land use practices can create legacy effects that persist for decades.
Soil microorganisms are essential components in the response of agricultural
ecosystems to climate change through their capacity to cycle nutrients and process
soil carbon. In order to fully understand and manage the impacts of climate change
on soil communities we must be sure to include assessments of their composition
and biomass in our studies, and design studies over the longer-term. Short-term
studies are inadequate to capture the impact of climate change on soil microbial
dynamics, and are inadequate. Finally, we must consider how our land use choices
impact microbial communities into the future.
Acknowledgments The authors would like to acknowledge the members of the Balser laboratory
at the University of Wisconsin-Madison, especially Dr. Harry Read and Kevin Budsberg, for their
untiring support and assistance. We would also like to thank Dr. Chris Field, Dr. Nona Chiariello,
and supporting staff at the Jasper Ridge Global Change Experiment where much of our own work
has taken place. Finally we thank the book editors for their valuable comments in strengthening
this chapter.
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Chapter 11
Evaluating the Economic and Social Impact
of Soil Microbes
Harpinder S. Sandhu, Vadakattu V.S.R. Gupta, and Stephen D. Wratten
Introduction
Developments in agriculture have been very successful in producing sufficient

amounts of food to meet the growing demand by human population in the twentieth
century. This was possible due to breakthroughs in the development of high yield-
ing cultivars and the combined efforts of agricultural research and public policy to
support high input-based farming in different parts of the world. The ‘Green
Revolution’ yielded desired outcomes due to agricultural intensification, accom-
plished through the use of input-responsive plant cultivars, increased chemical use
in the form of fertilisers and chemical based plant protection measures. These mod-
ern technologies are practised in agro-ecosystems which are the largest managed
ecosystems on earth and comprise 1.5 billion hectares of cropping area and about
3.5 billion hectares of pasture land area (FAO 2009).
Modern cropping and livestock farming practices have made agriculture a major
driver of land use change (Vitousek et al. 1997; Goldewijk and Ramankutty 2004;
UNEP 2005) which resulted in both gains and losses in several ecosystem services
(ES) (Heywood 1995; Costanza et al. 1997; Daily 1997; Tilman et al. 2001).
Ecosystem services are the benefits people obtain either directly or indirectly from
functioning ecological systems (Daily 1997; MA 2003; Reid et al. 2005). They
include products such as food, fuel, and fibre; regulating services such as climate
and water regulation and flood control; and nonmaterial assets such as recreational
or aesthetic benefits (de Groot et al. 2002). These ES support life on earth through
H.S. Sandhu (*)

CSIRO Sustainable Ecosystems, PMB No. 2, Glen Osmond, South Australia 5064, Australia
V.V.S.R. Gupta
CSIRO Entomology, PMB No. 2, Glen Osmond, South Australia 5064, Australia
S.D. Wratten
Bio-Protection Research Centre, Lincoln University, P.O. Box 84, Lincoln 7647, New Zealand
400 H.S. Sandhu et al.
a wide range of processes and functions (Myers 1996; Daily 1997; Daily et al.
1997) which have been demonstrated to be of very high economic value in natural
(Costanza et al. 1997) and agro-ecosystems (Porter et al. 2009). Current trends in
degradation of agro-ecosystems threaten to alter radically not only the capabilities
to produce food and fibre but also the delivery of ES by agro-ecosystems (Pretty
2002). The key challenge, therefore, is to meet the food demands of a growing
population by enhancing productivity and maintaining ES (UN 1992).
Agricultural intensification has also resulted in monocultures. The remarkable
scientific and technological achievements of the Green Revolution resulted in
reduced plant biodiversity in some regions and promoted planned diversity of crop
systems (Matson et al. 1997). Reduction in crop diversity (above ground plant
diversity) can result in a variety of effects on belowground (soil biota) diversity and
abundance of soil biota (Hooper et al. 2000), which in turn can affect soil function
or ES. Higher diversity of plant species can result in higher belowground biodiver-
sity through a broader range of crop residue types (resource heterogeneity) and
diverse root exudates. Thus, to maintain agricultural productivity and high resource
use efficiency, it is essential to maintain and even enhance the capacity of soils to
provide adequate ES via functions performed by soil biota. These ES provided by
soil, support crop production by providing growth media for seeds, aeration, plant
support, nutrients (timely supply of nutrients and biological N inputs through fixa-
tion), water and accumulation of carbon (Brady 1990; Daily et al. 1997; de Groot
et al. 2002). These bio-physical processes mediate necessary soil functions to sup-
port crop production. Biological components of soils include macro-, meso- and
micro-fauna as well as micro-flora (Nannipieri et al. 2003). They carry out ecosys-
tem functions in the form of supplying nutrients, biological control, organic matter
turn over, maintaining soil structure and support the provision of ES by soil
(Brussaard et al. 1997; Gupta et al. 2010). These ES are the result of complex
interactions between biotic (living) and abiotic (chemical and physical) components
of soil ecosystems through the universal driving forces of matter and energy
(de Groot et al. 2002). To capture the benefits of soil biological activity, a better
understanding of the linkages between soil biota and ecosystem function is
required. Detailed discussions on the roles of different soil biota groups are in pre-
vious chapters of this Book.
An increase by 50% of the world human population, expected by 2050, will
double the food demand thereby placing enormous pressures on agro-ecosystems
and their components especially on soils. This could have severe social and eco-
nomic impacts. Agricultural sustainability and productivity in coming decades
requires not only water and crop management for more efficient use of resources
but also the optimal use and management of soil fertility and physical properties to
improve soil health, which relies on soil biota to maintain biological processes and
biodiversity.
In the following sections, we discuss the social and economic impacts of soil
microbes in agriculture using specific case studies and examples from Australia and
other parts of the world.
11 Evaluating the Economic and Social Impact of Soil Microbes 401
Social and Economic Impacts of Soil Microbes
Soil microbes deliver substantial social and economic incentives annually to the
global economy and to society in general by supporting various ecosystem func-
tions and processes (Fig. 1). Soils are a reservoir of a large proportion of Earth’s
biodiversity and are considered as one of the last frontiers of unknown microbial
diversity (Wall and Virginia 2000). Biological processes in the soil are crucial in the
effective functioning and overall health of both the terrestrial (soil) and aquatic
ecosystems. There is a two-way relationship between the soil microbes and agricul-
tural activities as human activities are increasingly intervening, both positively and
negatively, in the microbial diversity and the biological processes they mediate both
through direct effects and indirectly by affecting the soil habitat.
Economic Impacts
Global economic growth is projected to remain in the 1.7% range while growth in
the developing-countries is expected to average 2.1% in 2009 (World Bank 2009).
This growth can change the demand side of the world food equation. High income
growth in developing and least developed countries may readily lead to increased
food consumption. The estimated world population by the end of the twenty-first
Global scale
Ecosystem Services Human Well-being

Supporting ES in soil Provisioning Social
Soil erosion Food Basic utilities
control Raw materials Health
Carbon Social relations
sequestration in
soils Security
Local scale Freedom of choice
Nutrient cycling and action
Regulating
Soil structure
modification Hydrological
flow
Nitrogen fixation Economic
Organic matter Globalization,
decomposition
Trade
Pest and disease Market
control Cultural
Aesthetics Policy framework
Recreation
Fig. 1 Linkages between ecosystem functions provided by soil microbes and human well-being
at local and global scale (Modified from Millennium Assessment, Reid et al. 2005)
century is approx. ten billion and to feed this population will require considerable
efforts and measures to improve and maintain soil health (Lal 2009) apart from
other measures such as improved resource use efficiency and improved formulation
of public policies.
Soils sustain global ecosystems as plants and animals both need soil or its
products for their survival. Terrestrial ecosystems provide both essential ES as well
as food supply for humans (FAO 2007). The living populations in soil (soil
microbes) are responsible for carrying out 90% of the soil processes (Coleman et
al. 2004). Thus soil biota provides major inputs to the global agricultural economy
through the functions they perform that lead to the provision of ES. These ES
provided by soil organisms have been demonstrated to be of very high economic
value (US$1.5 trillion/year globally) (Pimentel et al. 1997). An example is
biological nitrogen fixation (BNF) in agriculture by legume-rhizobia symbiosis.
The global estimates of biological nitrogen fixation into agriculture range between
33 and 46 million tonnes of nitrogen each year (Herridge et al. 2008), which
approximately equates (at current fertilizer prices) to $50–70 billion annually. But
the importance of legume and fertilizer nitrogen sources varies with the region and
is influenced by a range of socio-economic factors including patterns of land use,
farming traditions and population density. The use of nitrogenous fertilizers has
greatly increased global food production. The benefits of using these fertilisers are
numerous, but nitrogen fixing legumes are driven by solar energy and differentiated
from the fertiliser nitrogen which requires non-renewable fossil fuels (Peoples et al.
2008). Nitrogen fertilizers also have high external costs (Pretty 2005) largely
associated with losses of some of the nitrogen applied, high energy costs,
contributions to net carbon dioxide emissions. Recent increases with the costs of
nitrogenous fertilizers also highlight the need for this element to be supplied to
crops in the most efficient ways possible. Inefficient use of fertilizer inputs and
concomitantly increasing costs can threaten the economic sustainability of food
production practices that solely depend upon external inputs. However, legumes
offer to restore the functional biodiversity in soils and to yield a more sustainable
agriculture (Altieri 1999).
An economic assessment of the global microbial inoculant industry (Phillips
2004) embraces three types of technology: nitrogen fixing rhizobial bacteria,
phosphate-solubilising Penicillium fungi and insecticidal inoculants. These tech-
nologies can provide significant economic and social gains, e.g. legumes contribute
20% of the global food proteins (Montanez 2000). Legumes appear to fix on an
average 277 kg N ha−1 year−1 (FAO 1984). At this rate, total inoculants’ area of 43
million hectares will fix about 12 million tons of nitrogen, equal to about 13% of
current nitrogen use globally each year (Phillips 2004) which is worth US$7.8 bil-
lion annually at the price of nitrogen (US$0.65 kg−1 N; 2004). Recent increases in
the cost of nitrogen fertilizer (US$ > 1.00 kg−1 N) would only increase the impor-
tance of nitrogen inputs through biological fixation. In addition, there is a growing
push to utilize the antibiotic potential of soil microorganisms (bacteria, actinomy-
cetes and fungi) as biocontrol agents to reduce the impact of plant pathogens on
agricultural production (Pal and McSpadden-Gardener 2006).
Nutrient cycling (Brady and Weil 2004) is one of the most important functions
provided by soil microbes in soil. Sandhu and colleagues (Sandhu et al. 2008)
investigated the economic value of nutrient cycling carried out by soil microbes and
the role of land management practices in the maintenance and enhancement of this
ES (nutrient cycling) in agricultural land. They quantified the economic value of
this ES at the field level based on an experimental approach. The study sites
included 29 arable fields, distributed over the Canterbury Plains in New Zealand
and comprised 14 organic and 15 conventional fields. The economic value of this
ES carried out by soil microbes was in the range of US$25.60 to 425.50 ha−1year−1
(mean US$160.65 ha−1year−1) in organic fields and US$30.00–348.00 ha−1year−1
(mean US$142. ha−1year−1) in conventional ones (Sandhu et al. 2008).
Microorganisms play important roles in the degradation of the diverse range of
agrochemicals and the bioremediation of both soil and water environments. There
is an extensive effort to utilise biological processes for the removal of contami-
nants and take advantage of the amazing catabolic diversity and adaptability of
microorganisms to degrade toxic contaminants into non-toxic forms (Singh and
Ward 2004; Diaz 2008).
Social Impacts
For centuries many communities have survived by harvesting resources from

nature. In spite of the unprecedented increase in agricultural production during the
‘Green Revolution’, the majority of small farmers in developing countries are too
poor to purchase seeds and inputs and increase their farm productivity. Inadequate
access to appropriate technical support with which to harness benefits from the new
technology is a key bottleneck inhibiting improved productivity. Food insecurity
has been identified as a key issue by United Nations in its Millennium Development
Goals (MDGs) to be achieved by 2015 (UN 2005) which contains social, economic
and environmental objectives. The vast majority of the world’s poor farmers reside
in developing countries in South Asia and the African continent. The success of the
Green Revolution by managing soil constraints has not been realised by these
small-scale farmers. The United Nations led Millennium Assessment (Reid et al.
2005) concluded that natural and modified ecosystems and the services they pro-
vide are declining. It also pointed out the consequences for global stability if that
rate of decline continues. The livelihood of large populations in developing and
least developed countries depend on healthy natural and agro-ecosystems. There is
greater need to address land degradation and soil health so that agriculture can
sustain growing food demand and increasing human population.
Human livelihoods and social and economic well-being are inextricably linked
to our dependence on soil health (see Box 1). Therefore, environmental and social
sustainability can be achieved by maintaining soil. Soil health and fertility is critical
in improving impoverished agricultural communities, which constitute approxi-
mately 2.4 billion people worldwide (Lal 2009).
Box 1 Soil Health and Social and Economic Well-being

Haiti is one of the least-developed countries. It ranks very low in the United
Nations Human Development Index 2006 (UNDP 2006). Agriculture pro-
vides work to about two-thirds of its population but this makes up only one-
third of the GDP of low income countries. Per capita food production has
dropped 30% from 1991 to 2002. There are several constraints. Farmers are
unable to afford prime agricultural land, irrigation or inputs and increase farm
productivity.
Sustainable Organic Integrated Livelihoods (SOIL) is a non-profit organi-
zation which is dedicated to work with communities to increase agricultural
production and to provide livelihoods on farm. For this, SOIL dedicated its
efforts to protect soil resources and transform wastes into resources.
Composting toilets are built in rural communities to get much needed organic
matter and fertility back into fields. Composting of such organic materials is
performed by soil macro- and micro-fauna. Soil microbes like fungi and bac-
teria decompose the composted material resulting in the release nutrients for
plant use. Incorporating organic matter can also lead to improvements in the
soil structure and soil aggregation. Managing soil health by using this tech-
nology has potential to improve farm production and household income.
SOIL thus promotes integrated approaches that utilize biological activities
to tackle the socio-economic issues and help achieve sustainability. SOIL is
promoting close collaboration between local communities and international
academics and activists and channels resources to build the soil health,
strengthen and empower grassroots communities (http://www.oursoil.org).
Soils had profound social impacts in ancient civilizations (Marris 2006). In the
Amazon, terra preta is a soil rich in vital minerals such as: calcium, manganese,
phosphorus and zinc, which are scarce in most tropical soils. Terra preta is blacker
than the blackest coffee and is very deep soil extending from the surface down as
much as 2 m. In the Amazon region it is found where humans inhabited the area
and it is known to be artificially made by mankind between 450 BC and AD 950 to
support agriculture production. Unlike ordinary tropical soils, terra preta remains
fertile after centuries of exposure to tropical sun and rain. It has been suggested that
the charcoal component of the terra preta provides an important habitat for the
microbial populations hence improving its soil biological fertility.
The use of a variety of pesticides (e.g. insecticides, herbicides, fungicides) has
become an integral part of modern farming systems with the main aim of removing
constraints caused by insects and plant pathogens and competition from weeds to
achieve higher productivity and resource efficiency. Microorganisms play an
important role in the degradation of all types of pesticides in soil and water.
Chemical pollution of land and water resources has the potential to threaten
environmental sustainability and can result in harmful effects on human health.
In addition, there are a number of examples of the contamination of natural

environments by the pollutants transported from nearby agricultural fields (Young
et al. 1989; Manz et al. 2001). Although the benefits from judicious use of pesti-
cides have been demonstrated to provide economic benefits, long-term exposure of
farmers to pesticides may cause increased health costs (Pingali and Roger 1995)
and associated social impacts to their quality of life. Pingali and Roger (1995)
identified the potential for social gains from reducing pesticide use in Philippine
rice production. They suggested that productivity losses from reduced pest control
can be offset by the gains from improved farmer health.
Moderate and extreme poverty occur in many least developed and developing
countries, in which more than 25% of the population lives on less than US$2 per day.
The most important reason for declining economic growth of these countries is their
poor food productivity. Most of the poverty occurs in rural areas where populations
are increasing while food production is stagnant or declining in many cases. Resource
poor farmers cannot afford expensive inputs. Therefore, crop yields are adversely
affected by extractive farming. Technologies such as biological nitrogen fixation
(BNF), no-till husbandry, mulching with residues and cover cropping and integrated
nutrient management that enhance the functions performed by soil microorganisms
can play a crucial roles in reducing environmental pollution, raise yields and alleviate
poverty. Biofertilisers consisting of beneficial microorganisms can increase the
growth and yield of plants (Mulongoy et al. 1992). Use of biofertilisers helps in
reducing off-site environmental pollution and avoiding non-target interactions.
Soil erosion by wind is a natural process but its occurrence and severity has
increased along with the expansion of agriculture. The major affects of soil loss can
occur through dust storms affecting human health, reducing visibility, diminishing
air quality and disrupting electricity supplies (Leys 2003). Williams and Young
(1999) estimated the off-site impacts of dust storms in South Australia to be in
the range of $2–15 million annually, largely due to impacts on human health
(asthma and general respiratory problems). There has been significant decrease in
the annual frequency of dust storms in Australia since the 1970s (Hamblin 2001)
largely due to the improved control of rabbits, the spread of woody weeds such as
the invasive Acacia nilotica and the adoption of conservation tillage. Some farming
practices impair soil structure through tillage and that increases the likelihood of
dust storms. Soil microbes play crucial roles in maintaining soil structure through
their involvement in aggregate formation and subsequent stabilization and in turn
helping in decreasing soil erosion that contributes towards reduction in dust storms.
Ecosystem Functions
Soil organisms contribute to a wide range of services essential for the sustainable
functioning of all ecosystems. These functions include carbon and nutrient
(nitrogen, phosphorus and sulphur) cycling, turnover of soil organic matter, soil
carbon sequestration and reducing greenhouse gas emissions; modifying soil
physical structure and water regimes, enhancing the amount and efficiency of
nutrient acquisition by the vegetation, and enhancing plant health (Wall and
Virginia 2000; Brussaard et al. 2007). In addition, the diversity and complexity of
soil microbes is also attributed to the suppression of plant diseases (Hornby 1983;
Gupta and Neate 1999). Soils not only harbour the majority of plant pathogens,
they are the sources of several human pathogens (Ajello 1956). Soil organisms are
grouped into different functional and trophic groups in order to illustrate linkages
between various groups of biota and specific ecosystem processes (Lavelle and
Spain 2001; Coleman et al. 2004). The soil biota mediated services are not only
essential to the functioning of natural ecosystems but constitute an important
resource for the sustainable management of agricultural systems (Altieri 1999;
Roper and Gupta 2007).
The importance of ecosystem functions carried out by soil microbes in low-input
and organic systems is generally accepted however, their importance in high input
agro-ecosystems may be neglected because external inputs of fertilizers and pesti-
cides are expected to replace the products of biological processes (Barrios 2007).
Optimum functioning of biological processes is necessary, however, for the efficient
use of human inputs and reducing or eliminating any off-site non-target effects.
Recent studies have proposed an ES framework to examine the linkages between
biodiversity, ecosystem functions and services (Reid et al. 2005). According to the
Millennium Ecosystem Assessment (2003), ES can be classified into four categories
depending on their functions – provisioning, supporting, regulating and cultural ser-
vices. Soils contribute to all four categories of ES (Table 1). The ecosystem functions
carried by soil microorganisms contribute to the provision of ES that leads to improved
economic and ecological sustainability of agro-ecosystems. Consequently, it has poten-
tial to improve the existing socioeconomic conditions of the farming community
Table 1 Relationship between soil functions and socio-economic impacts

Ecosystem functions Social impacts Economic impacts
Regulating Functions
1 Maintenance of soil structure High High
2 Regulation of soil hydrological processes Low Medium
3 Gas exchanges and carbon sequestration High High
4 Soil detoxification Medium Low
Supporting functions
5 Nutrient cycling Medium High
6 Decomposition of organic matter Medium High
7 Suppression of pests, parasites and diseases Medium High
8 Symbiotic and asymbiotic relationships with plants Low High
and their roots
9 Plant growth control (positive and negative) Low Medium
Provisioning functions
10 Sources of food and medicines Low Medium
Cultural functions
11 Aesthetics/spiritual Low Low
(Altieri 1995). Linkages between ecosystem functions performed by soil microbes and
their social and economic impacts are summarised in Table 1. Two regulating functions
have high social and economic impacts through maintenance of soil structure and by
regulating gas exchanges and carbon sequestration. Whereas, three supporting functions
(nutrient cycling, decomposition of organic matter and suppression of pests, parasites
and pathogens) have medium social impacts but high economic impacts as food produc-
tion depends on these functions that are performed by soil microorganisms. Symbiotic
relationships have low social but high economic impacts. Soil microbes have low social
and economic impacts for provisioning and cultural functions.
Supporting Functions
The cycling of nutrients is a critical ecosystem function carried out by soil

microbes. Soil microbes facilitate biological nitrogen fixation (BNF) (e.g.
Rhizobium spp.) and phosphorus uptake through arbuscular mycorrhizal fungi
(AMF) (Smith and Read 1997). Nitrogen fixation through the legume-Rhizobium
symbiosis dominates the global inputs of biologically fixed nitrogen. It is estimated
that ~50 million tonnes of nitrogen is harvested globally each year in food crops
and microbial functions play an important role in making available of both biologi-
cally fixed and fertilizer nitrogen inputs. Soil microorganisms also perform the
decomposition of organic matter and its transformation and play essential roles in
biogeochemical cycles of essential plant nutrients, e.g. nitrogen, phosphorus and
sulphur (Coleman et al. 2004). Microorganisms play an important role in the deg-
radation of all types of pesticides, i.e. insecticides, herbicides and fungicides, in soil
and water. Soil microbial functions support the production of provisioning goods
and services in agriculture (Porter et al. 2009) by providing essential activities in
soil and resulting in food and fibre production along with other essential ES.
Regulating Functions
Soil biota regulate essential ecological processes through bio-geochemical cycles

and other biospheric processes. Soil organisms help in aggregate formation and
the maintenance of soil structure. Micro-aggregates, which are stabilized by per-
sistent organic binding agents of microbial origin, are bound together by tempo-
rary (roots and fungal hyphae) and transient (plant and microbial polysaccharides)
binding agents forming macro-aggregates (Tisdall and Oades 1982).
Soil organisms have profound influences on global crop losses due to soil-borne
pests and pathogens, as described elsewhere in this Book. For example, soil-borne
diseases are one of the major bottlenecks to achieving maximum yield potential in
rainfed and irrigated crops all over the World. Brennan and Murray (Murray and
Brennan 2009) estimated that yield losses resulting from the four major soil-borne
diseases of wheat crop in Australia would cost more than US$325 million annually.
In undisturbed natural ecosystems with high above-ground diversity, soil-borne
plant disease epidemics are rare when compared with disturbed agricultural sys-
tems (Cook and Baker 1983; Schisler and Linderman 1984). Poor nutrition can
accentuate the effects resulting from pathogen and pest incidences resulting in pro-
ductivity and economic losses. In general, there is a strong relationship between
soil biota, fertility and plant health (Altieri and Nichols 2003), and the diversity of
microbial communities is one of the critical factors contributing to the proper func-
tioning of soil suppressiveness (Gupta and Neate 1999; Garbeva et al. 2004).
Case Studies
Assessing the impacts of soil microbes is difficult as there are so many ways by
which their actions can have direct or indirect influence on societies or economies.
These effects are the product of complex interactions between microbial processes,
physical and chemical components of soils and are influenced by environmental
and management practices. This study includes two case studies which elaborate
the role of soil in general and soil microbes in particular and their socio-economic
impacts through their functions.
The first case study that we discuss below, deals with the adoption of new tech-
nology that enhances soil microbial population and/or the processes which they
mediate yielding multiple benefits and also having social and economic effects.
A recent study by Llewellyn and colleagues (Llewellyn et al. 2009) examined
trends in the adoption of no-till husbandry across southern cropping regions in
Australia using socio-economic data. It identified opportunities for the research, its
adoption through extension and changing policies to achieve improved farming
systems with advantages for both private and public good.
The second case study elaborates the role of soil microbes in improving soil
health in sub-Saharan Africa, through the discussion of various individual projects.
Adoption of No-Till and Conservation Farming

Practices in Australia
Conservation agriculture and no-till husbandry practices enhance soil biological

activity and modify the diversity and population structure of soil microflora and
fauna by reducing soil disturbance and improving the levels of biologically
available carbon (energy source) (Roper and Gupta 1995; Kladivko 2001). Long-
term adoption of these practices could lead to the removal of biological constraints
to plant growth and improve the overall soil quality. Thus soil biota plays a crucial
role in the successful adoption of no-till husbandry and conservation farming
(Stubbs et al. 2004). No-till farming also reduces fuel inputs in particular during
non-cropping season. According to one estimate (Derpsch 2005), no-tillage and

conservation farming practices cover 95 million hectare worldwide. In Australia,
it covers about 9 million hectares spread over the main cropping districts. Data in
this study (Llewellyn et al. 2009) were collected from 1,172 primary cropping
decision-makers in each household and only farms cropping greater than 200 ha
in a ‘normal’ season were included. The results show that no-tillage seeding
practices have now been adopted by the majority of grain growers across all
regions. The proportion of growers using at least some no-tillage is now peaking
at levels of around 90% in many regions. In regions with relatively low adoption
5 years ago, there have been very rapid increases in adoption, particularly in the
period 2003–2006.
In general, relatively few growers appear to have made the decision not to adopt
no-tillage in the near future and continue cropping without adopting it at all. The
proportion of growers using no-tillage is expected to exceed 80% in a majority of
regions by 2013. Substantial net gains in no-tillage adoption have been observed
and further net increases are expected in the future, except where no-tillage adop-
tion is already reaching a plateau as it approaches full adoption.
For no-tillage practices to be widespread in the landscape there needs to be both
high adoption rates and extensive use by the farmers. The results show that it
remains common for no-tillage adopters to still use some forms of cultivation.
However, the extent of no-tillage use by adopters is typically high. The average
percentage of crop sown using no-tillage by adopters exceeds 70% in all regions.
Because a majority of growers in most districts still use some tillage, it should
be expected that economic and agri-environmental factors may cause seasonal
shifts in extent of tillage use. Just as falls in the price of glyphosate herbicide led to
increased no-tillage adoption (D’Emden and Llewellyn 2006), growers in this study
indicated that recent rises in the price of this chemical have led to their increased
use of tillage in many regions. In regions such as those in Western Australia where
it is more common that 100% of crop area is sown without tillage, the rising price
of glyphosate has had little or no reported influence on tillage use. No-tillage adopt-
ers are more than twice as likely to use a paid cropping consultant. This suggests
that availability of high quality cropping advisory support may be an important
factor in encouraging further adoption and/or maintaining its extensive use. Use of
no-tillage husbandry is also strongly associated with larger farm sizes.
This study shows the remarkable diffusion of no-tillage across Australian crop-
ping regions, confirming that it is highly adoptable and that extensive use has so far
been sustained across a wide range of agro-ecosystems. The last 5 years have seen
many regions with previously lower no-tillage adoption rapidly adapt to levels
similar to early-adopting regions. It needs to be recognised however, that based on
current indicators, over the next 5 years, and possibly at peak adoption, there will
still be several regions with a combination of a relatively lower no-tillage adoption
levels and lower extent of use than the average, which stands at 70%. To achieve
the same reductions in cultivation use and erosion risk in these regions requires new
and innovative approaches which encourage adoption through research, develop-
ment and extension of soil conservation practices.
Long-term adoption of no-tillage practices by farmers in Australia has the poten-

tial for significantly improving soil physical, chemical and biological conditions in
particular when combined with retention of crop residues (Fig. 2a and b). This often
results in increases in microbial population and diversity (Stubbs et al. 2004). This
can lead to large improvements in productivity, profitability and sustainability in
agriculture (Williams and Young 1999) that has potential to enhance the socio-
economic conditions of the rural communities.
Fig. 2 (a) Extensive

cultivation with no stubble
protection at Loxton, South
Australia. (b) Intensive
cropping with zero-tillage and
stubble retention at Waikerie,
South Australia Soil type at
both sites is Arenic Calcisol
and a Mediterranean climate
Soil Improvement in Sub-Saharan Africa
Agricultural land in sub-Saharan Africa is considered to be severely degraded

(FAO 1994; GEF 2003). Increasing agricultural productivity to feed growing
population in these regions is possible only if substantial efforts are made to
restore and maintain the productivity of their soils (Lal 2009). Various technolo-
gies are available to restore and maintain the biophysical structure of soils (con-
servation tillage and no-tillage farming, controlled grazing, mulching with
natural materials, leaving crop residue in situ, application of manure and biosol-
ids, incorporation of cover crops in the rotation cycle), improve the efficiency of
water and nutrient use (integrated nutrient management, biological nitrogen
fixation) and manipulate the rhizosphere to enhance the soil and plant perfor-
mance (NRC 2008). Another useful technology is the incorporation of grain
legumes into rotations to improve nutritional components in the food and also
indirect benefits through nitrogen fixation.
Soil degradation is common in Sub-Saharan Africa due to the nutrient deficien-
cies in soils (Henao and Baanante 2006) which affects 95 million hectares of arable
land. This has serious implications on the social and economic conditions of the
farmers. Large investments and efforts are required for soil restoration to make
them productive again (IFDC 2006).
Social, political, and cultural factors have deep influences on the practices in
Sub-Sahara that affect soil quality. Soil quality can be restored with established
management practices by increasing carbon content, enhancing water infiltration,
reducing erosion, creating a nutrient surplus and encouraging beneficial organisms.
Research has shown that these management practices will increase crop productiv-
ity and improve soil health (Lal 1987, 2006; Kapkiyai et al. 1999; Sanchez 2002;
Wani et al. 2003; Singh et al. 2005).
There have been several barriers to the adoption of new technologies including
weak government institutions and extension, inadequate infrastructure, and resource-
poor agricultural systems.
In spite of these barriers, African Highland Initiative (AHI; http://www.african-
highlands.org) is working to improve livelihoods and reverse natural resource
degradation in the densely settled highlands of eastern and central Africa. The AHI
is an eco-regional programme of the Consultative Group for International
Agricultural Research (CGIAR) and a network of the Association for Strengthening
Agricultural Research in Eastern and Central Africa (ASARECA) hosted by the
World Agroforestry Centre (ICRAF). The AHI’s targeted beneficiaries and partners
in this work include national and international research organizations and networks,
development organizations, local government, civil society organizations, service
providers, policy makers, community-based organizations, and farmers.
In one of its studies, this initiative helped farmers of Tanzania to restore and
improve soil nutrient content by using native shrubs (Wickama and Mowo 2001).
Farmers in the Tanzanian village of Kwalei use very little mineral fertiliser to improve
their crop yields as it is expensive. In this region, farmers used leaves of certain local
Table 2 Inventory of useful shrubs identified by farmers to improve soil fertility in

Tanzania (Wickama and Mowo 2001)
Local name in Kisambaa Botanical name Family
Tughutu Vernonia subligera (O.Hoffn.) Compositae
Mhasha Vernonia amyridiantha (Hook, J.) Compositae
Mshai Albizia schiniperiana Mimosaceae
Mkuyu Ficus vallis-choudae (Del.) Moraceae
Sopolwa Kalanchoe crinata (Andrew) Haw. Crassulaceae
Tundashozi Justicia glabra (Roxb.) Acanthaceae
Boho Bothriocline tementosa (S. Moore) M. Gilbert Compositae
shrubs as green manure, these seemed to improve soil fertility when ploughed into the
fields. Researchers and extension workers did not know their taxonomic description or
nutrient value, and it was agreed that an inventory should be made of this important
local source of nutrients. Farmers identified seven shrubs that seemed to improve soil
nutrient content (Table 2). Their observations were confirmed by soil sampling and
mineralisation studies. Participatory research methods helped to identify the important
shrubs and shared local knowledge has helped to increase soil health.
In another study, Amede and Kirkby (2004) used socio-economic criteria to
develop guidelines for the integration of legumes into multiple cropping systems of
East African Highlands. They conducted participatory research to evaluate the per-
formance of six legume cover crops and two food crops in southern Ethiopian
Highlands, to be used for soil fertility improvement.
These examples show that using local knowledge combined with the scientific
and technical expertise it is possible to manage natural resources and to increase
food production more beneficially in Sub-Saharan African countries.
Summary
Soil microbes have social, environmental and economic impacts through the func-
tions they perform in soils. Improved management of soils and the functions per-
formed by soil microbes are imperative to ensure food security and achieve MDGs.
Modern agricultural technology has been very successful in reducing hunger and
poverty in some developing countries in the last 50 years (Smil 2000; Tilman et al.
2002; McNeeley and Scherr 2003; Federico 2005); however with increasing new
demand to food during the next 20–50 years and lack of additional land with which
to expand agriculture, a new approach is needed in order to achieve the next genera-
tion of the Green Revolution. This involves better utilization of ES (which requires
the services of soil microbes) and extension of technological developments both in
terms of cultivars and farming systems to all parts of the world. The need may be
greater in the regions that are dependant on low fertility soils and have poor eco-
nomic status. In such environments there is an even greater need to utilize micro-
bial services in support of higher productivity systems. The increasing world
population also puts pressure on the health of both soil and water systems in terms
of their abilities to provide the diverse range of ES. In addition there is a greater
urgency to utilize the enormous and wide ranging capabilities of microorganisms to
maintain or ameliorate and improve the health of soil and water systems for both
social and economic needs of the society.
Therefore developing sustainable agriculture by incorporating traditional and
modern methods of farming is the most appropriate choice for developing countries
(UN 2008). Management of soils by maintaining and enhancing soil microbial
functions will help to alleviate some of the barriers as explained in the case studies
discussed above. The challenge in promoting economic and social benefits from
biological processes to farming community is to allow the ecosystem functions in
farming systems to operate optimally with a minimum of external inputs. The cur-
rent and future challenge is to develop cost-effective, low-input eco-technologies,
for their rapid implementation and uptake by end-users (Swaminathan 2001).
Policy makers should aim for sustained economic growth within ecological con-
straints to eliminate hunger and poverty as agreed in the MDGs (UN 2008).
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Index
A Ammonium-nitrogen (NH4-N), 221, 222,

Abiotic-biotic interactions, 276, 400 226, 341
Above-ground diversity (plants), 88 Ammonium oxidising bacteria, 279
Above-ground/below-ground interactions, 93 Ammonium sulphate, 222, 339, 354
Acacia nilotica, 405 Anaerobic metabolism, 286
ACC deaminase, 159 Anastomosis, 124
Acidic soils, 217, 222, 340 Andisols/Andosols, 40, 211
Acidobacteria, 40 Andropogen gayanus, 19
Actinobacteria, 40 Animal husbandry, 284
Actinomycetes, 40, 41, 45, 46, 378 Animal manures, 68, 283–284
Adaptive evolution, 167–171 Animal wastes, 70, 77
Adenosine triphosphate content, 276 Antagonistic bacteria, 316, 326
Adenosine triphosphate determination, 289 Antagonistic microbes, 209, 318
Adhesive hyphae, 325 Antagonists, 354
Adoryphorus coulani, 321 Anthropogenic disturbance, 37
Aeration, 344 Antibiosis, 212, 316, 319, 324, 328
Aflatoxin, 211 Antibiosis/toxins, 324–325, 331–333
Africa, 32, 41–46 Antibiotic activity, 333
African highland initiative (AHI), 411 Antibiotic production, 338
Agricultural, donor, 175 Antibiotics, 212, 222, 284, 331, 340
Agricultural field trials, 283 Antifungal compounds, 332
Agricultural inputs, 281 Antifungal factors, 162
Agricultural intensification, 274, 383, 400 Antimicrobial compounds, 333, 340
Agricultural practices, 352 Ants, control of, 350
Agricultural revolution, 207 Aphanomyces, 225
Agrobacterium, 328 Aphanomyces
Agrobacterium tumefaciens, 331 euteiches, 214, 220, 229
Agrocin 84, 331 flavipes, 342
Agroecosystems, 2, 3, 6, 22, 50, 409 flavus, 211
Alkaline soils, 219, 340 radiobacter, 331, 350
Alkalinity, pH, 217–220 radiobacter strain K1026, 331
Allelochemicals, 326 ramarii, 342
Allium, 208 Aphanomyces root rot of peas, 332
Allo infections, 220 Apical nodule meristem, 140
Alopecurus myosuroides, 215 Apoplastic compartment, 129
Amazon, 32, 404 Apoplastic space, 164
Amber disease, 323 Aporrectodea caliginosa, 98
1-Aminocyclopropane–1-carboxylate, 159 Aporrectodea rosea, 95
Amino sugar analysis, 28 Apressorium, 128
419
420 Index
Arbuscular mycorrhiza, 123, 126–133, 299 thuringiensis (Bt), 305, 324, 326, 345, 346,
Arbuscular mycorrhiza ancestor, 167 355
Arbuscular mycorrhizal colonization, 147, 300 thuringiensis var. kurstaki, 347
Arbuscular mycorrhizal deficient mutants, 145 Bacillus-augmented compost, 317
Arbuscular mycorrhizal development, Bacteria, 27–30, 39–41, 45–47, 50, 279, 345,
137–142 354
Arbuscular mycorrhizal fungal (AMF) Gram-negative, 283, 378
diversity, 280 Gram-positive, 378
Arbuscule development, 147 Bacterial antagonists, 316, 325
Armillaria mellea, 206 Bacterial antibiotic substances, 160
Arthrobotrys, 325 Bacterial community composition/structure,
Arum-type morphology, 126 98, 281, 286, 300
Ascomycota, 125 Bacterial enzymes, 161
Aseptate hyphae, 123 Bacterial inoculum, 345–347
Aspergillus Bacterial siderophores, 160
flavus, 210 Bacterial surface molecules, 136
ustus, 342 Bacterial symbionts, 284
Asset value of land, 206 Bacterial wilt and canker of tomato (Ralstonia
Assimilative condition, 130 (Pseudomonas) solanacearum, 221
Association for Strengthening Agricultural Bacterial wilts, tomato, 224
Research in Eastern and Central Africa Bacteriocins, 331
(ASARECA), 411 Bacteroids, 134, 140
Asymptomatic colonization, 163 Baiting of soil, 352
Atmospheric carbon dioxide, 87, 98 Banana, 223, 335
Attachment to root surfaces, 161 Bark, 318
Australia, 400, 405, 408–410 Barley, 209
Autochthonous biomass, 69 Basidiomycota, 125
Auto infections, 220 Bassianolide, 324
Autoregulation of nodulation, 142 Beauveria bassiana, 324, 343
Autoregulation of symbioses, 156–157 Beauvericin, 324
Autotrophy, 171 Bell pepper, 317
Avirulence (Avr) genes, 337 Beneficial fungi, 320, 344, 354
Avirulent strains, 335 Beneficial microorganisms, 221, 316
Avocado, 316 Beneficial traits, 355
Avoidance response, 343 Benign microbial activity, 223
Azospirillum, 74, 158, 298 Bentonite, 15–17
Azotobacter, 74 Bifidobacterium, 346
Bioactive microbes, 315
Bioactives, augmentation, 319–337
B Bioactive secondary metabolites, 323
Bacillus, 73, 163, 232, 316, 326 Bioavailability, 82, 87
amyloliquefaciens IN937a, 354 Biodegradation, 233
bassiana, 320–322, 324, 338, 343 Biodiversity, 93, 203, 282, 400, 401
brongniartii, 320 Biofuel, 4, 285
cepacia, 326, 332 Biofumigation, 233–234, 303, 317
cinerea, 330 Biogeochemical carbon cycle, 63
lactucae, 206 Biogeochemical nitrogen cycle, 71–77
oleracea, 208 Bioinoculated composts, 318
popilliae, 347 Biological activity, 83
rapa, 208 Bio(logical) control, 160, 212, 222, 223, 230,
sorokiniana, 214 233, 282, 315–356, 400
subtilis, 305, 317 Bio(logical) control activity, 338, 340, 341
subtilis GB03, 354 Bio(logical) control agents, 307, 316, 326,
subtilis RB14-C, 332 340, 352, 354
Index 421
Bio(logical) control agents, characteristics Brussels sprouts, 231

of, 347–351 Bt toxins, 346
Bio(logical) control agents, effective Bulb onions, 208
population, 344 Bulk density, 35
Bio(logical) control agents, establishment Burkholderia cepacia, 326
of, 344 Butenoliches, 333
Bio(logical) control agents, fungal, 338
Bio(logical) control agents, identification
of, 347, 351 C
Bio(logical) control agents, tailored, 343–344 Cabbage, 221, 231
Bio(logical) control constraints, 315, Cabbage maggot, 322
337–347, 353 Ca2+ ion influx, 138
Bio(logical) control of plant diseases, 328 Calcium, 211, 217–221, 223, 224, 227, 234
Bio(logical) control, effect of fertilization, 354 Calcium carbonate, 218, 220, 227
Bio(logical) control efficacy, 335, 340, 352, 355 Calcium channels in plants, 151
Bio(logical) control potential, 336 Calcium cyanamide, 223, 232
Bio(ogical) control strategies, 317, 319, 343 Calcium nitrate, 218
Bio(logical) control successes, 315 Calcium oxide, 227
Biological drilling, 19 Calmodulin-dependent protein kinase,
Biological fitness, 351 151–152
BIOLOG MicroPlate™ 42, 46, 279 Canadian canola, 207, 208
Biomass, 213, 214, 233, 287 Candidatus glomeribacter gigasporarum, 134
bacterial, 375, 378 Capture organs, 326
fungal, 375, 378 Carbendazim resistant Trichoderma
microbial, 375, 377, 378, 384 harzianum, 233
Biomass-C, 274 Carbohydrates, 283
Bionematicide, 327 Carbon, 81, 129, 140, 283, 339–340, 354
Biopesticides market, 355 availability, 89, 94
Bioshield™, 323, 346 cycle, 88, 89
Biotic factors, 342–344 fixation, 63
Biotic legacies, 385 global cycle, 63–71
Biotrophic mycoparasitism, 329 limitation, 376
Bipolaris, 214 pools, 81
Bipolaris sorokinia, 207 sequestration, 87, 88
Blackgrass, 215 sink, 87
Black leg canker, 215, 216 source, 87
Black leg of canola, 330 stocks, 274
Black vine weevil, 318, 321, 354 storage, 93
Black vine weevil larvae, 343 substrates, 89, 279
Blight (Plectrosporium tabacinum), 233 Carbon dioxide, 81, 83, 87, 88
Boron, 222, 223, 226, 227 atmospheric, 373, 375, 379, 380
Botrytis cinerea, 206 elevated, 374–378, 380–382, 387
Brassica green manures, 233 Carbon stored, 20
Brassica juncea, 234 Carbon-to-nitrogen ratio, 72, 74, 76, 90, 93,
Brassicas, 208, 231, 233, 301, 304, 322 281, 340
Brazil, 35, 36, 41, 44 Carnation, 335, 337
Break crops, 209 Carrot (Daucus carota), 206, 220, 221, 354
Breeding protocol, 175 Carrot cavity spot, 220
Bremia lactucae, 206 Carter, 214
Broadacre cropping, 32, 37 Ca2+ spiking, 138
Broccoli, 234 Catalysed reporter deposition fluorescence in
Brome-grass (Bromus spp.), 215 situ hybridization (CARD-FISH), 277
Browning root rot, 224 Cation exchange capacity (CEC), 7–17,
Brown rust (Puccinia recondita), 216 283, 341
422 Index
Cattle ranching, 37 Commercial inocula, 172

Cavity spot, 220 Commercialization of biological control,
Cavity spot in carrots, 228 347, 355
Cell biology, 168 Common root rot, 214
Cell paste, 346 Common scab, 219, 222
Cellulase, 223 Common symbiosis pathway (CSP), 149
production, 340 Common symbiotic genes, 145
Cellulose, 68, 334 Community-level physiological profiling, 28
Central America, 37 Compaction, 5, 18, 212, 214, 229, 235, 286
Cepacin, 332 Competition for resources, 160, 328,
Cercospora arachidicola, 215 333–335, 340
Cercospora carotae, 221 Competitive environment, 350
Cereal root rot, 214 Competitive exclusion of pathogens, 160, 334
Cereals, 209, 213–215, 224, 230, 317 Competitive saprophytic ability, 338
ChaferGuard®, 321, Components of yield, 231
Charcoal, 283 Compost application, 318
Chemical compatibility, 352 Composted grass-clippings, 317
Chemical degradation of soil, 7 Composted green waste, 318
Chemical fungicide, 354 Composting process, 318
Chemotropism, 329 Compost nutrient content, 318
Chinese cabbage, 230 Compost-related suppression, 315
Chitinases, 161 Composts, 283, 316–318
Chlamydospores, 334, 344 Compost stability/maturity, 318
Chloride, 226 Congo Basin, 32, 41
Chloroform fumigation extraction, 38 Conidia, 334, 344
Citrus nematode, 234 germination, 340
Citrus root weevil, 354 production, 340
Civil engineering, 285–290 stability, 344
Clay content, 338 viability, 344
Climate, 30, 31, 47 yield, 344
Climate change, 274, 290, 373–388 Coniothyrium, 330, 350
Climate variation, 386 Coniothyrium minitans, 329, 330
Climatic modulators, 385–386 Conservation agriculture, 13, 21, 408–410
Climax ecosystems, 8, 9 Conserved genes, 167
Climax rain forest, 10 Construction, 286
Clostridium, 74 Consultative Group for International
Clover root weevil, 321 Agricultural Research (CGIAR), 411
Clubroot (Plasmodiophora brassicae), Consumer awareness, 356
206–208, 217, 218, 222, 223, 227, 228, Contaminated soils, 284
230, 231, 330 Contans®, 330
Coal spoil tips, 285 Conventional agriculture, 83, 280–281,
Cochliobolus sativus, 207 284, 403
Coconut, 225 Conventionally tilled system, 94
Cocoyam (Xathosoma sagittifolium), 211 Conversion to agriculture, 383–385
Coenocytic, 123 Conversion to pasture, 35, 36, 39, 41
Co-evolution, 168 Coordinated expression by plant and microbe
Coir, 318 genes, 157
Cold tolerance, 338 Copper, 222, 223, 225, 226, 284
Cold-tolerant isolates, 351 Corky root rot, 354
Collar rots, 210 Corn, 214
Collembola, 343 Cortex invasion, 146
Colonisation efficiency, 213 Cortical cells, 138
Colonization, 333 Cortical phases, 227
Colony forming units, 350 Corynebacterium michiganense, 221
Index 423
Costa Rica, 32–41, 43, 44, 46 Dehydrogenase, 276, 288

Costelytra zealandica, 321 Dehydrogenase activity, 289
Cotton, 204, 211, 229, 334, 335, 337 Delia
Cover crops, 233–234 antiqua, 322
Crop geometry, 228 radicum, 322
Crop health, 220, 221 Delivery systems, 355
Crop management practices, 341 Dematiaceae, 45
Crop plant species, 343 Denaturing gradient gel electrophoresis
Crop productivity, 229 (DGGE) 42, 44, 281
Crop residues, 11, 13, 84, 214, 223, 319, 410 Denitrification, 71, 73, 75–76, 281
Crop rotations, 173, 207, 214, 325, 327 Denitrifying bacteria, 75
Cross-inoculation groups (CIGs), 135 5-deoxy-strigol, 128
Crown, 214 Desired trait, 351
Cry proteins, 306, 307 Determinate nodule meristems, 137
Cucumber, 317 Deterrent chemicals, 350
Cucumber seedlings, 317 Detritus, 81, 82, 95, 96
Cultivars, 325 Developed countries, 404
Cultivar development, 175 Developing countries, 403, 405
Cultivation, 220, 280 Developmental genetics, 121, 168
Culturing, 28 Developmental genetics, symbioses, 142–157
Curling of root hairs, 138 Developmental stages, plants 144
Cyanogenesis, 225 2, 4-Diacetylphloroglucinol (DAPG), 319, 332
Cyclic b-glucans, 158 Dilution plate counts, 45
Cyclic lipopeptides, 160 Diplodia tubericola, 232
Cyst nematodes, 327 Diplotaxis tenuifolia, 232
Cytokinin receptor, 156 Dipterocarp forest, 9
Cytokinins, 155 Direct drilling, 214
Cytokinin signalling, 155–156 Direct incorporation, 344
Cytoskeleton, 129, 138 Direct-seeding, 213
Direct sowing, 215, 216
Disease burden, 224
D Disease control, 334
Dactylaria, 325 Disease expression, 218
Dactylellina candidum, 232 Disease incidence, 204, 214, 215, 229, 231
Damage threshold, 205 Disease index, 223
Damage to roots, 320 Disease management strategy, 224, 233
Damping-off, 210, 233 Disease progress curves, 220
Damping-off, cotton 332, 354 Disease severity, 217, 221, 224, 227
Damping-off, Pythium ultimum, 234 Disease suppression, 212, 222, 316–317
Damping-off, Rhizoctonia solani, 234 Disinfestation, 354
Damping-off, sugarbeet, 330 Dissimilative stage, 130
Damping-off, tomato and pepper, 354 Disturbance response, 284, 287, 375, 382–384,
Damping-off, vegetables, 330 387, 388
De-aforestation, 43 Diversity
Decision support, 204 bacterial, 30, 36, 39, 40, 42, 45
Decomposer activity, 87 microbial, 36, 39, 42, 50, 51, 88
Decomposer diversity, 92, 93 rhizosphere communities, 90
Decomposition, 61, 66–71, 75, 283 soil microorganisms, 27, 32, 36, 39, 41, 45
Decomposition activity, 91, 93 Double stranded RNA (dsRNA), 335
Decomposition, nitrogen, 71 Downy mildew (Bremia lactucae), 206
Dedifferentiation, 138 Drainage, 287
Defence reactions, 142 Drechslera (Pyrenophora) tritici-repentis, 214
Defence-related gene activation, 132 Drought, 374, 381, 382
Defensive associations, 159–163 Drought stress, 131
424 Index
Drying-wetting regimes, 382 Entomopathogens, 342

Dual culture test, 352 Environmental competence, 344, 350, 353
Durian, 225 Environmental concerns, 328
Dwarf beans, 206 Environmental impacts, 412
Enzyme activity
hydrolytic, 382
E peroxidase, 382
Earthworms, 69, 71, 82, 95–98, 286, 343 Enzyme assays, 276
Earthworms, aneic, 95, 97 acid phosphatase, 43
Earthworms, endogeic, 95, 96 alkaline phosphatase, 43
Earthworms, interactions with aryl sulfatase, 43,
microorganisms, 95 phosphatase, 33
East African highlands, 412 Enzymes, 75, 337
Ecological competence, 345 Epacridaceae, 125
Ecological data, 352–353 Epidermis, 128
Ecologically friendly plant cultivation, 173 Epizootic, 323, 350
Ecological niche, 333 Ergosterol, 289
Ecological obligation, 120 Ericaceae, 125
Economic impact, 412 Ericoid mycorrhiza, 125
Economic losses, 320, 328 Erwinia, 164, 328
Economic value, 403 Erysiphe cruciferarum, 231
Eco-regional, 411 Ethylene, 141, 159, 163
Ecosystem, 27–32, 36, 38, 40, 41, 44, 46–51, 290 Ethylene, negative regulation, 163
diversity, 89 European cockchafer, 321
functions, 87, 89, 93, 98 Evaluating losses, 203–206
goods and services, 274 Evolution, 119, 121
models, 88 Evolutionary convergence, 168
processes, 87, 89, 90, 93, 94, 98 Evolutionary divergence, 171
process rates, 93 Exchangeable acidity, 341
productivity, 83, 86 Excreta, 67
services, 31, 202, 282, 352, 385, 399 Exotic isolates, use in biological control 351
Ecosystems, 405 Experimental mutagenesis, 145
Eco-technologies, 413 Exploitative mycorrhizal associations, 125
Ectomycorrhiza, 121, 125, 342 Extraradical mycelium, 124, 129
Efficient mass production, 355 Eyespot, 215
Efrapeptins, 324 Eyespot (Tapezia yallundae), 207, 224, 334
Eisinia fetida, 97
Elmerrillia tsiampacca, 229
Elmerrillia ovalis, 229 F
Endocellular bacteria, 133 Fabaceae, 134
Endomycorrhiza, 121 Facultative pathogens, 325
Endophytes, 164, 230, 343 Faeces, 70
Endophytic bacteria, 163–164, 326 Farmyard manure, 284
Endophytic fungi, 324 Fatty acid methyl esters (FAMEs), 279, 289
English four-course rotation, 207 Faunal activity, 82
Enhanced biocontrol, 341 Faunal detritus, 83
Enhanced disease control, 354 Feeding deterrence, 324
Enhanced plant disease resistance, 316 Ferralsols, 211
Enterobacter, 316 Fertilisation, 280
Enterobacteriaceae, 324, 346 Fertilisers, 212, 229, 232
Entomopathogenic bacteria, 323 Fertility, 282
Entomopathogenic fungi, 317, 320–323, Festuca pratensis, 324
338, 343 Festuca arundinacea, 324
Entomopathogenic nematodes, 354 Flavobacterium, 316
Index 425
Flavonoids, 127, 135 Fusarium oxysporum f. sp. dianthii, 337

Flooding, 381, 382 Fusarium oxysporum f. sp. medicaginis, 226
Fluorescein diacetate hydrolysis test Fusarium oxysporum f. sp. pisi, 206, 214, 221
(FDA), 45 Fusarium oxysporum f.sp. radicis-lycopersici,
Fluorescent pseudomonads, 221, 223, 225, 232, 335, 354
226, 232, 234, 332 Fusarium root rot (Fusarium solani), 232
Fodder, 317 Fusarium solani, 232
Foliar aphids, 324 Fusarium spores, germination, 333
Foliar-feeding insects, 355 Fusarium suppressive soils, 319
Food availability crisis, 2 Fusarium wilt, 223, 225, 335, 354
Foot rot, 210, 214, 224 Fusarium wilt (Fusarium oxysporum f. sp.
Forage grasses, 324 cubense), 223
Forecasting model, 204 Fusarium wilt pathogens, 337
Forest, 283, 284, 287, 289 Fusarium wilt, suppression of, 319
Forest crops, 328 Fusarium wilt, tomato, 224
Forest soils, 91, 95
Formulations, 346, 355
Fossilised plants, 167 G
Frankia, 74 Gaeumannomyces, 225, 328
Frankliniella, 317 Gaeumannomyces graminis, 226, 234
Freeze-drying, 346 Gaeumannomyces graminis var. tritici, 207,
Fresh produce, 205 209, 211, 214, 222, 224, 226, 230, 302,
Fruit, 328 318, 332, 341
Fruit-fly, 322 Galegoid legumes, 136
Fumigants, 354 Gall formation, 218, 227
Functional comparative genomics, 168 Gross domestic product (GDP), 404
Functional diversity, 89, 90 Gene expression, 132
Functional redundancy hypothesis, 88 Generalist strains, of pathogens, 347
Fungal-to-bacterial biomass ratio, 279 General suppression, 319
Fungal biomass, 289 Genetic analysis, 144
Fungal inoculum, 344–345 Genetic determinants, 351
Fungal metabolites, 324 Genetic mapping, 152
Fungal storage organs, 129 Genetics, 168
Fungal-to-bacterial ratio, 30, 285, 289, 383 Genetic stability, 351
Fungal viruses, 335 Genetic systems, 142
Fungi, 27–29, 32, 40, 46, 47, 50, 51, 82, 87, Genetic variability, 174
90, 92, 93, 95, 96, 99, 121, 281, 286, Genome loci (QTL), 163
316, 320, 321, 326, Genomic libraries, 152
Fungicide resistance, 328 Genome size, 134
Fungicides, 281, 354 Genotype, 286
Fungi, cost-effective, 355 Geosiphon, 123
Fungistasis, 340, 342 Germination, inhibition of, 338
Fungi-static compounds, 338 Germ tube growth, 338, 339
Fungitoxic terpenoid phytoalexins, 337 Germ tubes, 126
Furrow irrigation, 227, 228 Gibberella zeae (stalk rot), 224
Fusarium, 209, 213, 214, 224, 232, 234, 317, Gigaspora, 124
328, 332, 335, 350, 354 Gliocladium, 316, 331, 333,
Fusarium avenaceum, 224 334, 351
Fusarium culmorum, wheat, 224 Gliocladium virens, 329, 330,
Fusarium oxysporum, 232, 319, 333–335, 354 333, 354
Fusarium oxysporum, chlamydospores, 319 Gliocladium virens G1–3, 354
Fusarium oxysporum Fo47, 335 Gliotoxin, 333, 338, 340
Fusarium oxysporum Fo-B2, 335 Gliotoxin production, 333
Fusarium oxysporum f. sp. cubense, 223 Gliovirin, 333
426 Index
Global change Host-mycoparasite interaction, 329

direct impacts, 373, 374, 387 Host plant, 328
indirect impacts, 373, 374, 387 Host population, 350
interacting factors, 375, 376, 382, 386 Host-specificity, 320
Glomalin, 131 Human population growth, 3
Glomeromycota, 123 Humic substances, 83, 86
Glomus, 123 Humus, 83
Glucose, 340 Hydrogen cyanide, 225, 234
Glucosinolates (GSL), 234, 303–305 Hydrogen ion, 217, 220
Glycogen, 129 Hymenoscyphus, 126
Grain yield, increased, 322 Hypernodulating mutants, 147
Granules, fungal propagules, 344 Hypersensitive reaction (HR), 337
Grapes, 318 Hypersensitive responses, 132
Grass grub, 323, 347, 354 Hyphae, 123
larvae, 338 Hyphal web, 124
Grassland, 289 Hypovirulence, 328, 335, 336
Grassland leys, 10, 13, 20, 208 Hypovirulent strains, 335–336
Grazing, 288
Greenbelt Legislation, 355
Green broccoli (calabrese), 234 I
Green cane trash blanketing, 13 Identification/selection of desired traits,
Greenhouse gas emissions, 405 351–352
Green manures, 234, 316 Idriella bolleyii, 334
Green revolution, 297, 399, 403, 412 Immobilization, 71–73
Grey mould (Botrytis cinerea), 206 Indeterminate nodules, 137
Gross margin, 212 India, 32, 42, 46
Guanine+cytosine content (G+C), 39 Indonesia, 44
Gypsum (calcium sulphate), 232 Induced resistance, 326, 328, 336–337
Induced systemic resistance (ISR), 132, 161,
316, 319
H Induce host plant defences, 336
Haber-Bosch, 74, 75 Industrial revolution, 207
Hairy vetch (Vicia villosa), 233 Infection court, 223
Haiti, 404 Infection droplets, 139
Hartig net, 125 Infection hours, 204
Harvest date, 228 Infection processes, 355
Hawaiian Islands, 39 Infection thread, 138
Hay, 234 Infectious microbes, 325
Heavea braziliensis (rubber), 229 Injury-damage-loss paradigm, 203
Heavy metals, 284 Inundative controls, 320
Helicobasidium purpureum, 206 Inoculants, 172, 402
Hemicellulose, 68 Inoculated composts, 317
Herbicides, 281 Inoculum, 220, 344
Heterobasidion root rot, 336 Inoculum density, 212
Heteroconium chaetospira, 230 Inoculum load, 219
Hexoses, 129 Inoculum potential, 208, 217
H+ ions, 219 Inorganic soil substrate (ISS), 7, 10
Hirsutella minnesotensis, 325, 328 Insect-active, 318
Hirsutella rhossiliensis, 325, 327, Insecticidal toxins, 323
Histological zones, 139 Insecticides, 281, 343
Homologous genes, 154 Insect-pathogenic fungi, 351
Honey fungus, 206 Insect pathogens, 354
Horizontal gene transfer, 170 Insect pest management, 343
Horticulture crops, 317 Insect pests, 320–325, 351–352
Index 427
Insect pests, biocontrol of, 328 Land degradation, 6

Integrated control, 225, 353–355 Land management practices
Integrated crop management, 353, 355 no-till, 50
Integrated crop production systems, 356 tillage, 50
Integrated management, 229, 232 Land quality, 223
Integrated pest management (IPM), 320 Land reclamation, 285, 289
Intercellular mycelia, 128 Landscape reinstatement, 285, 287
Interior homeostasis, 168 Land-use, 83, 274
Interspecies (reciprocal) altruism, 171 changes, 27, 32, 35, 37–39, 41–43, 46, 50,
Intraradical mycelium, 124, 129 383, 385
Intrinsic pathogenicity, 350 history, 380, 384
Intrinsic specificity, 347 policies, 214
Inundative control agents, 320, 352 Large-scale liquid fermentation systems, 345
Invade™, 323, 346 La Selva Biological Station, 37
Invasive species, 48, 380, 381 Late sowing, 231
Inversion husbandry, 215 LCO. See Lipo-chito-oligosaccharide (LCO)
Invertebrates, 69, 286, 342, 343 Leaching, 70, 72, 73, 76, 283
In vitro testing, 343, 352 Leaf blight (Cercospora carotae), 221
In vitro mass production systems, 344, 345, Leaf blotches, 214
351 Leaf spot (Cercospora arachidicola), 215
Iron, 222, 223, 225, 226, 341 Lectins, 137, 329
Iron chelation, 334 Leeks, 207, 208
Iron competition, 334–335 Leghaemoglobin, 140
Iron uptake, 334 Legume breeding symbiotic effectiveness, 173
Irrigation, 220, 223, 227–229, 234 Legume-rhizobia symbiosis, 402
Isocyane metabolites, 333 Leguminous plants, 134, 284
Isothiocyanates, 233, 234, 303, 304 Leptosphaeria, 330
ISR. See Induced systemic resistance Leptosphaeria (Phoma) maculans, 215, 216
Lettuce, 206, 234
Lettuce, bacterial leaf spot, 317
J Lettuce corky root (Sphingomonas
Japanese beetle, 323, 324 suberifaciens), 234
Jasmonate, 132 Lettuce drop, 228
Leucine-rich kinase, 156
Lignin, 67, 70, 83, 226, 303
K Lime, 217, 223, 227, 281
Key nutrients, 86 Liming, 222, 281
Kilauea volcano, 40 Lipids, 83
Kiwifruits, 318 Lipo-chito-oligosaccharide (LCO), 135
Klebsiella, 74 Lipopeptide antibiotic iturin A, 332
Koch’s postulates, 220 Liquid fermentation, 346, 355
Koni®, 330 Litter feeders, 95
K-selection, 69 Litter quality, 93, 380, 384
Kwalei, 411 Livelihoods, 403
Locally sourced isolates, 166
Logging, 35, 44
L Loline alkaloids, 324
Labile organic matter, 91 Longevity of biological control agents, 346
Laccaria laccata, 342 Longevity, disease suppression, 318
Lactobacillus, 346 Lotus japonicus, 149
Laetisaria arvalis, 329 Low-input agriculture, 173
Lanarkshire disease (Phytopthora Low temperature storage, 346
fragariae), 206 Lucerne (alfalfa, Medicago sativa), 203, 232
Land conversion, 31, 38, 40, 43, 47 Lumbricus terrestris, 95
428 Index
Lumbricus rubellus, 95 Microbial community, size, 285

Lysis, hyphae, 330 Microbial diversity, 87–89, 91, 93, 94, 96, 98,
Lysozyme, 305 277, 282, 284, 401
Lytic enzymes, 340 Microbial-faunal interactions, 87, 94–98
Microbial flora, 225
Microbial growth, 317, 339, 353
M Microbial metabolic activity, 374, 376
Macroaggregate formation, 385 Microbial pathogens, 352
Macroconidia destruction, 319 Microbial phosphorus, 300
Macroinvertebrates, 69 Microbial plant pathogens, 328–337
Macro-nutrient, 222 Microbial populations, 171, 222, 353
Macroporosity, 19–21, 283 Microbial reservoir, 350
Magnesium, 211, 218, 222, 223, 225 Microbial response, 374–378, 386
Maize (Corn), 213, 214, 224, 322 Microbial turnover, 301
Management, 288–290 Microbiostasis, 316
practices, 383, 384 Microbivorus nematodes, 232
strategies, 274 Micro-elements, 223, 225
Manganese, 222, 225, 226, 302, 341 Micro-nutrients, 222, 225
Manganese-efficient genotypes, 302, 303 Milky disease, 347
Manganese sulphate, 226 bacteria, 323
Manipulating husbandry, 207–240 Millennium development goals (MDGs), 403,
Manures, 68, 70, 77, 283–284 412, 413
Meadows, 288, 289 Millennium ecosystem assessment, 403, 406
Medicago truncatula, 149 Mineral extraction, 285–290
Melolontha melolontha, 321 Mineralization, 72–74, 77
Membrane permeability, 227 Mineralization-immobilization, 71
Metabolic integration, 129 Mineralization rates, 385
Metabolites, 333, 342 Mineral nutrition, 339
Metabolites, production of, 324 Mine restoration, 287
Metagenomics, 28 Minimal/no tillage agriculture, 215, 216, 282
Metam sodium, 233 Mining, 287
Metarhizium, 318, 342 Microdochium nivale, 214
Metarhizium anisopliae, 320–323, 338, 339, Mitochondrial mutations, 335
343, 345, 354 Mitotic reactivation, 138
Metarhizium anisopliae DAT F001, 350 Microorganism, safety, 346
Methyl bromide, 233, 234 Modes of action, 326, 350
Methyl isothiocyanate, 233 Model legumes, 148
Microaggregates, 131, 407 Model plants, 149
Micro-arthropods, 282 Mole crickets, 343, 347
Microbes, 69, 121 Molecular genetics of symbioses, 148
Microbial abundance, 88, 90 Molecular methods, 28, 35, 36, 39, 40,
Microbial activity, 82, 86, 88, 95, 98, 289, 42, 43, 353
308, 316 Molecular phylogeny, 167
Microbial biocontrol agents, 325, 352, 353 Molecular tools, 220, 281, 353, 355
Microbial biological control, 315 Mollisols, 38
Microbial biomass, 29, 31, 38, 42, 43, 46, 50, Monocultures, 400
86, 90, 96, 211, 274, 282, 286, 287, 289 Mono-inoculation, 172
Microbial biomass, activity, 284 Monophyletic group, 167
Microbial colonisation, 318 Mulches, 229, 315
Microbial community, 279, 280, 288, 373–388 Multi-component preparation, 175
activity, 29, 36, 42, 283 Multi-dimensional networks, 326
structure, 29, 40, 42, 44, 45, 50, 51, 88, 89, Multi-microbial inoculants, 173
98, 276, 284, 285, 301, 308, 353, 375, Multi-microbial systems, 166
383–387 Multi-partite symbioses, 166
Index 429
Mustard, 233, 234 Nitrate inhibition of nodulation, 142

Mustard oil, 234 Nitrate leaching, 234
Mutualism, 119 Nitrate tolerant symbiosis, 141
Mutualistic (mutually beneficial) symbioses, 120 Nitrification, 71, 76, 281
Mycelial granules, 344 Nitrification inhibitors, 76
Mycelial growth, 338–340 Nitrifying bacteria, 76
Mycelial pellets, 344 Nitrobacter, 76
Myc factor, 128 Nitrococcus, 76
Myco-heterotrophic orchids, 125 Nitrogen, 61, 63, 66, 70–77, 81, 82, 89, 94, 96,
Myconematicide, 327 99, 174, 211, 215, 216, 221, 223, 224,
Mycoparasites, 329, 330, 351 226, 230, 232, 234, 281, 283, 284,
Mycoparasites, biological control of, 329 339–341, 402, 407
Mycoparasitism, 222 Nitrogenase, 73, 134
Mycorrhiza, 29, 47, 121 Nitrogen cycling, 276, 279
Mycorrhiza-helper-bacteria, 165 Nitrogen deposition, 375, 376
Mycorrhizal fungi, 77, 82, 342 Nitrogen fixation, 71, 73–75, 134, 158, 298
abundance, 378 Nitrogen fixation, biological, 402, 405, 407
arbuscular, 48 Nitrogen immobilization, 71, 73
indicator (lipid), 378 Nitrogen limitation, 376
Mycorrhizal symbionts, 68 Nitrogen metabolism, 140
Mycorrhizins, 143 Nitrogen mineralization, 71–73, 298
Mycorrhizosphere, 165 Nitrogen turnover, 94
Mycosphaerella graminicola, 215–216 Nitrosococcus, 76
Myrosinase, 303 Nitrosolobus, 76
Nitrosomonas, 76
Nitrosovibrio, 76
N Nitrospina, 76
Narcissus, 205 Nitrospira, 76
Narrow specificity, 136 Nitrous oxide, 72, 75, 87
Natural disease suppression, 318 Nod factors, 136
Natural pathogens, 321 Nodulation genes, 136
Natural population, 352 Nodulation programmes, 138, 145
Necotrophic mycoparasitism, 329 Nodule primordium, 138
Nematode-associated bacteria, Nodule-specific isozymes, 140
Xenorhabdus, 324 Nodulins, 143
Nematode parasitism, 327 NoGallTM, 331
Nematodes, 282, 317, 326 Non-humic substances, 84
Nematode suppression, 326 Non-legumes, 147
Nematode-suppressive soils, 327 Non-pathogenic fusaria, 319
Nematode-trapping fungi, 325 Non-pathogenic strains, 335–336
Nematode-trapping fungus (Dactylellina Non-spore forming microbes, 346, 350
candidum), 232 Non-target invertebrates, 321
Nematophagous species, 326 Non-target species, 320, 347
Neotyphodium, 324 Nostoc, 74
Neotyphodium uncinatum, 324 No-till, 83, 93, 94, 213, 215, 408–410
Neutron scanning, 277 No-tillage regimes, 213
New biocontrol agents, 347 Novel tissue, 168
New Caledonia, 44–46 Nuclear magnetic resonance, 277
New Zealand grass grub, 321 Nuclear mutations, 335
Niche, endophytes, 164 Nucleoporins, 151
Niche exclusion, 212 Nutrient availability, 86, 93
Nil-tillage, 214, 233 Nutrient competition, 212
Nitrate nitrogen (NO3-N), 75, 211, Nutrient cycles, 94, 98
221–223, 340 Nutrient cycling, 283, 298, 403, 407
430 Index
Nutrient losses, 283 Paris-type morphology, 126

Nutrients, 286, 333, 334, 351 Parsnips, 206
Nutrient status, 220–227 Pasteuria, 327
Nutritional associations, 158–159 Pasteuria penetrans, 325, 327
Pasture, 285, 328
Pasture conversion, 37, 38, 51
O Pastures, Livestock management, 281
Oats, 209, 214 Pasture soil, 350
Obligate parasites, 325 Pathogen density, 230
Oceania, 32, 41–46 Pathogenesis related proteins, 132
Oil seed rape, 204, 205, 215, 216, 231 Pathogenic bacteria, 323
Oligosaccharides, 337 Pathogenic fungi, 132
Olpidium brassicae, 228 Pathogenicity, 351
Onion maggot, 322 Pathogenic microbes, 210, 221, 226
Onions, 207, 221 Pathogenic potential, 219
Onion thrips (Thrips tabaci), 221 Pathogen incidence, 224
Onion white rot (Sclerotium cepivorum), 206 Pathogen recolonisation, 232
Oosporein, 324 Pathogens, 282, 406
Opencast coal mines, 288 Pathogens of insects, 320
Orchid mycorrhiza, 125 Pawpaw, 224
Orchids, 125 Pea, 229
Organic acids, 159, 338, 343 cultivar “Triumph,” 175
Organic agriculture, 282 Peanut, 215
Organic amendments, 212, 232, 315, 319, 327 Peat, 318
Organic carbon, 81, 83, 86, 97, 99 Pecan weevil, 354
Organic fields, 403, 406 Pedogenesis, 6–8
Organic matter, 63–69, 71, 213, 225, 232, 276, Penetrometer resistance, 18
283, 287, 400, 404, 407 Penicillium, 316, 331, 403
Organic matter decomposition, 69, 70 Penicillium chrysogenum, 342
Organic matter dynamics, 283 Penicillium citrinum, 342
Organic matter stocks, 282 Penicillium griseoroseum, 342
Organic mulches, 225 Penicillium miczynskii, 342
Organic nitrogen, 86 6-pentyl–2H-pyran–2-one, 333
Organic soil amendments, 315 Peptaibols, 333
Osa Peninsula, 38 Peptides, 334
Osmotolerance, 339 Perennation of inoculum, 203
Otiorhynchus, 317 Perennial ryegrass, 208
Otirorhynchus sulcatus, 321 Periarbuscular membrane, 129
Oxalic acid, 324 Periarbuscular space, 129
Oxidative burst reactions, 132 Peribacteroid membranes, 139
Oxisols, 38, 40 Peronospora viciae, 206
Oxygen, 89, 91, 286 Persistence of biological control agents, 338,
339, 343, 350, 351, 355
Pest/disease control, 354
P Pesticides, 280, 281
Pacific Southeast Asia, 44 reduced use, 322, 355
Paecilomyces, 322 pH, 217–219, 222226, 230, 234, 339, 344
Paecilomyces lilacinus, 325, 327 Phenazine–1-carboxylic acid (PCA), 341
Paenibacillus polymyxa, 336 Phenazines, 332
Paenibacillus popilliae, 323 Phenolics, 226
Paenibacillus validus, 167 Phenotypic characterization, 144
Pantoea, 346 Phenylalamine, 340
Parallel evolution, 168 2-Phenylethylisothiocyanate (PEITC), 304, 305
Parasitism, 317, 328, 329–331, 350, 353 Phialophora, 209
Index 431
Phoma medicaginis var. pinodella (foot rot), 209 Plant receptors, 152–155
Phosphatase, 276, 301 Plant resistance to pathogens, 132–133
Phosphate, 211, 222, 224 Plants, 121, 342
Phosphate transport proteins, 130 Plant-soil systems, 274
Phospholipid fatty acid analysis (PLFA), 33, Plant symbiotrophy, 171
40, 279, 289 Plasma membrane, 129
Phospholipid fatty acid biomarkers, 284, 289 Plasmodiophora brassicae (clubroot),
Phospholipid fatty acid profiles, 276, 283 207–208, 217–219, 223, 226–228,
Phosphonate (potassium phosphite), 225 230–232, 330
Phosphorus, 61, 77, 81, 82, 86, 99, 130, 174, boron, 227
222, 225, 226, 230, 231, 407 Plasmodiophorales, 217, 219
Phosphorus availability, 301 Plastic sheeting, 225, 231, 232
Phosphorus cycle, 276 Pochonia chlamydosporia, 325, 327
Phosphorus-fertilisation, 281 Poecilus versicolor, 321
Phosphorus uptake, 130, 280, 299 Poinsettia, 332
Phosphorus-use efficiency, 300 Pollution, 287
Phosphorylation substrate, 151 Polyamines, 127
Photorhabdus, 324 Polymerase chain reaction (PCR), 353
Phylogenetic analysis, 35, 36, 40 Polymorphism analyses, 167
Physiological disorders, 220 Polynesia/Micronesia, 44
Phytase, 301 Polyphosphate, 130
Phytate, 301 Polyphyletic origins, 168
Phytoalexins, 133 Polyversum, 330
Phytoalexins, dianthalexin (2-phenyl–7- Popillia japonica, 323
hydroxy–1,3-benzoxazin–4H-one), 337 Pore continuity, 283
Phytohormone (auxin) synthesis, 158 Pore networks, 213
Phytophthora, 211, 214, 317, 328 Pore space, 131
botryose, 229 Positional cloning, 152
cinnamomi, 206 Potassium, 211, 224, 225, 234
fragariae var. rubi, 206 Potassium phosphonate, 224
palmivora, 224 Potato common scab (S. scabies), 222
ramorum (sudden oak death), 207 Potatoes, 219
Phytophthora root rot, 316 Potato scab, 225
Phytophthora root rot of raspberry Potential determining ions, 10
(Phytophthora rubi), 232 Pre-adaptation to symbiosis, 168
Phytoremediation, 284 Predatory carabid, 321
Pine plantations, 44 Pre-infection threads, 138
Pisum sativum, 144 Pre-penetration processes, 128, 148
Plant bioprotection, 326 Pre-symbiotic stage, 127
Plant breeding, 175 Primary cultivation: tillage, 213–215
Plant defence reactions, 132 Primary infections, 220
Plant disease control, 329, 350 Primary tropical forest, 35, 44
Plant endophytic microbes, 352 Priming of decomposition, 336
Plant genes, 143 Principle biotic components, 342
Plant growth promoting rhizobacteria (PGRP), Production and formulation,
157, 284, 354 344–347
Plant health, 226, 353 Production systems, 353
Planting, 231 Profitability, 410
Planting density, 220 Proliferation within roots, 130, 139
Plant-microbe symbioses, 124 Propagule lysis, 319
Plant mutants, 144 Proteins, 154, 337
Plant parasitic nematodes, 325–328 Proteobacteria, 35, 36, 40, 44
Plant pathogens, 402 Protozoa, 281, 282
Plant productivity, 283 Pseudobactins, 334
432 Index
Pseudomonas, 39, 157, 225, 316, 326, 328, Rhizobacteria, 157, 232, 336, 354
331, 332, 335, 336, 340, 342, 346, 354 Rhizobia, 134, 402
chlororaphis PCL1391, 340 Rhizobiaceae, 135
fluorescens, 226, 233, 319, 332, 337 Rhizobium, 74, 298, 407
fluorescens 2–79 RN10, 341 Rhizobium-Agrobacterium, 39, 44
oryzihabitans, 325 Rhizoctonia, 125, 213, 215, 307, 317, 328,
putida, 335 332, 336, 350, 354
Pseudo-nodules, 169 cerealis, 214, 224
Public policy, 399 oryzae, 215
Puccinia melano-cephala, 225 solani, 213–215, 229, 234, 317, 329, 330,
Pumpkin, 233 332, 333, 336, 337, 350, 354
Pyoluteorin, 332 Rhizodeposition, 75, 88
Pyoverdines, 334 Rhizoplane, 232
Pyrones, 333 Rhizosphere, 29, 66, 83, 129, 162, 212,
Pyrrolnitrin, 332 221–223, 226, 227, 232, 233, 298, 300,
Pythium, 213, 214, 224, 233, 317, 328, 330, 301, 316
350, 354 Rhizosphere activity, 88
myriotylum, 211 Rhizosphere-colonizing bacteria, 319, 326
oligandrum, 329, 330 Rhizosphere competence, 326, 334, 343, 352
sulcatum, 220 Rhizosphere pH, 222, 223
ultimum, 330, 333 Rhodospirillum, 39
violae, 220, 228 Rice, 229, 230
Pythium, cotton, 316 Risk assessment, 353
Pythium-induced damping-off, 332 Root adhesion, surface components, 160
Pythium inhibition, 335 Root aphids, 324
Root colonization, 160, 299, 353
Root exudates, 82, 83, 88, 127, 159, 212,
Q 334, 343
Quantitative variation, 163 Root-exuded organic acids, 163
Quorum sensing, 162 Root-feeding Coleoptera, 321
Root growth, 19
Root hairs, 218, 219, 227, 228, 231
R Root-hair infection, 218
Radish, 234, 335 Root-knot nematodes, 326–328, 354
Radish, bacterial leaf spot, 317 Root nodules, 134
Ralstonia, 164 Root parasitic nematodes, 336
Rapeseed, 204 Root pathogens, 336, 352
Raspberry, 206 Root penetration, 147
Receptor kinases, 152, 155 Root rots, 211, 214, 224, 232
Red core disease, 206 Root rot, peas, 214, 220, 229
Red-headed cockchafer (Adoryphorus Rosid I clade, 135
coulani), 321 Rotations, 207–210, 212, 229
Redox potential, 225 Rothamsted experiment station, 207
Registration of biological control agents, 352 rRNA intergenic spacer analysis (RISA), 35
Regulatory environment, 355 Rr-selected, 69
Regulatory symbiotic genes, 142 Ruminant grazing animals, 67
Remediation of contaminated soil, 124 Rust, 225
Resilience threshold, 6 Rye (Secale cereale), 233
Resistance, 352
Resource competition, 333–335
Respirometry, 276 S
Resting spores, 231 Salicylic acid, 132, 337
Restoration, of land, 285, 287 Salinas Valley, California, 234
rhi genes, 162 Sand, 341
Index 433
Saprotrophic fungi, 91 Social/economic well-being, 404

SAR. See Systemic acquired resistance Social impacts, 412
Scab, 222 Social insects, 322, 350
Scarabaeidae, 323, 347 Socio-economic issues, 404
Scarab pests, 324 Sodium, 224, 341
Sclerotia, 330 Sodium tetraborate, 227
Sclerotia, degradation of, 338 Soft or vine fruits, 318
Sclerotial mycoparasites, 330 Soil abiotic factors, 341–342
Sclerotial parasitism, 350, 352 Soil acidification, 340
Sclerotia mycoparasitism, 338 Soil acidity, 29, 46, 217–220
Sclerotinia, 328, 330 Soil aggregates, 29, 97, 98, 131, 213, 278,
Sclerotinia homeocarpa, 336 282, 283, 288
Sclerotinia minor, 227, 228, 232, 330, 331, 336 Soil aggregrate dynamics, 97, 98
Sclerotinia root rot, 330 Soil aggregate stability, 83
Sclerotinia sclerotiorum, 204, 223, 330, 331, Soil amendments, biochar, 282–283
338, 351 Soil bacteria, 276, 286, 324
Sclerotinia trifoliorum, 330 Soil biodiversity, 93
Sclerotinia stem rot, 205 Soil biological communities, 290
Sclerotium, 328, 330 Soil biology, 281
cepivorum (white mould), 206, 215, 232, Soil biomass, 286
330, 331, 339, 354 Soil biota, 353
rolfsii, 329 Soil-borne diseases, 209, 212, 223, 315,
Secale cereale, 233 316, 328
Secondary cultivation, 215–217 Soil-borne fungi, 329
Secondary infections, 219 Soil-borne inoculum, 207
Secondary metabolites, 326, 339 Soil-borne microbes, 223, 231
Seed-borne, 164 Soil-borne nematode, 205
Seed germination, 125, 127 Soil-borne pathogens, 203, 206, 207, 214, 232,
Seed productivity, 174 233, 282, 316, 340, 350
Seed treatment, 334, 337, 354 Soil-borne pests, 315, 316
Selection, 347 Soil-borne propagules, 204
Selection pressures, 352 Soil-borne saprophytic fungi, 334
Septoria tritici (Mycosphaerella graminicola), Soil-borne wilt, 203
215, 216 Soil carbon, 234, 283, 405
Serology-based risk assessment, 220 Soil carbon storage, 20, 89
Serpentine, 31, 45 Soil compaction, 5, 17, 18, 214, 286, 288
Serratia, 323, 347 Soil degradation, 5–7, 9
Serratia entomophila, 345, 354 Soil disinfection, 223
Sewage sludge, 283–284 Soil drainage, 229
Sharp eyespot (Rhizoctonia cerealis), 214, 224 Soil dwelling species, 347
Siderophores, 212, 335, 354 Soil, ecology, 352
Signal molecules, 152, 337 Soil ecosystems, 277, 320
Signal perception, 132 Soil environment, 208
Signal transduction, 132 Soil enzyme measurements, 288
Silt, 341 Soil erosion, 213, 283, 405
Silurian and Devonian flora, 167 Soil evaporation, 229
Single continuum of radical growth phases, 124 Soil fertility, 212, 223
Site-specific recombinase, 161 Soil food web, 82, 281
Sitona lepidus, 321 Soil formation, 8, 30
SkleroPro, 204 Soil fungistatic effect, 342
Slash and burn system, 35, 36 Soil habitat, 29
Slurries, 59, 67, 70, 77 Soil health, 274, 402, 404
Small cereal grains, 344 Soil heterogeneity, 277
Snow moulds in turf grass, 317 Soil-inhabiting insects, 324
434 Index
Soil-less potting media, 317 Specific antagonism, 319

Soil microbes, 229, 315–356, 403 Specificity, 135–137, 351
Soil microbial biomass, 234, 284, 289 Specific selective pressures, 171
Soil microbial biomass-carbon, 286 Sphingomonas suberifaciens, 234
Soil microbial communities, 274–281, Spontaneously nodulating mutant, 155
283–288, 290 Sporangial maturation, 227
Soil microbial community composition, Spores, 130, 324, 350
282, 284 Spore densities, 230
Soil microbial functions, 413 Spore-forming, 350
Soil microbial “genome,” 279 Spore germination, 126, 208, 218
Soil minerals, 225 Spore load, 217
Soil moisture, 220, 225, 227–231, 318, 376, Sporidesmium, 350
377, 381–383 Sporidesmium sclerotivorum, 329, 330,
Soil, moisture characteristics, 131 336, 350
Soil nutrients, 47, 225 Sporocarps, 130
Soil organic carbon, 6, 8–10, 13, 20, 29 Sporulation, 339, 353
Soil organic matter, 63, 65, 67–69, 225, Spray-drying, 346
274, 338 Spring barley, 214
Soil organic matter, pools, 83, 98 Stable carbon isotopes, 277
Soil organic matter stocks, 281 Stagnospora (Phaeosphaeria) nodorum, 214
Soil pests, 345, 351 Stalk rot, 224
Soil pH, 208, 340–341 Steinernema carpocapsae, 350
Soil physical architecture, 276 Steinernema scapterisci, 347, 348
Soil physical disturbance, 50, 51 Stem/root white rot, 224
Soil porosity, 286 Stem rot, 204
Soil quality, 234, 285, 290 Stomatal conductance, 131
Soil respiration, 91, 288 Strawberry, 234, 318
Soil sandwich assay, 339 Streptomyces, 163, 225, 331
Soil saprophytes, 351 griseoviridis, 354
Soil solarisation, 231–233, 354 scabies, 219, 222
Soil structure, 29, 50, 131, 210–215, 277, 280, Streptomycetes, 336
281, 283, 287, 400, 405 Strigolactones, 127
Soil structure formation, 124 Strip tillage, 215
Soil temperature, 338–339 Stylosanthes hamata, 19
Soil texture, 29–31, 210, 211, 287 Sub-Saharan Africa, 408, 411–412
Soil types, 210–212, 224, 234, 338 Subsistence agriculture, 42
Soil water potential, 229, 339 Substrate induced growth response (SIGR), 38
Soil–water retention, 229 Substrate-induced respiration (SIR), 28, 38
Solanaceous plants, 336 Substrate quality, 88, 90, 91
Solar gain, 231 Substrate quantity, 89, 90
Solarisation, 231–233 Substrate utilization, 42
Soluble carbon, 82, 95, 96 Subsurface drip irrigation, 228
Soluble organic compounds, 82 Subterranean pests, 320
South Australia, 405 Subterranean termites, 322
Southeast Asia, 32, 41–46 Succession, 285
Southern blight of tomatoes, 354 Sugarbeet root maggot, 321
Sowing density, 228 Sugarcane, 339
Sowing times, 230–231 Sugarcane rust (Puccinia melano-cephala), 225
Soybean, 214, 234 Sugars, 159
Soybean cyst, 328 Sulphatase, 276
Spatial variability, 290 Sulphate-sulphur, 341
Species diversity, 90 Sulphonamides, 284
Species identity, 90 Sulphur, 61, 81, 86, 219, 225, 407
Species richness, 90, 91, 95 Sulphur cycle, 276
Index 435
Suppression of insects, 317 Terra Preta, 36, 282, 404

Suppression, 223, 233, 282, 316–319, 343 Test for biocontrol, 352
Suppressive soils, 318–320 Testing under field conditions, 355
Suppression of root-knot nematodes, 327 Tetanops myopaeformis, 321
Surface charge finger printing (SCF), 7 Thermotolerant isolates, 339
Sustainable agriculture, 87, 121, 171–175, Thielaviopsis basicola, 225
229, 282–285, 290, 410 Thin sections, 277
Sustainable organic integrated livelihoods Threshold, 220, 225
(SOIL), 404 Thrips tabaci, 221
Sustainable pest and disease management, 355 Thyme (Thymus vulgaris), 232
Swedes (Brassica napus), 208 Tillage, 94, 212, 213–216, 227, 228, 234, 280,
Sweet potatoes, 232 327, 343, 383
Symbiosins, 143 Tilletia indica, 232
Symbiosis, 73, 74, 119, 127, 174, 407 Tobacco, 225
Symbiosis-specific cytokinin receptors, 152 Tolypocladium, 324
Symbiosomes, 140 Tomato, 233, 234, 317, 332, 335, 354
Symbiotically effective strains, 171, 174 Tomato damping-off, 233
Symbiotic bacteria, 326 Tomato wilt (Fusarium oxysporum f. sp.
Symbiotic compartments, 168 lycopersici), 221, 224
Symbiotic effectiveness trait, donor, 175 Topsoil, 286–287
Symbiotic nitrogen, 75 Total nitrogen, 86, 341
Symbiotic receptor kinase, 151 Toxic compounds, 316
Sym genes, 143 Toxic effects, 324
Synchitrium endobioticum, 228 Trait stability, 345
Synergistic effects, 165–176, 354 Transcellular growth, 138
Syntenic, 154 Transcription activator, 157
Systemic acquired resistance (SAR), 132, 212, Transformants, 353
316, 326, 336–337 Trap crop, 233, 234
Systemic autoregulation, 141, 147 Trash management, 10
Trehalose, 129
T-RFLP, 279, 281, 286
T Trichoderma, 222, 316, 317, 326,
Take-all, 207, 209, 211, 214, 222–224, 226, 329–331, 333, 334, 336, 337,
228, 303, 319 340, 342, 351, 354
Take-all disease (Gaeumannomyces graminis Trichoderma-amended compost, 317
var. tritici), 209, 211, 216, 228, 230, 334 Trichoderma hamatum, 317, 392
Take-all decline, 209, 211, 212, 222 Trichoderma harzianum, 226, 229, 233, 329,
Take-all disease in wheat, 210 330, 338, 342, 343
Take-all suppression, 222, 226, 318–319 T. harzarium T–35, 334
Take-all, wheat, 332, 341 T. harzianum T–22, 350
Talaromyces flavus, 329, 350 T. koningii, 222, 223, 340, 341
Tanzania, 412 Trichoderma hyphae, 329
Tapezia yallundae, 207, 215, 224 Trichoderma lignorum, 329
Target pest, 347 Trichoderma-mediated suppression, 340
Tc toxin complex, 324 Trichoderma-Rhizoctonia interaction, 329
Tea plantations, 43, 44, 46 Trichoderma viride, 338
Temperate Trophic interference, 166
forests, 29, 30 Tropical
grasslands, 31 ecosystems, 32, 51
Temperature, 338, 344 forests, 30–46, 50
increase, 373, 376 Tuberculariaceae, 45
optima, 338 Turfgrass, 317, 324, 339
sensitivity, 379 Turf pests, 347
Termites, 322, 350 Trichoderma virens, 337, 338, 340, 343, 354
436 Index
Trichoderma virens G6, G6–5, G–11, 337 Water-logging, 286, 288

Twin-scale repropagation, 205 Water management, 227–230
Water potential, 339
Water productivity, 229
U Water retention, pF, 230
Ultramafic, 45, 46 Water-use efficiency, 228, 229
Uncultured bacteria, 133 Weak saprophytes, 342
United Nations, 404 Weathering rate, 31
University of Wisconsin, 221 Weed control, 229
Urease, 276 Weeds, 317
Western flower thrips (Frankliniella
occidentalis), 318, 322
V Western Ghats, 43
Vegetables, 317, 326 Wetland, 31, 47–49
Verticillium, 204, 214, 224, 232, 328, 337, 354 Wheat, 209, 211, 214, 216, 222, 228,
Verticillium albo-atrum, 203, 221, 224 229, 232
Verticillium chlamydosporium, 325, 327 Wild barley, 324
Verticillium dahliae, 204, 209, 215, 232, 234 Wild rocket (Diplotaxis tenuifolia), 232
Verticillium, wilt of potato, 209 Wilting, 204, 337
Vesicles, 129 World Agroforestry Centre (ICRAF), 412
Vesicular-arbuscular mycorrhizas (VAM), 214
Veterinary medicines, 280, 284
Vicia villosa, 233 X
Violet root rot (Helicobasidium purpureum), Xathosoma sagittifolium, 211
206 X-ray tomography, 277
Viridiol, 340 Xylanase, 337
Virulence, 344
Virulence factors, 164
Virus-tested Narcissus (Narcissus Y
pseudonarcissus), 205 Yellow Cabbage (Fusarium oxysporum f. sp.
Volatile terpenes, 333 conglutinans), 221
Yersinia entomophaga, 325
Yield losses, 325
W
Waste management, 284
Water activity (aw), 339 Z
Water, availability, 86 Zinc, 222, 225, 226, 284, 341
Water holding capacity, 6, 12, 13, 15, 17 Zymogenous biomass, 68

Soil Microbiology and Sustainable Crop Production

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Soil Microbiology and Sustainable

Soil Microbiology and

ISBN 978-90-481-9478-0 e-ISBN 978-90-481-9479-7

© Springer Science+Business Media B.V. 2010

Printed on acid-free paper

Springer is part of Springer Science+Business Media (www.springer.com)

objectives. Detailed studies of beneficial and pathogenic soil- and root-borne

integral part of the biosphere. It has been written by an international team of

1 The Nature of Sustainable Agriculture.................................................. 1

2 The Microbiology of Natural Soils......................................................... 27

3 Soil Microbiology and Nutrient Cycling................................................ 59

4 The Role of Microbial Communities in the Formation

5 Intimate Associations of Beneficial Soil Microbes

6 Soil-Borne Pathogens and Their Interactions

7 The Impact of Land-Use Practices on Soil Microbes........................... 273

8 The Effects of Plant Breeding on Soil Microbes.................................... 297

9 Utilizing Soil Microbes for Biocontrol................................................... 315

10 How Will Climate Change Impact Soil

11 Evaluating the Economic and Social Impact

Jessica L.M. Gutknecht

Andrew D. Noble and Sawaeng Ruaysoongnern

A.D. Noble (*)

240 Food Price Index

Recent Challenges in the Global Foodscape

Land Degradation and It Impact on Sustainability

Central to the discussion of the notion of sustainable agriculture is the impact of a

chemical attributes. The properties of an Oxisol cleared of climax rain forest 53

CECT (cmolc kg−1) 19

Amelioration of Charge Degradation

Declining productivity on degraded soils can in part be ascribed to a reduction in

Fig. 4 Surface charge fingerprints a

in productivity before significant increases are achieved. In an effort to mitigate this

Cumulative yield (g/pot) 4

14000 Yield 2002

Dry matter production (kg ha−1)

Addressing Soil Physical Degradation

Penetrometer resistance (MPa)

Root frequency impact (%)

resulting in the development of permanent stable macropores and the storage of

Transforming the Agricultural Landscape

While there is considerable uncertainty about the impacts of climate change,

Teri C. Balser, Devin Wixon, Lindsey K. Moritz, and Laura Lipps

Soil microorganisms, such as bacteria and fungi, control ecosystem functioning

T.C. Balser (*) and L.K. Moritz

Any treatment of microbial communities or ecology must include consideration of how

Soil formation is generally considered to be driven by five factors: parent material,

Neotropics: Amazonia, Costa Rica, Hawaii

Authors Location Study scheme Sampling depth Microbiological methods

Nevertheless, both studies showed that soil microbial communities in Amazonia

Tropics of Africa, Southeast Asia, and Oceania

but was not documented in phylogenetic analyses of tropical soils by Borneman

to land-use conversion from primary forest to pineapple plantations in Tahiti

Conclusions: Old World Tropics

Wetlands are transient or permanently saturated terrestrial habitats. The term

Soil Microbial Communities and Changing Agricultural

Humans have converted one-quarter of the Earth’s surface to cultivated ­systems,

particular, become limiting factors to microorganisms, and thus species-specific

Sessitsch A, Weilharter A, Gerzabek MH, Kirchmann H, Kandeler E (2001) Microbial population

David W. Hopkins and Jennifer A.J. Dungait

D.W. Hopkins (*)

Humans have converted one-quarter of the Earth’s surface to cultivated systems,