FAO SOILS BULLETIN 49

application
of nitrogen-fixing systems
in soil management

F000 AND AGRICULTURE ORGANIZATION

OF THE UWITED NATION'S ROW L
 PREFACE

In view of the continuing increase in cost and scarcity of

mineral fertilizers resulting from the use of high-cost fossil
energy, there is renewed interest in organic recycling and biological
nitrogen-fixation to improve soil fertility and productivity.

FAO, in cooperation with the Swedish International Development

Authority (SIDA) has assumed a leading role in this respect, including
the sponsoring of a series of important international meetings to
promote the use of organic materials and biofertilizers. Examples of
these meetings include: the Expert Consultation on Organic Materials
as Fertilizers held in Rome in 1974; the Workshop on Organic Recycling
in Asia held in Bangkok in 1976 and the Workshop on Organic Materials
and Soil Productivity in the Near East held in Alexandria in 1978.

The workshop in Alexandria recommended the further promotion

of research, development, application and dissemination of information
available on various aspects of biological nitrogen-fixation, including
symbiotic systems of rhizobia/legume and Azolla/blue-green algae, and
free-living nitrogen-fixing bacteria and blue-green algae.

Dr Y.A. Hamdi, a delegate and invited speaker at the workshop

was requested to prepare the present Bulletin, which reviews the
literature and provides information on the characteristics, production
and use of biofertilizers in agriculture.

Although some of the methods described are being continually

improved upon, it is hoped that the compilation of various aspects of
nitrogen-fixation under one cover in this Bulletin will be of interest
and assistance not only to research workers, but also to extension
workers and planners concerned with the further development and refine-
ment of these natural systems for soil improvement and management
throughout the various agro-ecological zones of the world.
 ACKNOWLEDGEMENTS

The Food and Agriculture Organization of the United Nations

and the author wish to gratefully acknowledge all organizations
and individuals who have assisted directly or indirectly in the
preparation and processing of this Bulletin.

The author also wishes to express his deep appreciation of

assistance from all those scientists and colleagues who made their
research papers and photographs available for use as required in
the Bulletin. More specific acknowledgements of contributions and
sources of information are given in the text, supported by a list
of references for further reading as required on these important
subjects.
 TABLE OF CONTENTS

Page
1. INTRODUCTION 1
1.1 General 1
1.2 Global Status of Nitrogen 1
1.3 Fixed Nitrogen 2
1.4 Fertilizer Nitrogen 3
2. BIOLOGICAL NITROGEN-FIXATION 5
2.1 Introduction 5
2.2 Free-Living Nitrogen-Fixing Organisms 5
2.2.1 Bacteria 5
2.2.2 Blue-green algae 5
2.3 Associative Symbiosis Between Free-Living
Diazotrophs and Other Organisms 6
2.3.1 Loose associations 6
2.3.2 Associations with animals 6
2.3.3 Associations involving morphological
modification or accommodation 8
2.4 Biochemistry of Biological Nitrogen Fixation 9
2.4.1 Nitrogenase enzyme system 9
2.4.2 Requirements for nitrogen-fixation 9
3. RHIZOBIA-LEGUME SYMBIOSIS 11
3.1 The Microsymbiont.Rhizohium 11
3.1.1 Taxonomy 11
3.1.2 Morphology and cytology of Rhizobium 11
3.1.3 Cultural characteristics of Rhizobium 11
3.1.4 Physiology of 12
3.1.5 Ecology of rhizobia in soil 14
3.2 The Macrosymbiont. The Legumes 14
3.2.1 Cross-inoculation groups 14
3.2.2 Nodules 16
3.3 Genetic Aspects of Nodulation and Nitrogen-Fixation
by Legume-Rhizobia Association 22
3.3.1 The macrosymbiont. Legume host 22
3.3.2 The microsymbiont. Rhizobium 22
3.4 Legume Inoculation 24
3.4.1 Strain selection 24
3.4.2 Collection of strains 24
3.4.3 Evaluation of strains 24
3.4.4 Bacteria propagation 25
3.4.5 Carriers of rhizobia 25
3.4.6 Inoculant preparation 28
 Legume Inoculation (Cont.) Page

3.4.7 Standards of inoculants 28

3.4.8 Multistrain inoculants 28
3.4.9 Tests for control of inoculants 28
3.4.10 Inoculant application 29
3.4.11 Response to inoculation 31

3.4.12 Factors affecting the success of inoculation 36

3.4.13 Amount of nitrogen fixed by different legumes 40
4. BLUE-GREEN ALGAE 45
4.1 Introduction 45
4.2 Nitrogen-Fixing Blue-Green Algae 45
4.3 Classification 45
4.4 Genetics of Blue-Green Algae 48
4.5 The Site of the Nitrogenase Enzyme in Blue-Green Algae 48
4.6 Isolation and Purification of Blue-Green Algae 48
4.6.1 Unialgal cultures 48
4.6.2 Bacteria-free cultures of algae 49
4.6.3 Tests for purity of algal cultures 49
4.6.4 Methods for testing the ability of blue-green
algae to fix nitrogen 50
4.7 Growth Rate of Blue-Green Algae 51
4.8 Ecology and Factors Affecting Nitrogen-Fixing
Blue-Green Algae 51
4.8.1 Distribution 51
4.8.2 Nutrients for blue-green algae 51
4.8.3 Light 53
4.8.4 Temperature 54
4.8.5 pH 54
4.8.6 Desiccation 54
4.8.7 Oxidation reduction potential 54
4.8.8 Pesticides 55
4.8.9 Algal pathogens 56
4.8.10 Salinity 56
4.9 Amounts of Nitrogen Fixed by Blue-Green Algae 56
4.9.1 Under laboratory conditions 56
4.9.2 Under field conditions 56
4.10 Transfer of Fixed Nitrogen 58
4.11 Production of Vitamins and Growth Substances by
Blue-Green Algae 59
4.12 Mass Cultivation of Blue-Green Algae 59
4.12.1 Japanese technology 59
4.12.2 Indian technology 60
4.12.3 Chinese technology 62
4.12.4 Other methods 62
4.13 Algalization or the Application of Blue-Green Algae
to Soil 63
4.13.1 Methods of inoculating blue-green algae 63
4.13.2 Effect of blue-green algae on soil and rice yield 64
4.13.3 Effect of blue-green algae on crops other
than rice 73
 Page
5. FREE-LIVING NITROGEN-FIXING BACTERIA 75
5.1 Azotobacteriaceae. Taxonomy 75
5.2 Genus Azotobacter 75
5.3 Azotobacterin 75
5.3.1 Historical background 75
5.3.2 Production of azotobacterin 75
5.3.3 Methods of application of azotobacterin 77
5.3.4 Effect of azotobacterin on plant yield 78
5.4 Azotobacter paspali 80
5.4.1 Potentiality of nitrogen-fixation 81
5.4.2 Location of nitrogenase activity 82
5.4.3 Amounts of nitrogen fixed in the field 82
5.5 Beijerinckia 82
5.5.1 Taxonomy 82
5.5.2 Geographic distribution 82
5.5.3 Soil pH 84
5.5.4 Vegetation 84
5.5.5 Significance in soil improvement 84
5.5.6 Efficiency of nitrogen-fixation 84
5.6 Azospirillum 84
5.6.1 Isolation of Spirillum lipoferum 85
5.6.2 Taxonomy 88
5.6.3 Location of A. lipoferum 88
5.6.4 Nitrogen-fixation potential 88
5.6.5 Nitrogen-fixation rates 88
5.6.6 Temperature 88
5.6.7 pH 90
5.6.8 Oxygen 90
5.6.9 Response to inoculation 90

6. AZOLLA 93
6.1 Introduction 93
6.2 Taxonomy 93
6.3 Azolla-Azolla anabaena association 94
6.4 Reproduction 96
6.5 Nitrogen-Fixation 96
6.5.1 Effect of combined nitrogen 99
6.5.2 Nitrogen-fixation efficiency 99
6.5.3 Nitrogen-fixation and conversion factors 99
6.5.4 Release of fixed nitrogen by the algae 99
6.5.5 Photosynthesis of Azolla 99
6.5.6 Growth of Azolla 99
6.6 Total Nitrogen Input Estimates of Azolla 100
6.7 Environmental Factors Affecting Azolla 101
6.7.1 Water 101
6.7.2 Light 101
6.7.3 Temperature 101
6.7.4 pH 102
6.7.5 Season and soil conditions 102
 Environmental Factors Affecting Azolla (Cont.) Page
6.7.6 Inoculum density 103
6.7.7 Nutrition 103
6.7.8 Salinity and salt concentration 105
6.7.9 Pests of Azolla 106
6.7.10 Pesticides 106
6.8 Control of Azolla Pests 106
6.9 Fungal Diseases 107
6.10 Role of Azolla in Agriculture1 107
6.10.1 Azolla as green manure 107
6.10.2 Azolla grown in the field 108
6.10.3 Crop response to Azolla 109
6.10.4 Availability of Azolla nitrogen 110
6.10.5 Azolla cultivated under rice plants 111
6.10.6 Place of Azolla in rice rotation in Vietnam 111
6.10.7 Double narrow row method 112
6.10.8 Recommendations for Azolla cultivation 114
6.10.9 Nutrients for Azolla during summer114
6.10.10 Azolla and rice yields 115
6.10.11 Azolla as feed 116
6.10.12 Azolla as weed 117
6.10.13 Azolla as means of improving water quality 117
6.10.14 Other potential uses of Azolla 117

7. NODULATED NON-LEGUMINOUS PLANTS 119

7.1 The Macrosymbiont. The Plants 119
7.2 The Endophyte. The Microsymbionts 120
7.3 Cross Inoculation Groups 122
7.4 Nodules 122
7.5 Extent of Nitrogen-Fixation in some Non-Legumes 124
7.6 Other Non-Legume Nitrogen-Fixing Plants 126

8. SOME COSTS OF BIOFERTILIZERS 127

8.1 Legume Inoculants 127
8.2 Algal Inoculants 127

APPENDIXES

A. METHODS OF ASSESSING BIOLOGICAL NITROGEN-FIXATION 129

A.1 Introduction 129
A.2 Kjeldahl Method 129
15
A.3 N Method 129
A.4 Acetylene Reduction 129
B. RHIZOBIA 134
B.1 Recognition and Characterization of Rhizobia 134
B.2 Litmus Milk Reaction 135
B.3 Legume Seed Pelleting 135
B.4 Techniques for Quality Control of Legume Inoculants 137
 Page
C. FREE-LIVING NITROGEN FIXERS 143

D. BLUE-GREEN ALGAE 147

15
D.1 N Tracer 147
D.2 Determination of Ammonia in Cultures of
Blue-Green Algae 147
D.3 In Situ Assay of Nitrogen-Fixing Activity 148
D.4 Nitrogenase Activity of Blue-Green Algae 148
D.5 Silica Gel Plates 148
D.6 Chlorophyll (a) Content 148
D.7 Algal Biomass in Soil 149
D.8 Culture Solutions for Blue-Green Algae 149
D.9 Management of Algal Producation for Paddy Inoculation 154
E. AZOLLA 157
E.1 Guidelines of Azolla multiplication and
Utilization 157
E.2 Multiplication of Azolla on Large Areas 158
E.3 Utilization of Azolla 158
E.4 Nutritient Solutions used for Azolla Growth 159
F. CULTURE COLLECTIONS OF NITROGEN-FIXING ORGANISMS 161
F.1 Culture Collections of Rhizobium 162
F.2 Culture Collections of Blue-Green Algae 164
F.3 Culture Collections of Azotobacter and
Azospirillum 166
F.4 Culture Collection of Azolla 167

LIST OF REFERENCES 169

 LIST OF FIGURES
Page
1 Biological nitrogen-fixing relations 7
2 R. trifolii on the root hair of clover 17
3 Infection thread in cowpea nodule 19
4 Cross section of Vicia faba nodule 20
5 Cross section of soybean nodules 20
6 Bacteroids of R. leguminosarum in V. faba cell 21
7 Clover plants inoculated with single auxotrophic
mutants of R. trifolii 23
8 Diagram of simple fermentation unit for production of
rhizobial broth cultures 26
9 Response of broad bean to inoculation with R. leguminosarum 33
10 Response of berseem clover to inoculation with R. trifolii 35
11 Some nitrogen-fixing blue-green algae 47
12 Algal cultivation in trays containing soil 62
13 Effect of soil inoculation with blue-green algae on growth
of rice plants 69
14 Cells of A. chroococcum isolated from Egyptian soils 76
15 B. indica isolated from Sudanese soils 83
16 Pellicles of Azospirillum enrichments from maize roots and
colonies of A. lipoferum 86
17 Different types of Azospirillum sp isolated from Egyptian soils 87
18 Cells of A. lipoferum isolated from maize rhizosphere 87
19 Nitrogenase activity in non-sterile rice (IR8) soil systems,
inoculated with 3 strains of nitrogen-fixing bacteria 92
20 Azolla fern floating on water 93
21 Azolla pinnata fern 95
22 Blue-green algae Anabaena azollae 96
23 Life cycle of Azolla 97
24 Crop response to Azolla inoculation 110
25 Double narrow row method of rice cultivation 112
26 Infected cell from Myrica gale nodules showing endophyte
vesicles 121
27 Types of nodules of Purshia tridentata and Alnus glutinosa 123
28 Primary nodules (2 weeks after inoculation) in cortex and
initiation cof side vascular bundles 124
29 Azolla multiplication 157
 LIST OF TABLES

Page
1 Estimates of total nitrogen-fixationfor the year 1974 2
2 World nitrogen fertilizer production 3
3 Nitrogen-fixing bacteria 5
4 Inoculation rate and minimum number of rhizobia per seed
for Australia 29
5 Response of clover to R. trifolii and granulated inoculant 31
6 Effect of inoculation in presence of superphosphate on
broad bean yield and the total nitrogen content of the seeds 33
7 Grain yield (kg/ha) of Cajanus cajan in field experiments at
different sites in India 34
8 Weight of foliage (g) from plants inoculated with selected
Rhizobium strains in glasshouse trials 35
9 Effect of water stress on nodule number, size and acetylene
reducing activity of P. vulgaris plants inoculated with
two strains of R. phaseoli 38
10 Nitrogen fixed by pulses, forage, green manure and shade trees 41
11 Nitrogen fixed by different legumes under Egyptian conditions 41
12 Amount of nitrogen fixed by leguminous crops, and their
influence on a following cereal crop 42
13 Changes in soil nitrogen cultivated with legumes and non-legumes 42
14 Nitrogen fixed by three legumes in plastic-lined cinder plots 43
15 Nitrogen contribution by two Desmodium forage legumes in
association with pangola grass 44
16 Effect of green manure incorporated immediately before planting
on the yield of field beans (Phaseolus vulgaris) in
Sao Paulo State, Brazil 44
17 Heterocystous blue-green algae reported to fix nitrogen in
pure cultures 46
18 Non-heterocystous algae reported to fix nitrogen in pure cultures 46
19 Number of doublings per day of some blue-green algae 51
20 Effect of herbicides on dry weight, nitrogen and chlorophyll
content of T. tenuis 55
21 Amounts of nitrogen fixed by different blue-green algae
in liquid cultures 57
22 Build up of organic matter due to algae inoculation 58
23 Culture media used to propagate blue-green algae 60
24 Effect of inoculation of Aulosira fertilissima on average
growth and yield of a variety of paddy (T9) 64
25 Effect of T. tenuis on grain yield (kg/ha) of 498-2
“Aman” variety of rice 65
26 Grain yield of paddy (IR 8) due to algal inoculation at
different levels of nitrogen 65
 Page
27 Grain yield of rice variety ASD 5 due to algal inoculation 66
28 Effect of T. tenuis on the grain yield (kg/ha) of rice variety
BR 34 at Sabour in Bihar 66
29 Effect of inoculation with alga T. tenuis on the yield of rice 68
30 Effect of nitrogen and algal inoculation on rice yield in
the absence or presence of phosphate 70
31 Effect of certain fertilizers and the preceding crops on the
yield of rice 71
32 Effect of inoculation with algae and nitrogen fertilization
on nitrogen contents of rice plants in different soils 72
33 Yield response to algalization with T. tenuis and nitrogen
and phosphorus fertilizers 72
34 Effect of Azotobacterin on crop yields in various regions
of U.S.S.R. 78
35 Effect of Azotobacterin on crop yields on collective farms
in U.S.S.R. in 1949-1951 79
36 Effect of seed inoculation on grain yields of wheat in
field trials 81
37 Occurrence of nitrogen-fixing Spirillum in roots and soils
under various climatic conditions 85
38 Occurrence of nitrogen-fixing spirilla in malate enrichments
prepared from rhizosphere soil and roots of various plants 85
39 Nitrogenase activity on roots and in the rhizosphere soil
of tropical forage grasses 89
40 Nitrogenase (C H ) activity of S. lipoferum enrichment
2 2
cultures obtained with roots and rhizosphere soil from
various tropical crops 90
41 Field work on the inoculation of different crops with
S. lipoferum 90
42 Dry matter forage yields inoculated with S. lipoferum
compared to uninoculated 91
43 Field trials on 19 varieties or species of other plants
with and without inoculation with S. lipoferum 92
44 Characteristics of Azolla varieties common in Vietnam 94
45 Effect of combined nitrogen sources on acetylene reduction 98
46 Effect of various concentrations of nitrogen on the yield and
nitrogen-fixation capacity of Azolla 98
47 Nitrogen increase and growth rate of Azolla 100
48 Influence of pH on Azolla growth in a rice field water
solution under different conditions 102
49 Growth of Azolla in soils of varied pH 102
50 Azolla growth in different seasons and types of soils 103
51 Fresh weight, total nitrogen and acetylene reduction activity
of Azolla in P, K, C, and Mg deficient media and N-ammended
medium 104
 Page

52 Some principal insecticides for control of AzoZZa pests 107

53 Yield of Azolla and nitrogen in field and concrete tanks
in terms of fresh weight 109
54 Yield of AzoZZa spp in different localities
55 Effect of AzoZZa on yield of IR 8 and Supriya rice varieties
during Rabi 1977 110
56 Yields of rice and Azolla in paddy fields with Azolla
cultivated most of the year 113
57 Effect of various fertilizers during the summer period upon
the propagation and nitrogen-fixing capacity of Azolla 114
58 Straw and grain yield of rice variety IR 30 treated with
phosphorus, puddled and inoculated with Azolla 115
59 Chemical composition of Azolla 116
60 Effect of various proportions of Azolla along with commercial
feed on the body weight of chicks 117
61 Classification of non-leguminous nitrogen-fixing angiosperms 119
62 Number of species and distribution of nodulated non-leguminous
angiosperms 120
63 Cross inoculation in various Alnus sp 122
64 Accumulation of nitrogen by nodulated plants in rooting
media free of combined nitrogen 125
65 Field measurements of nitrogen-fixation in some non-legumes 125
66 Nitrogen-fixation assays 130
67 Cultural and biochemical characteristics of the major groups
of rhizobia and distinction from agrobacterium 134
68 Nodulation specificity of rhizobial species 135
 l. INTRODUCTION
1.1 GENERAL
In order to help meet the excalating demand for food resulting from
the dramatic expansion of the world’s population, mineral fertilizers and
pesticides have been used extensively to increase the yield of crops from
arable land. However, intensive agricultural methods have introduced
undesirable and sometimes catastrophic consequences by polluting air, soil
and aquatic systems as well as foodstuffs. Furthermore a matter of concern
is the high cost, in terms of fossil energy, of mineral fertilizers,
especially those supplying nitrogen, and the wasting of fertilizers.
It is recognized that methods based on the combined use of chemical
or physical factors and biological agents should improve soil production.
However, chemicals applied in agriculture as nutrients, amendments, growth
regulators, herbicides and disease control agents may alter soil and plant
microorganisms favourably or unfavourably.
The microbiological activity in the rhizosphere is partially dependent
upon climate and soil physical factors, and the latter can be manipulated to
alter soil and plant microorganism associations. The prevention of harmful
factors such as waterlogging, by good drainage, is a prerequisite for
efficient nitrogen-fixing symbiosis between Rhizobium and legumes. Tillage
methods affect many soil characteristics such as aeration, structure,
temperature and water regime, all of which affect the microbiological
balance.
Altering the rhizosphere flora by inoculation with certain organisms
has long been recognized as a practical possibility. The beneficial effect
of rhizobial inoculants on the yield and quality of crops and on soil
fertility is widely accepted, as also mycorhizal inoculation, provided the
proper environmental conditions are met. The use of other bacterial
fertilizers, e.g. azotobacter and phosphate-dissolving bacteria, has been
widely accepted in the Soviet Union and Eastern Europe.
Furthermore inoculation of non-legume crops by associative nitrogen-
fixing Spirilla is promising. Algalization and application of
azolla-anabaena systems have been evaluated and accepted for application
as biofertilizers in India, the Philippines and Vietnam.
The purpose of this bulletin is to describe methods applied to
utilize nitrogen-fixing systems in soil improvement and management and the
consequent increases in crop yields obtainable by these methods. The rest
of the integrated systems involved i.e. chemical and physical manipulations,
are characterized with respect to their interaction with biological
nitrogen-fixing systems.

1.2 GLOBAL STATUS OF NITROGEN

The most important element for all forms of life is nitrogen as it is
found in nucleic acids, proteins and chlorophyll. Accordingly, all forms of
live bacteria, fungi, green plants, and animals of all kinds cannot grow and
function unless they aquire nitrogen in an acceptable form.
 There is of course a vast amount of elemental nitrogen in gaseous
form in the earth's atmosphere, dissolved in the oceans, and in both forms
in soil and certain rocks. However, plants and animals cannot of themselves
make use of elemental nitrogen. Thus gaseous nitrogen is always present in
air spaces in plant tissues, but not utilized. Likewise animals inhale air
and reject the nitrogen.

It is only in the combined forms that most organisms can utilize

nitrogen; green plants grow satisfactorily if their roots are supplied with
nitrate or ammonium salts and under natural conditions they can usually
obtain these from the soil or from water if they are aquatics. Plants can
synthesize enzymes and hence proteins and other complex nitrogenous compounds
required for growth. Animals do not have this power and they acquire their
nitrogen chiefly by consuming proteins originally synthesized by plants.

1.3 FIXED NITROGEN

Fixation of atmospheric nitrogen is the major factor in the nitrogen
cycle. The natural processes though continuous are generally too slow in
localized areas of intensive crop production. Physical fixation by
lightening and rain, chemical fixation from volcanoes, natural or man-made
smog, effluents from industry and mechanical transport and biological
fixation has recently been estimated (Cast, 1976) and is presented in
Table 1.

From these data it appears that the amount of nitrogen which is

artificially fixed (i.e. man-fixed) is only about half of that fixed by
natural means. Nevertheless artificial fixation of nitrogen causes a
substantial modification of the nitrogen cycle in the natural ecosystem.

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of fixed nitrogen, some of which is derived from legume fixation. Non-
legumes also contribute a substantial proportion of naturally fixed nitrogen.

1.4 FERTILIZER NITROGEN

The world production of nitrogen fertilizers (Table 2) shows a trend
towards increased production, mainly in developed countries.

In Asia, the production is only half that of Europe, and of this,

Japan contributes approximately a third of the total. Fertilizer production
is low or nil in many regions of South East Asia and Africa.

For “bag” nitrogen i.e. industrially fixed nitrogen, it has been

estimated that for one unit of nitrogen two units of oil are required. Thus
if the cost of transport is included, the fertilizer nitrogen becomes too
expensive especially for developing countries. In addition, environmental
pollution resulting from the excessive use of fertilizer nitrogen, indicates
that the greater exploitation of biological nitrogen-fixation is essential.

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 2. BIOLOGICAL NITROGEN-FIXATION

2.1 INTRODUCTION
The importance of legumes in building and conserving soil fertility
has been recognized since the beginning of agriculture. With the development
of new techniques for detecting the ability to fix nitrogen such as Kjeldahl,
15N and acetylene (C2H2) reduction methods, an increased number of species
and systems able to fix nitrogen have been identified.

Such species and systems include free-living ones e.g. bacteria and
blue-green algae; symbiotic systems e.g. rhizobia-legume, angiosperm-
actinomycetes, and blue-green algae associations and associative systems e.g.
rhizosphere, phyllosphere and lichens. All these systems have one character-
istic in common: the ability to utilize nitrogen, but they differ in physiology,
metabolic structure and genetic characteristics.

2.2 FREE-LIVING NITROGEN-FIXING ORGANISMS

2.2.1 Bacteria
The free-living bacteria known to fix nitrogen can be classified as in
Table 3 (La Rue, 1977). The list contains organisms of soil, aquatic,
phyllosphere and rhizosphere habitats, photosynthetic aerobic, anaerobic and
facultative species. Collectively, they are capable of metabolizing a wide
range of carbon compounds. Some can grow at pH 3 others at pH 10.

Table 3 NITROGEN-FIXING BACTERIA

(La Rue, 1977)

Family Genera

Thiorhodaceae (Chromatiaceae) Thiocapsa, Chromatium

Athiorhodaceae (Rhodospirillaceae) Rhodospirillum, Rhodopseumonas
Hyphomicrobiaceae Rhodomicrobium
Chlorobacteriaceae Chlorobium
Spirillaceae Desulfovibrio, Desulfotomaculum
Azotobacteriaceae Azotobacter, Beijerinckii,Derxii
Entrobacteriaceae Klebsiella,Escherichia,Enterobacter
Corynebacteriaceae Corynebacterium
Bacillaceae Bacillus, Clostridium

2.2.2 Blue-Green Algae

Nitrogen-fixing blue-green algae (Cyanophyceae) use light energy
captured through photosynthetic processes to meet the energy requirements of
nitrogen-fixation. They are the only organisms that exhibit a higher plant
type of photosynthesis, i.e. oxygen production and carbon dioxide fixation,
and have species capable of nitrogen-fixation. (See chapter 4).
2.3 ASSOCIATIVE SYMBIOSIS BETWEEN FREE-LIVING DIAZOTROPHS AND OTHER
ORGANISMS
A great variety of associations exists between free-living diazotrophs
and other organisms (Fig. 1) (Burns and Hardy, 1975). Although many such
associations have been documented, there may still be some that exist
unnoticed or overlooked.

2.3.1 Loose Associations

Many free-living microorganisms participate in what may be considered
a loose association with other organisms. These do not involve structural
or morphological accommodation of the nitrogen-fixing organism, but consist
more of close physical contacts in which natural influence can be exerted
between the symbionts.
i. Interaction among microorganisms
This type of symbiosis is difficult to define with precision because the
Intensity and nature of the interaction is itself often subtle and transient.
A consensus of the literature however, testifies to an enhancement in
activity of free-living diazotrophs by microorganisms which do not fix
nitrogen. For example organisms capable of decomposing polysaccharides
have been observed to stimulate nitrogen-fixation by associated Azotobacter,
Beijerinckia and Clostridium, possibly supplying carbon sources.
ii. Phyllosphere
The phyllosphere association involving free-living diazotrophs and
aerial parts of higher plants have long been overlooked in nitrogen-
fixation studies. Yet as early as 1956 it was shown that the leaves
of tropical plants generally supported extensive surface populations of
microorganisms, among which aerobic diazotrophs were prominent (Ruinen, 1975).
Studies of tropical grasses show that these organisms exude biologically
significant concentrations of carbohydrates, but little or no nitrogenous
nutrients, thus providing a particularly suitable habitat for diazotrophs.
These associations have also been indicated in temperate crops and have
been shown to have the potential for a significant contribution to the
nitrogen economy of temperate zone woodlands.
Ruinen (1975) also enumerated the common genera of microorganisms detected
in the phyllosphere in different parts of the world e.g. Azotobacter,
Beijerinckia, Derxia, Agrobacterium, Pseudomonas, Xanthomonas, Azotomonas,
Mycoblana rubra, Spirillum, Myconostoc, Flavobacterium, Klebsiella,
Aerobacter, Bacillus, Clostridium, Nocardia, Cyanophyceae, Chlorophyceae,
lichens, bryophyta, and protozoa.

iii. Rhizosphere
The rhizosphere of various plants harbours high levels of nitrogen-fixing
bacteria and evidence for significant nitrogenase activity has been reported
for the rhizosphere of sugar cane, rice, grasses and marine angiosperms.

2.3.2 Associations With Animals

The scope of the participants in nitrogen-fixing associations has
recently been extended beyond the limits of the plant kingdom. The involve-
ment of higher animals is becoming increasingly well documented in reports
of nitrogen-fixing activity, using the acetylene reduction technique, by
the intestinal flora of both ruminants and non-ruminants. The animals
implicated include swine, guinea pig, humans, reindeer, goat, sheep, steer
and camel (Hardy et al, 1973).
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 It appears that Klebsiella may be involved in the nitrogen-fixation
found in human and swine faecal matter. However, possibly Escherichia coli
and Enterobacter cloacae are also involved.

2.3.3 Associations Involving Morphological Modification Or Accommodation

i. Leaf nodules
In these nodules, a high concentration of nitrogen-fixing Klebsiella
occurs
extracellularly in the crude subepidermal leaf protuberances of tropical
plants Pavetta, Psychotria, Ardisia and Dioscorea.

ii. Blue-green algae associations

Nitrogen-fixing blue-green algae are present in symbiotic association
with fungi (lichens), several bryophytes (liverworts) and pteridophytes
(water ferns), gymnosperms (cycads) and the angiosperm Gunnera.

The algal symbiont is always a Nostic or Anabaena species and, except for
Gunnera, it is extracellular though often localized in morphologically
specialized structures of the host plant. Moreover, the symbionts are
often morphologically and physiologically distinct from the free-living
species.

iii. Obligatory symbiosis

a. Non-legume angiosperms
These root nodule symbioses are sometimes referred to as an older
type, and are the most studied member of the group. They share
many characteristics with legume symbiosis but possess unique
qualities which clearly warrant separate classification.

Symbioses are known among 14 genera of angiosperms, in 8 families

and 7 orders. About one third of the more than 300 species are
reported to be nodulated and probably nitrogen-fixing, but the
actual fraction may be greater because many species have not been
examined.

b. Legumes
The family Leguminosae comprises three subfamilies: Mimosoideae,
Caesalpinoideae, and Papilionoideae with over 12 000 to 14 000
species. The exact incidence of nodulation and nitrogen-fixation
is not known but reports indicate that about 90o/ of the subfamilies
Mimosoideae and Papilionoideae and 33% of the Caesalpinoideae are
nodulated.

These species are associated with bacteria of the genus Rhizobium

and form characteristic nodules.

c. Tissue culture symbiosis

In an application of tissue culture technique to legume symbiosis,
Holsten et al (1971) described conditions for effective rhizobia
infection of callus tissue of soybean root origin. The resulting
symbiosis provides a possible test system for investigating various
facets of the legume symbiosis with minimal interference from plant
influence.
 The morphological changes which accompany the onset of nitrogenase
activity in callus tissue were found to parallel closely the changes
observed in intact nodule systems. Electron micrographs revealed
the presence of a structure similar in appearance to an infection
thread and also showed some of the intra-cellular bacteria to be
enclosed in membranous sheaths or vesicles. With time, infected
callus cells displayed the same dense population of microorganisms
typical of intact nodules.

2.4 BIOCHEMISTRY OF BIOLOGICAL NITROGEN FIXATION

2.4.1 Nitrogenase Enzyme System

The nitrogen-fixing enzyme, nitrogenase, which reduces nitrogen to
ammonia consists of two fractions:

i· the iron protein (Fe-protein or azo-ferrodoxin or component I),

ii. the iron-molybdenum protein (Fe-Mo-protein, or component II).
The two components catalyse nitrogen reduction only when combined and
do not do so alone. It is possible to cross-react nitrogenase Fe-protein
with Fe-Mo-protein from another organism to give active nitrogenase.

The Fe-protein is the smaller of the two and has a molecular weight
of 50 000 - 70 000 and is extremely sensitive to inactivation by oxygen,
losing over 70% of its activity after one minute of exposure to air. It is
cold labile and is made up of two identified subunits, and contains 4 iron
atoms and 4 acid labile sulphide groups, similar to ferrodoxin.

The Fe-Mo-protein is larger with a molecular weight of 100 000 to

300 000. It is less sensitive to oxygen than the Fe-protein. It is not cold
labile but is composed of two subunits. The Fe-Mo-protein of A. vinelandii
is crystallized.

2.4.2 Requirements For Nitrogen-Fixation

For nitrogen-fixation to occur the following requirements must be
Satisfied:
- a nitrogenase enzyme system,
- a source of adenosive triphosphate (ATP),
- a source of reducing power,
- a protective system for the enzyme from oxygen inactivation
- the rapid removal of nitrogen fixed from the site of nitrogen-
fixation to avoid inhibition of the nitrogenase.
 3. RHIZOBIA-LEGUME SYMBIOSIS

3.1 THE MICROSYMBIONT RHIZOBIUM

The genus Rhizobium contains those bacteria able to form nodules on

the roots of the family leguminosae. It also includes cultures which are no
longer invasive, but which have an authentic history or origin from an
invasive strain.

3.1.1 Taxonomy
In the 8th edition of Bergys' manual of determinative bacteriology
Rhizobium is given as one of two genera making up the family Rhizobiaceae
(Jordan and Allen, 1974). The second genus is Agrobacterium. Both genera
produce cortical hypertrophies on plants. Rhizobium forms nodules mainly on
roots of legumes and evidence is presented that rhizobia also produce
nodules on non-legumes e.g. Trema and Zygophyllum. Agrobacterium produces
disorganized galls on many kinds of plants.

The type species for the genus Rhizobium is R. leguminosarum Frank.

Species are recognized on the basis of host preferences. The following
species are recognized:

Species Preferred hosts

Rhizobium leguminosarum Pisum, Vicia, Lathyrus, Lens
R. trifolii Trifolium
R. phaseoli Phaseolous vulgaris, P. angustifolia
R. meliloti Medicago, Melilotus, Trigonella
R. lupini Lupinus, Ornithopus
R. japonicum Glycine max

In addition many strains of rhizobia which associate with cowpea and

lotus are now recognized as cowpea rhizobia and lotus rhizobia.

3.1.2 Morphology and Cytology of Rhizobium

Younger cells stain evenly with simple basic dyes (except that strains
of R. leguminosarum and R. trifolii often contain metachromatic granules).
Older cells generally take longer to absorb dye and have unstained areas of
polyhydroxy butyrate (PHB) which give the cells a banded morphology

Young cells are motile with flagella which are either polar or
peritrictious. Young rhizobia, while in culture media, are rod-shaped and
become bacteroid under certain conditions, similar to the forms of rhizobia
in the nodule.

Attempts to find heat resistant endospores among rhizobia, as claimed

at times to exist, have not been successful.

3.1.3 Cultural Characteristics of Rhizobium

Much of the growth of most fast-growing rhizobia after 3-5 days
consists of extracellular gum which may, in some cases, flow over the
larger part of the agar surface. The growth of R. leguminosarum, R. trifolii
and R. phaseoli is often clear as water or has differentiated into more
opaque areas set in clear surroundings. Some are evenly mucoid throughout.
Growth of R. meliloti is often more evenly opaque, even chalky white rather
than clear or milky.
 Colonies of slow growing strains do not become apparent for several
days; they require about 10 days to reach maximum size and do not produce
free-flowing gum. Pink pigmented rhizobia have been isolated from
Lotononis bainesii.

The incorporation of congo red dye (at a concentration of 0.0025%)

has been generally used (Vincent, 1970) as a differential media for rhizobia
and contaminants including agrobacteria. Generally, rhizobia do not absorb
the dye but the others do. But there are exceptions as some rhizobia strains
absorb the dye and the extent of absorption may be affected by the nature of
the medium and conditions of cultivation. Similarly, the addition of 0.0025%
bromothymol-blue provides a useful indicator of relatively small changes in
acidity or alkalinity.

Variation and dissociation in the colony type of rhizobia have been

encountered. Variants of "R" and "S" forms have been found to be either
invasive or not.

Yeast extract mannitol medium, generally known as medium 79 (Allen

1959), is suitable for growth of rhizobia. Most strains of rhizobia grow,
poorly on glucose peptone agar but some, R. meliloti and Agrobacterium,
make a reasonable degree of growth on this medium.

Growth of rhizobia in liquid media is generally evenly turbid and may

become extremely viscous due to the production of gum.

Reactions in litmus milk have often been taken to be distinctive

between R. meliloti (acid reaction) and the slightly alkaline reaction
found with other species. The production of a serum zone appears as a strain
specific rather than a species characteristic.

3.1.4 Physiology of Rhizobium

Detailed accounts of the microbiology (Vincent, 1977) and physiology

(Bergesen,1978) of rhizobia are reported. A few points of practical
significance are mentioned here.
i. Carbon source
In general, R. leguminosarum, R. trifolii, R. phaseoli and R. meliloti
make good use of all carbon sources. The slow growers fail to use
rhamnose, sucrose, trehalose, raffinose and dulcitol. Maltose, lactose
and organic acids vary a good deal between strains.

ii. Nitrogen source

That rhizobia cannot utilize free nitrogen has been invalidated, as
certain rhizobia strains fix nitrogen freely under defined conditions
(Pagan et al,1975).

Many strains can utilize nitrate or ammonium as the sole or supplementary

source of nitrogen but care should be taken to avoid a change in pH.

Strains unable to use inorganic combined nitrogen are generally satisfied

by the addition of a single amino acid. In strains that differ in their
ability to utilize certain amino acids, glutamic acid is generally
acceptable.
About 5 - 10 ppm nitrogen are required for optimum growth of rhizobia;
multiplication and respiration rates are augmented in media containing 108
ppm.
Urea is utilized by a large collection of representative strains and
species, and most show a moderate degree of growth on biuret.
iii. Vitamin requirements.
Thirty five out of 63 representative rhizobial strains need the addition
of one or more vitamins to the basal medium. The R. leguminosarum,
R. trifolii and R. phaseoli group (26 out of 31 strains) require one or
more of the vitamins biotin, thiamin and calcium pantothenate. When
R. meliloti, R. lupini and cowpea rhizobia need vitamin (9 out of 23)
they are satisfied with biotin. None of the R. japonicum strains tested
(6 strains) required any vitamin (Graham, 1963 b).

Nicotinic acid, pyridoxin, folic acid, p-amino benzoic acid, inositol,

B and riboflavine generally have no effect. Synthesis of certain vitamins
12
has been confirmed, e.g. B .
12

iv. Mineral elements

Iron is essential for rhizobia. Low concentrations of salts are also
beneficial.

Rhizobia generally require a total concentration of 0.5 m mol of calcium

and magnesium. The distinctive effects due to a shortage of each ion
become particularly apparent when levels of calcium are below 0.025 m mol
or magnesium 0.1 µ mol. The viability of the culture is reduced when
either element is deficient. Elongated and sometimes branched cells are
developed in magnesium-deficient cells. Cells with low calcium content are
irregular, swollen and roughly spherical (Vincent, 1977).

Strontium is a less efficient substitute for calcium for growth; neither

barium nor magnesium induce growth.

The need for cobalt for growth has been confirmed: 0.01 m mol is required
for maximum growth of R. japonicum. Cobalt deficiency prevents sufficient
synthesis of vitamin B which is necessary for the normal function of
12
methy-malonyl-Co-enzyme A mutase and leads to excessive production of
ribonucleotide reductase apo-enzyme.

Low concentrations, e.g. 0.1-1.0 m mol of zinc and 0.1-1.0 m mol of

manganese are required for rhizobia.

v. Oxygen
Graham (1963 a) indicated that none of the rhizobia tested grew
anaerobically.
However, although excellent growth was obtained at less than 0.01 atm of
oxygen, none grew without it at all.

vi. pH
In general all rhizobia grow within a pH range of 5.5-7.5. Acid tolerance
in decreasing order, is (R. lupini, R. japonicum, cowpea) >
(R.leguminosarum, R. trifolii , R. phaseoli) > (R. meliloti). Tolerance to
high pH is in the reverse order.

vii. Temperature
From data available it appears rhizobia grow over the range 0-50°C with
the optimum near 20-28°C. Among temperate strains, those of R. meliloti
are the most tolerant (36.5-42.5°C), being 8°C higher on the average than
those of R. leguminosarum and R. trifolii (31-38°C). In the collection of
rhizobia from tropical legumes (cowpea miscellany) the range is from
30-42°C.
3.1.5 Ecology of Rhizobia in Soil
For the greater part of their existence, rhizobia survive as free-
living bacteria in soil. They will survive and even grow in the absence of a
host legume. A remarkable phenomenon occurs when the root of an appropriate
legume is interposed into the environment of the free-living rhizobia. It is
a phenomenon of mutual recognition between the two compatible partners. The
legume plant root recognizes just the right kind of Rhizobium among all other
bacteria, including other rhizobia in the vicinity, and the Rhizobium in turn
recognizes just the right kind of legume root among all the other roots which
may occur in that environment (Schmidt, 1978).

3.2 THE MACROSYMBIONT. THE LEGUMES

The family Leguminosae is a group of flowering plants found in both
temperate and tropical zones. They range from small but widespread plants
such as clover, through flowering plants such as lupins and bushes such as
brooms, to shrubs and trees (e.g. Acacia). The family has been divided into
3 major subfamilies, the Papilionaceae, the Mimosaceae and Caesalpinaceae.

3.2.1 Cross-inoculation Groups

Groups of legume types that can exchange rhizobia with each other are
known as cross-inoculation groups. Seven of these groups are now recognized.
They are given below, with a list of the most important legumes in each.

i. Alfalfa group

Common name Scientific name

Alfalfa Medicago sativa
Button-clover M. orbicularis
California bur-clover M. denticulata
Spotted bur-clover M. arabica
Black medic M. lupulina
Snail bur-clover M. scutellata
Tubercle bur-clover M. tuberculata
Little bur-clover M. minima
Tifton bur-clover M. rigidula
Yellow alfalfa M. falcata
White sweet clover Melilotus alba
Huban sweet clover M. alba annua
Yellow sweet clover M. officinalis
Bitter clover (sour clover) M. indica
Fenugreek Trigonella foenumgraceum

ii. Clover group

Alsike clover Trifolium hybridum

Crimson clover T. incarnatum
Hop clover T. agrarium
Small hop clover T. dubium
Large hop clover T. procumbens
Rabbit foot clover T. arvense
Red clover T. pratense
White clover T. repens
Ladino clover T. repens (giganteum)
Subclover T. subterraneum
Strawberry clover T. fragiferum
 Berseem clover T. alexandrinum
Cluster clover T. glomeratum
Zigzag clover T. medium
Ball clover T. nigrescens
Persian clover T. resupinatum
Carolina clover T. carolinianum
Rose clover T. hirtum
Buffalo clover T. reflexum
Hungarian clover T. pannonicum
Seaside clover T. wormskjoldii
Lappa clover T. lappaceum
Bigflower clover T. michelianum
Puff clover T. fucatum

iii. Pea and vetch group

Field pea Pisum arvercse
Garden pea P. sativum
Australian winter pea P. sativum (var arvense)
Common vetch Vicia sativa
Hairy or winter vetch V. villosa
Horse or broad bean V. faba
Narrow leaf vetch V. angustiffolia
Purple vetch V. atropurpurea
Monantha vetch V. articulata
Sweet pea Lathyrus odoratus
Rough pea L. hirsutus
Tangier pea L. tingitanus
Flat pea L. sylvestris
Lentil Lens culinaris (esculenta)

iv. Cowpea group

Cowpea Vigna sinensis

Asparagus bean V. sesquipedalis
Common lespedeza Lespedeza striata
Korean lespedeza L. stipulacea
Sericea lespedeza L. cuneata
Slender bush clover L. virginica
Striped crotalaria Crotalaria mucronata
Sun crotalaria (Sunnhemp) C. juncea
Winged crotalaria C. sagittalis
Florida beggarweed Desmodium tortuosum
Tick trefoil D. illinoense
Hoary tickclover D. canescens
Kudzu Pueraria thunbergiana
Alyce clover Alysicarpus vaginalis
(no common name) Erythrina indica
Pigeon pea Cajanus cajan (indicus)
Guar (cluster bean) Cyamopsis tetragonoloba
Jackbean (horse bean) Canavalia ensiformis
Groundnut (peanut) Arachis hypogaea
Velvet bean Stizolobium deeringianum
Lima bean Phaseolus lunatus (macrocarpus)
Adzuki bean P. angularis
Mat bean P. aconitifolius
Mung bean P. aureus
Tepary bean P. acutifolius var latifolius
 Acacia Acacia linifolia
Kangaroo-horn A. armata
Wild indigo Baptisia tinctoria
Hairy indigo Indigofera hirsuta
Partridge-pea Chamaecrista fasciculata

v. Bean group

Garden bean, kidney bean,) Phaseolus vulgaris

navy bean, pinto bean )
Scarlet runner bean P. coccineus (multiflorus)

vi. Lupine group

Blue lupine Lupinus angustifolius

Yellow lupine L. luteus
White lupine L. albus
Washington lupine L. polyphyllus
Sundial L. perennis
Texas bluebonnet L. subcarnosus
Serradella Ornithopus sativus

vii. Soybean group

All varieties of soybean Glycine max (Soja max)

There are certain legumes that appear to require specific strains of legume
bacteria for effective inoculation. Some of these legumes are:

Common name Scientific name

Birdsfoot trefoil Lotus corniculatus

Big trefoil L. uliginosus
Foxtail dalea Dalea alopecuroides
Black locust Robinia pseudoacacia
Trailing wild bean Strophostyles helvola
Hemp sesbania Sesbania exaltata
Kura clover Trifolium ambiguum
Sanfoin Onobrychis vulgaris (sativus)
Crown vetch Coronilla varia
Siberian pea shrub Caragana arborescens
Garbanzo (chick pea) Cicer arietinum
Lead plant Amorpha canescens

3.2.2 Nodules
Dart (1977) traced the recognition of nodules to drawings of legume
roots in the sixteenth century, when Dalechamp (in 1586) remarked on the
abundant nodules on Ornithopodium tuberosum.

i. Infection
b. Pre-infection stage
The point of chief interest and dispute is the nature and
specificity of the curling reaction. Yao and Vincent (1969)
distinguished degrees of curling and found that the
characteristic shepherd's crook curling is only produced by
bacteria able to infect the root, whereas indole-acetic acid
causes a more generalized kind of deformation.
 Some workers claim that polygalacturonase plays a role in the
infection process, while others have questioned this premise.
Small amounts of the enzyme produced by the host and detected
in the rhizosphere are induced only by the homologous bacteria,
or bacterial polysaccharide fractions, and this enzyme plays a
part in infection.

Phytohaemagglutenins (or lectins) may be involved in the infection

process. These substances are proteins with specific affinities
for polysaccharides and with certain lytic properties, and they
are commonly found in legume seeds. Those from soybean,
agglutinate most soybean bacteria but not other rhizobia, and
could therefore possibly function in the specific attachment
of the bacterium to its host. However, the specificity of
other legumes is not general. Some workers have claimed that
lectins strip the bacterium of its polysaccharide envelop,
thus allowing a closer association between host and bacterium.

Recently, the involvement of the polysaccharide callose in the

infection process has been demonstrated. This B 1-3 linked
glucan is known to be formed at the tips of growing root hairs
and to be involved in some responses of plants to fungal invasion.

b. Zones of infection on roots

Infection of the legume root only occurs in certain transiently
susceptible zones. Nutman (1962) indicated that infection is
restricted at first to a few broad zones along the root, but
areas of much denser infection occur within such zones. Fig. 2
shows the attachment of a R. trifolii cell onto a root hair.

1.
4;

Lis- sW turApm ou rpc. i.00t wum O CJOAGL (X i?0 000)

(cours4pritaq b-2- linfugu)
c. Infection threads
Infection of root hairs, following the initial penetration
or invagination of the hair cell primary wall, proceeds by
the development of a tube whose inner surface is lined with
cellulose. It contains rhizobia lying end to end, usually
in a polysaccharide matrix. The development of this tube,
the infection thread, has been documented by time-lapse
cinematography (Nutman, 1971). It can be regarded as an
invagination of the root hairs and developing nodules.

Infection thread development in clover is associated with

increased density and streaming. In about 4 hours the
refractile infection thread sheath is visible and the root
hair nucleus, now doubled in size and with a prominent nucleus,
remains close to the tip of the growing thread. Should the
nucleus move away from the tip of the thread it will cease
growth and abort.

d. Nodule initiation
The infection thread penetrates through and between the root
cortex cell towards the stele (Fig. 3). Actual nodule initiation,
usually at the inner cortical tetrophoid cells, takes place near
diploid cells in the pericycle that has the potentiality for
lateral root formation. The close identity of nodule and lateral
root initials is shown by common genetic control and parallel
physiology. By changing root temperature or by delaying inoculation,
nodules can be induced to form at positions that would otherwise form
laterals. Nodules differ from roots in their requirement for some
substances elaborated in the shoot, whereas roots have simpler
needs. Nodules are integrated with the whole plant physiology
and are therefore susceptible to changes affecting nutrition
photosynthesis, translocation, photoperiodic and photochemical
effects.

e. Nodule structure
Histologically, the term "mature nodule" refers to that stage
when all tissues essential for nodule function are well defined
structurally. Four distinct zones of tissue differentiation
characterize the mature nodule. Figures 4 and 5 show cross-
sections in nodules of Vicia faba and soybean.

- The nodule cortex: This tissue comprises the outermost four

to ten cell layers of non-infected parenchyma. It is readily
distinguished from the root cortex by the compactness and
smaller size of the cells.

- The meristematic zone: This area constitutes the growing

zone of the nodule. It consists of cells in a state of
active division and growth. Within this region the process
of tissue formation is continuous as long as the nodule is
functional. All specialized tissues within the nodule
originate from cell differentiation within the meristem.

The position, size and duration of this area vary according

to the host species. The relative position of the area
determines the shape and to some extent the size of the nodule.
Three types of nodule meristem are commonly recognized with respect
to location:
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(1) hemispherical: such as in bean, cowpea and soybean
nodule where an equal rate of cell division throughout
the peripheral area produces radial enlargement;

(2) apical: as in the elongated, irregular shaped nodules of

alfalfa and clover;

(3) bilateral: exemplified by nodules on Lupinus spp wherein

horizontal growth in opposite directions produces tissue
hypertrophy which tends to girdle the root.

- The vascular system: This tissue serves for the transport of

plant nutrients to the nodule, and, in turn, the translocation of
nitrogenous substances and other metabolic by-products of the
rhizobia to the host plant.

Fully developed vascular bundles consist of xylem tracheids,

phloem fibres, sieve tubes and companion cells enclosed in
a parenchymatous sheath surrounded by a Casparian endodermis.

- The bacteroidal zone: This area, located in the central portion

of the nodule, is literally the bacterial tissue. It is the site
of the nitrogen-fixation process. Although non-infected cells are
included, the term applies principally to the infected host cells,
and stems from the fact that rhizobia therein often assume
pleomorphic (bacteroidal) shapes (Fig. 6).

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(cotALOW 2bLsuc)
 3.3 GENETIC ASPECTS OF NODULATION AND NITROGEN-FIXATION BY
LEGUME-RHIZOBIA ASSOCIATION
3.3.1 The Macrosymbiont. Legume Host
The host is involved in the processes of infection,nodule development
and fixation. A conservative estimate is that there are 10 host genes in the
plant involved in fixation and they are liable to mutate (Holl and La Rue,
1976).Most genetic analyses have been carried out on soybean (Glycine max),
clovers (Trifolium spp) and pea (Pisum sativum).The genes rj1, rj2, rj3 and
rj4 are known in soybeans. In red clover, genetic analysis showed the presence
of the genes r, i1, ie, n and d. The genes Sym.1, Sym.2, Sym.3, no, and nod
were identified in peas. These genes modify the response of host to rhizobia,
ranging from reduction of efficienty to absence of nodulation.

3.3.2 The Microsymbiont. Rhizobium

i. Loss of symbiotic ability

a. Resistance to certain factors

Partial or full loss of symbiotic ability in some strains

of rhizobia during growth in culture media has been observed
by many investigators. The presence in the medium of
inhibitory levels of amino acids has usually led to
ineffective strains of rhizobia (Hamdi, 1968, 1969).
The prevalence of ineffective clover-nodulating rhizobia
in sandy acid soils has been ascribed to a build-up
of ineffective mutants that are tolerent to metal ions
of manganese, copper or aluminium (Holding and King, 1963).

Bacteriophages exert selective effects on variant types

during growth of rhizobia, e.g. R. trifoli where a high
portion of phage-resistant mutants of effective strains
have been shown to be ineffective (Kleczkowska, 1971).

b. Antibiotic resistance
A streptomycin-resistant marker has been the one most
frequently used in genetic or ecological experiments, or
for biochemical study of associated blocks in symbiosis
(Schwinghamer, 1977). Resistance to streptomycin occurs

independently of the change in symbiotic ability (Obaton,

1971).

c. Auxotrophy
Lorkiewicz et al (1971) noted a wide range of amino acid
or vitamin requirements among auxotrophs isolated from an
irradiated effective strain of R. trifioli, but all the
auxotrophs were non-infective, parasitic or ineffective
and all prototrophic revertants examined remained defective.

Abdel-Wahab (1977) showed that auxotrophic mutants of

R. trifioli of histidine, leucine. lysine, adenine and
uracil were ineffective (Fig. 7). However, auxotrophic
mutants of arginine, methionine or tryptophane were effective.

ii. Genetic transfer within the genus Rhizobium

Several reports dealing with evidence of transformation, transduction
conjugation,and plasmid transfer within the genus Rhizobium were reviewed
by Schwinghamer (1977).
 N,

N
. . Nit-
i
m-
lior ".

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-id_

_.,
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4
-

7 -
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Fig. 7 Clover plants inoculated with single auxotrophic mutants of R. trifolii

C. control (non-inoculated).
W. inoculated with R. trifolii wild type strain
A. " " " Agrginine-less mutant
T. " " " Tryptophane-less mutant
M. " " " Methionine-less mutant
H. " " " Histidine-less mutant
Le. " " " Leucine-less mutant
Ly. " " " Lysine-less mutant
D. " " " Adenine-less mutant
U. " " " Uracil-less mutant
(contributed by S.M. Abdel-Wahab)
3.4 LEGUME INOCULATION
Inoculation is defined as the addition of effective rhizobia to
leguminous seeds prior to planting for the purpose of promoting symbiotic
nitrogen-fixation. Pure cultures on agar were first introduced in 1896
by Nobe and Hiltner (Date and Roughly, 1977), but later sterilized soil
impregnated with rhizobia was used and then impregnated peat.

There is little doubt that inoculation is necessary in the majority

of agricultural soils throughout the world. In particular inoculation is
necessary under the following four conditions.
i. when a leguminous plant has not been previously cultivated;
ii. if the leguminous crop that was grown previously on the land
lacked good nodulation;
iii. in a rotation cycle accompanying, or following, a non leguminous
plant, and
iv. when the land has been abused or reclaimed from degraded conditions.

There are now six types of inoculants in general use:

(1) moist peat powder, (2) liquid or broth, (3) agar or bottle culture,
(4) oil dried rhizobia in vermiculite, (5) lyophilized rhizobia in talc, and
(6) a granular type of peat inoculant.

3.4.1 Strain Selection

The selection of highly effective rhizobia strains is one of the
principal obligations of the inoculant producer. A strain must be able to
form effective nitrogen-fixing nodules on the hosts for which it is
recommended under a wide range of field conditions. Other necessary
characteristics include competitiveness in nodule formation, survival and
multiplication in the soil, particularly in the absence of the host.
Additional criteria include prompt and effective nodulation over a range of
root temperatures; ability to grow well in culture; to grow and survive in
peat and to survive on the seed; tolerance to pH and pesticides, and
nodulation in the presence of combined nitrogen.

3.4.2 Collection of Strains

Date (1976) indicated two approaches to the collection of suitable
strains of Rhizobium for new legumes or the establishment of a new
collection of strains.
i. if the legume species is a new introduction, nodules should be collected
at the same time as the plants so that microbiologists can isolate the
Rhizobvum and subsequently provide it in the form of inoculum;
ii. uninoculated seed of the new species can be planted at a number of sites
in the hope that native strains will form effective nodules from which
suitable Rhizobium strains can be isolated.

3.4.3 Evaluation of Strains

Rhizobia for legume inoculants should be evaluated by:

i. screening for effectiveness in nitrogen-fixation;

ii. assay of the selected effective strain for nitrogen-fixing ability
under field conditions, including competition for nodule sites,
persistence and colonizing ability;
iii. testing for pH and pesticide tolerance, survival in culture and on seed.
 For controlled environment and glasshouse conditions aseptic culture
of plants in tubes or sand jars, as described by Vincent (1970), provides a
satisfactory means of ranking strains for effectiveness. Undisturbed soil
core samples (collected by driving an empty can into the ground) can also be
used.

Field trials provide the ultimate test of a strain’s ability, since

the proportion of nodules formed and the amount of nitrogen fixed are the
end result of many interacting factors. Effectiveness is measured either
directly by determining the amount of nitrogen fixed or indirectly by
measuring plant dry weight.

3.4.4 Bacteria Propagation

The submerged culture technique of growing rhizobia is now used by
many producers of legume inoculants. Culture vessels or fermenters range
from pyrex carboys to specially built tanks holding a few thousand litres
of liquid medium. The starters are prepared as follows. Growth from 3-5
days of test tube cultures of the various strains is transferred into special
flasks containing sterile yeast extract or mannitol agar medium. After 3-5
days incubation at 25°C, the starter flask is ready for use. The bacteria are
washed from the surface and this suspension constitutes the seed or starter
for transfer to a 45 litre (10 gallon) fermenter.

The fermentation medium for growth of rhizobia varies depending upon

the species of rhizobia to be grown. Basically, it is a yeast extract or
sucrose, and mineral salts medium. The medium is prepared and sterilized
in the individual fermenters. After cooling to 27°C, the starter culture is
added. Sterile air is introduced and dispersed into the bottom of the vessel
through small perforations in a stainless steel sparger. The average
fermentation cycle is 72 hours. At the end of this period the liquid culture
usually contains approximately 5 000 x 106 cells/ml.

The liquid cultures from the 45 litre fermenters are used as starter
or seed cultures for 1 800 litre fermenters. Again, 72 hours are required
to attain the desired bacteria count of 5 000 x 106 cells/ml.

Fermenters vary from elaborate large ones (1 000-2 000 litres) to

simple flasks or drums (10-100 litres). Good serviceable fermenters can be
made from steel drums fitted with inlet and outlet tubes, inoculation and
sampling ports (Fig. 8) (Date and Roughly, 1977).

3.4.5 Carriers of Rhizobia

The first artificial inoculants were liquid bacterial cultures added
to seed or directly to soil. Although the results were frequently unsatis-
factory, broth cultures of Rhizobium suspensions washed from agar medium
continued to be used. However, critical comparisons of liquid cultures
with peat inoculants consistently confirmed the superiority of the latter.
Liquid cultures seem to lack the protection afforded by peat to rhizobia on
seed following inoculation.

Many attempts ahve been made to produce inoculants other than peat
based ones such as: 1) peat containing carbon black; 2) soil plus wood
charcoal; 3) peat or soil ammended with materials such as lucerne meal,
ground straw, yeast and sugar; 4) Nile silt supplemented with nutrients;
5) soil plus coir dust or soybean meal. Other materials have been tested
as carriers of rhizobia: vermiculite, decomposed perlite and rice husk
composts, ground rock phosphate, coffee husk compost; cob-earth (milled and
decomposed), ground maize supplemented with nutrients; finely ground bagasse
which has been found suitable for soybean rhizobia; coal-based inoculant,
(the best results were obtained with coal) bentonite, lucerne(CBL at 2:2:1)
at a moisture content of 50%; and eat moss discs.

In spite of all attempts to substitute the peat inoculants they still

remain the best carriers. Most of the other potential carriers have been
studied in detail but they do not seem to possess superior properties to
peat. Also some of the other materials are unavailable in many countries.
However, it is clear that the search for new carriers has often been prompted
by the lack of suitable local peat, and that the materials investigated were
cheap and readily available in the areas concerned.

Fig. 8 Diagram of simple fermentation unit for production

of rhizobial broth cultures
(Date and Roughley, 1977)

1 Compressor for air supply. 2 Filter for sterilization.

3 Fermentation unit. 4 Outlet. 5 Inoculation port.
6 Broth stirred by air bubbles. 7 Sampling port.
8 Pressure reducing diaphragm.
i. Prior treatment of carriers
a. Source and character of peat
The most important factors to consider before selecting a
particular peat as a carrier are the source and character
of the material. Whereas the choice may be aided by
chemical analysis, actual multiplications of Rhizobium
strains in the peat are essential.

Most peat carriers have a high organic matter content but

this is not an essential feature for good inoculant.

b. Drying of peat and particle size.

Drying of the harvested peat is an important step in its
preparation. It has been shown, with Australia peat, that
the final drying temperature should not exceed l00°C in
order to avoid the formation of toxic degradation products
and excessive rises in temperature when the peat is
subsequently wetted by the addition of broth culture.
Burton (1967) described the production of high quality
inoculants by flash drying the peat carrier with hot air
at 65°C to a moisture content of 7%. Dried peat is
usually ground in a hammer mill. In Australia, the best
results are obtained with peat carriers that pass through
a 200 mesh sieve (Roughley, 1970). Whereas a small particle
size is considered important by many inoculant producers,
good results have been reported by Van Schreven (1970) with
peat sieved through a 2 mm screen. Peat and soil carriers
used by Indian workers, were ground to pass a 60 mesh sieve
(Iswaran 1969)
c. pH and moisture content
Most peat deposits, with the exception of the Mount Gambier
peats used in Australia, have a low pH and require the addition
of a neutralizing agent before use. Peat carriers are usually
adjusted to pH 6.0-7.0 with calcium carbonate. Calcium carbonate
is better than sodium or potassium carbonates.

A final peat moisture content of 40-50% after adding rhizobia

appears favourable for most peats used as carriers. Moisture
contents below 30% or above 60% are generally unfavourable for
survival. However, for sterile peat a 60% moisture content
was recommended by Roughley (1970).

ii. Containers
The containers in which carriers are packed are important since their
gas exchange and moisture properties may have a marked effect on the
quality of inoculants. Bottles plugged with cotton wool and covered
with cellophane have been used in Holland. Bags of low density polyethylene
are used in Australia (0.038-051 mm gauge), while high density ones of
0.31-0.32 mm gauge are considered suitable for carriers in New Zealand
and South Africa.

iii. Sterilization of peat

There is evidence that slow growing rhizobia, e.g. cowpea, soybean and
lotononis may survive only poorly in a non-sterile peat that will never -
theless support survival of fast growing strains (Roughley and Vincent,
1967). Whereas sterilization may not be essential for all peats, for
some it is a pre-requisite in order to ensure success with various
Rhizobium strains.
 Steam is most commonly used for sterilization which requires 1-4 hours
at about 120°C or 3 hours at 125°C. Dry heat can also be used for 1 hour
at 120°C. (It should be noted however that Australian workers have found
excessive heat treatment of peat detrimental to subsequent growth and
survival of rhizobia).

Roughley and Vincent (1967) reported excellent survival of rhizobia

in gamma-irradiated peat (5 x 106 rad) and this method is now used for
all inoculants produced in Australia. However, gamma-irradiation does
not appear to completely sterilize the peat and certain microorganisms
seem remarkably resistant to doses up to 2.4-2.8 x 106 rad (Anellis et al,
1973).

Gaseous sterilization of peat has been tested in South Africa using

Etox (90% ethylene oxide + l0% carbon dioxide) at 500, 750 and 1 000 mg/l
for 8 and 16 hours respectively. A common characteristic of all Etox-
treated peat is a marked decrease in number of rhizobia shortly after
inoculation
(Strijdom and Deschodt, 1976).

3.4.6 Inoculant Preparation

The next step in the production of legume inoculants is to blend the
liquid culture with the carrier medium. The freshly made inoculant is placed
in shallow layers and cured at room temperature for several hours before
packing. This dissipates heat and allows more intimate wetting. Super-
ficial drying of the peat lessens the risk of mould growth, but if the peat
is to be held longer than a few days at this stage, it should be covered
loosely with polyethylene sheeting or transferred to large polyethylene bags
or covered containers until packed.

3.4.7 Standards of Inoculants

The relationship between the number of viable cells put on the seed
and success in securing nodulation is not simple. When conditions are
extremely favourable, the application of a few rhizobia can result in rapid
colonization of the developing root and plenty of nodules. On the other
hand when conditions are unfavourable or there are numerous other rhizobia
already present, an extremely heavy inoculum may be necessary to achieve
success.

Table 4 illustrates accepted standards for different inoculants in

Australia (Date and Roughley, 1977).

3.4.8 Multistrain Inoculants

Multistrain inoculants contain either strains from two inoculation
groups, e.g. strains for clovers and medics, or a mixture of strains from
the one group. The first type is used to simplify distribution but it is
not recommended because of the reduced numbers of rhizobia of each host group.
The second type is used to provide an effective inoculant for the wider range
of host plants, to offset the dangers of undesirable variation resulting in
loss of essential characteristics.

3.4.9 Tests for Control of Inoculants

Several tests are applied to control the quality of inoculants:

i. qualitative tests for immediate detection of contamination or wrong strain,

in the broth culture (pH, agglutination, Gram smear, glucose, peptone agar
and yeast mannitol agar);
ii. quantitative tests in broth: total and viable count;
iii. quantitative tests on peat culture: surface plate count on Congo red
yeast
mannitol agar and plant infection counts.

Table 4 INOCULATION RATE AND MINIMUM NUMBER OF RHIZOBIA PER SEED FOR AUSTRALIA1
(Date and Roughley, 1977)

Inoculation
Seeds rate Rhizobia
Host species number/kg Kg seed/ minimum
70g number/
Peat2 seed3

Lotonis bainesii 3 840 000 5 110

Lotus pedunculatus 1 940 000 5 720
Trifolium repens 1 650 000 5 830
Stylosanthes humilis 440 000 5 3 180
Medicago sativa 430 000 10 1 640
Trifolium subterraneum 154 000 10 4 540
Macroptilium atropurpureum 88 000 10 8 000
Vigna sinensis 15 000 25 18 700
Glycine max 11 000 25 25 400
Lupinus angustifolius 7 500 25 37 400
Phaseolus vulgaris 4 000 25 70 000
Pisum sativum 3 100 25 90 000

1/ Inoculant Research and Control Service tested peat cultures.

2/ These values are higher than reported by Vincent (1970).
3/ For cultures in sterilized peat meeting the current standard at the
end of the expiry period, 100 x 106 rhizobia/g, except for Lotononis
culture which is 30 x 106/g.
For cultures in non-sterilized peat (min.standard expiry 30 x 106/
g),
the number of rhizobia/seed would be 10 fold fewer than listed.

3.4.10 Inoculant Application

There are two main methods of inoculant application: direct and
indirect. Direct methods include putting inoculant into seed before sowing
by dusting, slurry or pelleting. Indirect methods involve sowing seeds
without inoculation and the inoculant is then applied into the soil in liquid
or granular form.

i. Dusting
Manufacturers used to recommend that inoculant need only be mixed with
the seed immediately before sowing. By this method some cultures adhere
to the seed by lodging in the micropyle and in scratches and
irregularities
on the testa, and by electrostatic attraction. However, much of the
inoculant tended to fall off especially when the seed was passed through
machinery. Therefore dusting is no longer used.
ii. Slurry inoculation
By this method the culture is applied as a water suspension, or
alternatively, the inoculant is mixed with moistened seed. However the
seed must then be dried (not in direct sunlight), and while it is drying
part of the inoculant tends to fall off. A large amount of the inoculant
can be attached to seed by using an adhesive in the slurry, e.g. l0%
sugar solution, gum arabic or cellofas A (carboxymethyl cellulose).

iii. Seed pelleting

Pelleting legume seed with lime provides a means to protect rhizobia from
both acid soils and fertiizers. Pelleting involves mixing the inoculum
with an adhesive, then putting the mixture onto the seed. The seeds
are then rolled in a coating material. Adhesives usually used include
synthetic or vegetable glues, gelatin, various sugars and honey.
Whatever adhesive is used, it should have sufficient tenacity to
prevent the coating material from sloughing off, which could lead
to blockage in sowing machinery and also be detrimental to plant
establishment. Coating materials tested include calcium carbonate in
many forms, dolomite, various grades of gypsum, bentonite and other
clay minerals, superphosphate, rock phosphate and other phosphorous
compounds, titanium oxide, soil humus, talc and activated charcoal. The
main requirements for a good general purpose pelleting material are that
it should be relatively close to neutrality and be finely ground (90% at
least should pass through a 300 mesh sieve according to Brockwell, 1977).

iv. Pre-inoculation
Seeds can be pre-inoculated by seed merchants prior to sale or
Distribution to farmers. This implies a period of storage of the
inoculated seed before it is sown.
A seed impregnation technique has been devised to extend the life of
inoculum by introducing rhizobia beneath the seed coat where they are
protected from drying and other adverse environmental conditions.
Thompson, Brockwell and Roughley (1975) reviewed legume seed pre-
inoculation in the USA and Australia, as a result of which they
recommended that the date of preparation be displayed on all batches
of pre-inoculated seed. From that date the expiry period of one month
should be adhered to by all concerned.

v. Drenching method of inoculation (liquid inoculants)

Hely et al (1976) described a pressure controlled, commercial-scale,
spray as a legume inoculation technique. It was developed for the
accurate placement of suspensions of nodule bacteria into soil beneath
seeds and for the application of greater amounts of inoculum than can
be carried on seed inoculated according to the present practice.

vi. Granular inoculants

Fraser (1966, 1975) developed a granular type inoculant. Columnar beds
of the granules together with a synthetic medium, were inoculated with
R. meliloti or R. trifolii and allowed to incubate for 7 days. A mixture
of milk and sucrose was then percolated through them, and after draining
off the excess liquid they were spread out to dry at room temperature.
Granules with counts ranging from 200 to 500 x 106/g are produced by
this method, and storage tests indicate that at 20°C, a shelf life of
two years is possible before the number of rhizobia falls below the
required 300/seed. There are relatively few contaminants on the
granules, and owing to the low moisture content they are unable to
multiply during storage. In air tight containers, however, moisture
accumulates on the surface of the granules and this allows moulds to
develop. Comparative data on response of clover to the inoculant is
presented in Table 5.
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In another study, Aziz (1978) developed a granulated inoculant for

R. phaseoli, using a pharmaceutical tablet coating machine, sodium
carboxymethyl, cellulose and calcium sulphate. Response to the
granulated inoculant was significantly higher than to other methods,
e.g. seed pelleting.

Burton (1976) developed granulated inoculants from peat. With this

method chemical treatments can be applied to the seeds without fear
of killing the rhizobia. It is suitable for culturing soybeans in
hot sandy soils or for aerial sowing of legumes.

3.4.11 Response to Inoculation

In short, the beneficial effects from inoculation are summarized as
follows:
early nitrogen starvation is prevented; the demand by leguminous crops upon
soil nitrogen is reduced; crop yield as measured in foliage or fruit is
increased; the quality of the crop is improved; and finally, readily
decomposable, rich, succulent green manure is made available for soil
improvement.
Some examples of the response of different crop legumes to inoculation
are given below.

i. Glycine max (Soybean)

Inoculation of soybeans with effective strains of R. japonicum may increase
yields and seed protein percentage, when rhizobia are not already present.
Kang et al (1975) showed that for maximum yield and to avoid stunting in
early growth, the addition of 30 kg N/ha was necessary, even in well
nodulated plants in Nigeria. Without inoculation, 60 kg/ha were required.

Various reports give the response of soybean to inoculation in Cameroon,

Cuba, Egypt, Ghana, India, Iraq, Madagascar, Romania, Rwanda, Sierra Leone,
Tanzania and Zaire. It is of interest to note that in Tanzania, Chowdhury
(1975) found that although soybean had never been planted in certain fields
near Morogoro, nodules were found on uninoculated plants. Nodulation was
improved by inoculation but in some cases nodule weight was decreased.
In another series of studies Ham, Lawn and Brun, (1976) showed that no
significant response to inoculation was obtained when soybean was
inoculated and planted in soils containing naturalized R. japonicum.
Comparison of seed yields of non-nodulating isolines indicated that 20-41%
of the yield of nodulating isolines was attributable to nodulation.
ii. Arachis hypogea (groundnut)
Dart (1978) indicated that Arachis hypogea has a great potential for
nitrogen fixation. The following is the result of an investigation into
the symbiotic characteristics of 48 groundnut germplasm entries 85 days
from planting.

Range
Nodule number 247 - 628
Nodule weight 0.3 to 0.75 g/plant

Nitrogenase activity

mmol C H plant n-1 36 to 176

2 4
mmol C H g/wt nodule n-1 95 to 386
2 4

iii. Vigna unguiculata (cowpea)

Inoculation of cowpea is seldom necessary in the major growing areas,
e.g. Western Nigeria, as there is usually an abundance of indigenous
strains capable of good nodulation. The results of Kang et al (1975)
support this view. However, other reports (Gargantini and Wutke, 1960)
indicate that a well nodulated cowpea crop gained about 73 kg N/ha from
nitrogen fixation.

iv. Phaseolus vulgaris (green bean)

These plants and especially their symbiotic associations are sensitive
to water stress, soil acidity and many pests and diseases. The host has
specific Rhizobium requirements and inoculation is often essential.
Inoculation with appropriate rhizobia may double the yield and produce
15% more than crops provided with 50 kg N/ha (Drobereiner and Campelo,
1976).

v. Cicer arietinum (chickpea)

A significant response of Cicer arietinum to inoculation has been
reported; for instance, increases in yield of up to 31% under rainfed
conditions. However, in some locations, no significant response has been
observed.

In the U.S.S.R. inoculation of chickpea has increased its areal

mass by 25-36%. The nitrogen contribution from the symbiosis has been
calculated to reach 41-60% for effective strains of rhizobia.

Brockwell and Gault (1972) evaluated the response of 21 lines of

Cicer arietinum: all plant lines responded to inoculation with CB 1189;
in terms of dry matter, 16 of the 21 responses were significant; on the
basis of nitrogen content, all of the resonses except one, (line 19)
were significant.

vi. Vicia faba (broad bean)

Broad bean showed a response (Table 6) to inoculation under Egyptian
Field conditions (Taha et al, 1967).Figure 9 illustrates the type of
Response obtained under greenhouse conditions.
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Subba Rao (1976) indicated that inoculation of Lens culinaris increased
the yield by over 70% in some locations, but not in others.
Applications of P O with or without inoculation significantly increased
2 5
yield. In Egypt, Taha et al (1967) reported a significant response by
lentils to inoculation.

viii. Desmodium
Under conditions in western Kenya, Desmodium uncinatum did not respond
well to inoculation (Keya and Van Eijnatten, 1975). Seeds pelleted with
lime, rock phosphate and gypsum were planted in potted field soil and in
plots. In the pot experiment, inoculated seeds showed an initial
advantage, but did not maintain it beyond 20 weeks. The field
experiment showed no advantage from inoculation because of infection by
indigenous, ineffective strains of rhizobia.

ix. Cajanus cajan

In field experiments conducted in 1967 at Pantnagar and in 1972 at
Kanpur, neither Rhizobium inoculation nor application of nitrogen (40-80
kg/ha), P O (50-150 kg/ha), or K 0 (25-50 kg/ha), affected yield
2 5 2
whether individually or in combinations (Subba Rao, 1976). Pelleting
with lime increased the yield significantly only at one location
(Table 7).

x. Clover
Hastings and Drake (1972) isolated the rhizobia R. trifolii from five
different New Zealand soils. Eleven strains of rhizobia were inoculated
into white and subterranean clovers grown in the different soils in pot
experiments. The results (Table 8) show that all strains were effective
for red and white clover, the best strains being 2666, 2153, 2163 and
2641. With subterranean clover, strain 2121 was considerably better
than any other strain. Figure 10 shows the type of response of berseem
clover to inoculation under greenhouse conditions.

Table 7 GRAIN YIELD (kg/ha) OF CAJANUS CAJAN IN FIELD

EXPERIMENTS AT DIFFERENT SITES IN INDIA, WINTER 1972-3
(Subba Rao, 1976)

LOCATION
Hissar Badnopur Bangolore Indhiana
Treatment soil pH 8.0 soil ph 8.0 soil pH 6.2 soil pH 7.6
Var. T-21 not reported Var. S-5 Var. T-21

Uninoculated 2 405 786 536 1 730

(control)
Rhizobium 2 847 882 569 1 720
Rhizobium + talc 3 030 861 639 1 410
pelleting
Rhizobium + charcoal 2 638 852 549 1 510
pelleting
Rhizobium + lime 2 880 849 511 2 010
pelleting
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3.4.12 Factors Affecting the Success of Inoculation
There are many factors which can interfere with the success of
inoculation and hence nitrogen fixation. The amount of nitrogen fixed by the
legume rhizobia symbiotic system is influenced by many chemical and physical
factors of the environment which may affect the host plant, the Rhizobium
and the development and effective functioning of nodules.

i. Soil atmosphere
The composition of the gas phase may differ considerably from that of
the atmosphere, because there is more carbon dioxide and less oxygen
in the soil due to respiration of microorganisms and plant roots.

ii. Atmospheric nitrogen

In general the concentration of nitrogen gas in the soil is
Sufficient to allow nitrogen fixation to take place without
limitation. Under waterlogged conditions, there will be a lower
concentration of nitrogen gas and, at the same time, the oxygen level
will be too low to permit normal plant growth.

iii. Oxygen
For the formation and functioning of nodules, a liberal supply of
oxygen is necessary. In the soil, the oxygen level is usually well
below 20% and at this level both nodule formation and functioning
will be suboptimal. Moreover, when there is excessive water, there
may be a temporary reduction in nitrogen-fixing activity because the
thin layers of water surrounding the nodules will cause a reduction
in the oxygen supply.

iv. Carbon dioxide

Wilson (1940) reported that an increase of carbon dioxide in the
atmosphere stimulated nitrogen fixation by nodulated clover plants
and this was attributed to an increase in photosynthesis. The
beneficial effect on nodulation and nitrogen fixation of aerating
legume roots growing in water cultures, with air containing 4% carbon
dioxide was reported by Mulder and Van Veen (1960).

Hardy and Havelka (1973) showed that carbon dioxide enrichment for
soybean increased nitrogen fixation more than five-fold by doubling
nodule specific activity, doubling average nodule mass, and extending
the exponential phase.
v. Soil pH
A great deal of variability in pH tolerance has been found among
rhizobia. Vincent (1965) reviewed the effect of pH on rhizobia and
concluded that R. meliloti is acid sensitive, R. trifolii is less
so, and R. japonicum is acid tolerant (e.g. as low as pH 3.5).

vi Temperature
Temperature is one of the important factors affecting the survival of
rhizobia in soil. R. meliloti strains are relatively tolerant to
high temperatures and limiting ranges are reported to be between
36.5-42.5°C.
R. meliloti in peat has survived for 21 days at 40°C in the
Laboratory and for longer periods at intermittently higher
temperature in the field. Chowdhury et al (1968) indicated that
temperatures higher than 35-40°C were lethal for both R. trifolii
and R. lupini species grown in sterile unfertile soil. Salem and
Szegi (1971) showed that optimum growth of R. phaseoli in three
Hungarian soils was at 30°C, but incubation at 40°C limited its
growth in all three soil types.
vii. Moisture
As is the case for all soil bacteria, rhizobia must be surrounded by a
film of water in which the solutes are not so concentrated as to raise
osmotic problems for the cell. Too little water (rather than too much)
is a threat to their survival and function (Schmidt, 1978).

Foulds (1971) examined the survival of rhizobia during air drying of

three soils by plant infection techniques. R. trifolii proved to be
more tolerant of drought than R. meliloti and Rhizobium from the
Lotus group.

Hamdi (1971) studied the movement of R. trifolii in sterilized soils.

With suction applied to soils of varying textures, zones of movement
were sharply decreased by increased water tension, and ceased when
water-filled pores became discontinuous.

viii. Salinity
Several reports indicate that growth of rhizobia, and nitrogen fixation
by various legumes, is reduced with increasing levels of salts (Pilai
and Sen, 1966; Hamdi and Al-Tai, 1976). Steinborn and Roughley (1974)
showed that the death rate of R. trifolii increased with increasing
salt concentration in peat. An increase in the salt concentration in
irrigation water led to a significant decrease in weight of soybean
plant, pods and nodules (Ala el-Din, 1976).

Growth of soybean was totally inhibited at 0.8% or more of sodium

Chloride and at 1.5% of sodium sulphate. However, at 0.1% of sodium
sulphate a stimulation of growth was noted (Abd el-Gaffar 1976).

ix. Water and light stresses

Sprent (1976) examined the effect of water stress on nitrogen fixation
by Trifolium repens, Glycine max, Vicia faba and Lupinus arboreus.
Water stress affects the growth of young nodules, and the formation
of new nodules (Table 9), but whether the reduction in nodule number
resulted from few rhizobia being available for infection for bacteria
or from effects on the infection process or both is not clear.

Shading by trees is a major factor affecting the nodulation and

nitrogen fixation of the lupin, and its survival under a forest
canopy (Sprent, 1976). Shading mainly reduces nodule number and
size, but only the deepest shade appears to affect nodule activity per
unit weight of tissue of P. vulgaris.

x. Seed coat diffusates

Toxic, water soluble, seed coat diffusates have been demonstrated to
inhibit the growth of rhizobia. The seeds of several legumes prepared
in this manner have shown considerable variation in their toxicity to
various Rhizobium spp.

xi. Combined nitrogen

Forms of combined nitrogen are reported to decrease the nodulation of
legumes and to reduce nitrogen fixation. The degree of inhibition
depends on many factors including the concentration and form of
nitrogen, the time of application and strains used.

Vigne et al (1977) observed that growing soybean hydroponically on

urea allowed effective nodule development and function. This suggests
that if this form of nitrogen could be stabilized, i.e. if urea
hydrolysis and subsequent nitrification could be prevented, soybean
could simultaneously fix nitrogen and absorb combined nitrogen.
Table 9 EFFECT OF WATER STRESS ON NODULE NUMBER, SIZE AND ACETYLENE
REDUCING ACTIVITY OF P. VULGARIS PLANTS (44 DAYS) INOCULATED WITH
TWO STRAINS OF R. PHASEOLI
(Sprent, 1976)

Rhizobium strains
3601 3605
Control Stress Control Stress
Pmoles C H /mg/min 16.45 1.75 37.45 3.15
2 2
Nodule number 28.40 8.3 18.50 4.5
Average nodule weight (mg) 1.44 0.95 1.72 1.16
Water content of sand (% DW) 6.54 0.71. 6.34 0.81

xii. Trace elements

Many disorders due to deficiencies of trace elements have been shown
to affect the growth of legumes. Although most of these deficiencies
do not specifically inhibit the fixation of nitrogen, they limit the
growth of the host. A more specific role has been demonstrated for
molybdenum, cobalt, and boron.

- Molybdenum
The specific role of molybdenum in the efficient functioning of the
formed nodule has been studied intensively. 10-15 ppm molybdenum
appeared necessary for maximum nitrogen fixing ability in lucerne,
4 to 8 ppm sufficed subterranean clover.

- Cobalt
The essentiality of cobalt for symbiotic nitrogen fixation has been
demonstrated in soybean, lucerne and subterranean clover under
controlled conditions.

- Boron
Boron deficiencies that interfere with nodule function have been
reported from time to time, establishing the role of this element,
a deficiency of which affects the vascular system leading to the
nodule and hence causes a shortage of carbohydrate at the fixation
site.

xiii. Pesticides
All fungicides are likely to be toxic to rhizobia to some degree.
Thus when the host has to be both treated against fungal infection
and inoculated with rhizobia, a balance has to be achieved between
the two effects, by the careful selection of fungicides. Compounds
containing toxic metals such as mercury, copper and zinc, are always
a serious risk, as also halogen substituted aromatic compounds,
although reports differ as to the degree of risk in relation to
formulations used.
Various fungicides based on thuram are generally the least toxic to
rhizobia and this advantage has in fact been accepted for use in the
field.
Insecticides have not been studied in any detail in the laboratory, but
there are clear indications of toxic effects with inoculated seed.
 Some herbicides appear to have no effect on rhizobia, while some are
harmful. Information on inhibiting levels is confused by the fact that
species, and strains within species, differ considerably in their
sensitivity to even one herbicide, and because results are likely to be
affected by local conditions and time of inoculation.

xiv. Competition
One important attribute of a given strain of Rhizobium is its ability
to compete in nodule formation with closely related soil rhizobia.
The ability to colonize the rhizosphere in high numbers is essential
in all instances.
An example of the results of studies on this subject is the analysis
of the competitive ability of R. japonicum strains 110, 38 and 76
introduced into rhizobia-free soil. Strain 110 produced 98% of the
nodules when applied with strain 38, and 76% when combined with strain
76. Strain 76 produced 95% of the nodules when combined with strain
38 (Caldwell, 1969).

xv. Biotic factors

Many hypotheses have been advanced to account for the failure of
rhizobia to colonize readily or for the decline of the populations
naturally present or those deliberately added to soils. The hazards
to colonization include toxin-producing microorganisms, protozoa and
nematodes.
Actinomycetes introduced into sterile soil with R. japonicum reduced
the number of nodules by 35 and 53% when isolate E8 was introduced into
the soil, at the time of planting or 28 days before, respectively
(Damirgi and Johnson, 1966).
Extracts of certain recently cleared soils, in which subterranean clover
failed to nodulate, were frequently toxic to R. trifolii and it was
proposed that antibiotic-producing fungi, which proliferate on the
organic material left after soil clearing, could lead to nodulation
failure (Holland and Parker, 1966).
It has been suggested that bacteriophages which lyse rhizobia, are
important in reduction of nitrogen fixation. However, no convincing
evidence exists that the ecology of Rhizobium is in any significant
way affected by soil-borne bacteriophages.
The fungus Rhizoctonia is reported to cause a 63% decrease in fixed
nitrogen per plant in inoculated soybean (Orellana et al, 1976).
Mycorrhiza are reported to improve seed yield and other traits of the
nodulating isoline Hardee, but not the non-nodulating isoline (Shenk
and Hinson, 1978).
Bdellovibrio is an inhabitant of soil which probably exists in nature
as an obligate parasite. Strains feeding on R. meliloti, R. trifolii
and cowpea rhizobia have been found in many soils. When these vibrios
are provided with cowpea rhizobia in culture, they kill many host cells,
so they might be responsible for the death of rhizobia in soil
(Alexander, 1975).
Protozoa are suspected as predators of rhizobia in soil. Alexander
(1975) reported that R. japonicum introduced into a soil declined in
number and that protozoa were the cause. Each protozoa appeared to
consume thousands of bacteria, but nevertheless 6.3 x 107 rhizobia/g
were left after 12 days. It was further shown that ciliate Tetrahymena
eliminated R. japonicum if replication of rhizobia was to be prevented
by chloramphenicol.
Tetrahymena feeding on rhizobia in the absence of the inhibitor did not
reduce their number below 106/ml, but reduced the number of non-
replicative rhizobia to 270/ml.
 Nematodes were shown to interfere with the rhizobia-legume association.
A large proportion of cowpea nodules were occupied in varying degrees,
from those showing larval penetration only to conditions where nematodes
predominated (Robinson, 1961). Soybeans infected with Heterodera glycines
(race 1) showed reduced nodulation and nitrogen fixation, whereas plants
infected with other races did not show these symptoms when cyst nematodes
were introduced 14 days after rhizobia inoculation (Barker et al, 1972).
The greatest inhibition occurred when soybeans were inoculated
Simultaneously with R. japonicum and race 1 of the soybean cyst nematode.

Two insects are known to attack legume nodules: Sitona lineata and
Rivellia sp. Sitona larvae mature within nodules of a number of legumes,
such as Vicia faba, peas and sometimes lupine in Europe. Larvae of
Rivellia spp attack nodules of Glycine javanica resulting in 50-70%
of the nodules being damaged (Diatloff, 1965).

3.4.13 Amount of Nitrogen Fixed by Different Legumes

Various reports deal with the amounts of nitrogen fixed by legumes.
In general, pulses fix nitrogen less than forage legumes; however, this
depends largely on the crop itself and on factors interfering with the growth
of the crop. See Table 10 (Nutman 1976).

Rizk (1968) studied the amounts of nitrogen fixed by legumes under

Egyptian conditions (Table 11). It is clear that clover is in the lead
followed by the pulses.

Russell (1961) presented the results of a two year rotation of legume-

cereal, the cereal being either rye or barley (Table 12). They show that the
yield of the following cereal crop largely depends on the amount of nitrogen
the legumes add to the soil. The amount of nitrogen fixed varies from 79 to
505 kg/ha. Leguminous crops grown for seed (peas, field beans, soybeans and
groundnuts) show a tendency to reduce the nitrogen content of the soil,
whereas legumes grown for their leaf (clovers, sweet clover, and lucerne)
increase the nitrogen content.

Rizk (1968) reported that the nitrogen content of the soil varies
according to the crop cultivated. As indicated in Table 13, the increase in
nitrogen of soil varies between 34 and 259 kg/ha with legumes, but, with
non-legumes like barley, sesame and chickoria, the nitrogen content varies
between 7 and 38 kg/ha. It is apparent from the table that plants reduce the
nitrogen contents in the soil during the growth period.

Inoculation of seeds, and field inoculation with soil from a field

where inoculated soybean had previously been grown, both produced 73-94%
greater yield compared to the control (Saxena and Tilak, 1975). Without
application of nitrogen, following an inoculated soybean crop the yield of
wheat was more than 65% higher than when it followed an uninoculed soybean
crop. The response was equivalent to about 30 kg N/ha.

A series of field experiments on nitrogen fertilization of paddy

transplanted after non-leguminous crops and after berseem showed that paddy
transplanted in soils previously cropped with non-legumes was more responsive
to added nitrogen than after clover. Yields of rice after non-legumes
gradually increased as the rate of nitrogen increased up to 96 kg/ha, whereas
the yield of paddy after clover reached its maximum at 24 kg N/ha. (Serry
et al, 1970).
Table 10 NITROGEN FIXED BY PULSES, FORAGE, GREEN MANURE
AND SHADE TREES
(kg N/ha/yr; Nutman, 1976)

P L A N T Average Ranges

A. PULSES
Vicia faba 210 45-552
Pisum sativum 65 52- 77
Lupinus spp. 176 145-208
Phaseolus aureus (green gram) 202 63-342
Phaseolus aureus (mung) 61 -
Cajanus cajan (pigeon pea) 224 168-280
Vigna sinensis (cowpea) 198 73-354
Canavalia ensiformis 49 -
Cicer arietinum (chickpea) 103 -
Lens culinaris (lentil) 101 88-114
Arachis hypogaea (groundnut) 124 72-124
Cyamopsis tetragonoloba (guar) 130 41-220
Calpogonium mucunoides (calapo) 202 370-450

B. FORAGE, GREEN MANURE, SHADE TREES

Centrosema pubescens (centro) 259 126-395
Desmodium intortum and D. canum (tick clover) 897 -
Leucaena glauca 277 74-584
Lotononis bainesii 62 -
Sesbania cannabina 542 -
Stylosanthes sp (stylo) 124 -
Mixes of centro and stylo 115 -
Phaseolus atropurpureus (siratro) 291 -
Mikanea cordata 120 -
Pueraria phaseoloides (kudzu) 99 -
Enterolobium saman 150 -

Table 11 NITROGEN FIXED BY DIFFERENT LEGUMES UNDER EGYPTIAN CONDITIONS

(after Rizk, 1968)

Stage of Nitrogen fixed

C r o p growth kg/ha g/kg dry wt.

Berseem Fahl Single cut 67 17

Berseem Miskawy 1st cut 38
2nd cut 88
3rd cut 62 18
4th cut 31
Seeds 19
Lupine (termis) Mature 138 20
Broadbean Mature 136 17
Fenugreek Mature 105 23
Chickpea Mature 98 23
Lentils Mature 83 22
Groundnut Mature 79 16
Soybean Mature 40 14

Note.Original figures in kg/feddan converted to kg/ha to nearest kg.

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Table 13 CHANGES IN SOIL NITROGEN CULTIVATED WITH LEGUMES AND NON-LEGUMES

(after Rizk, 1968)

C r o p s Stage of growth Change, Kg N/ha

A. LEGUMES
Broad bean Immature - 10
Lentil Mature + 34
Lentil “ + 86
Fenugreek “ + 166
Termis (lupine) “ + 122
Chickpea “ + 91
Groundnut “ + 82
Soybean “ + 58
Berseem fahl Single cut + 101
Berseem miskawy 4 cuts + 259

B. NON-LEGUMES
Barley Immature - 26
Barley Mature + 7
Sesame Mature + 38
Chickoria 4 cuts + seeds + 22

Note - Conversion factor used for feddan to ha = 2.4 (figures rounded)

i. Transfer of nitrogen to companion plant
There are four possible routes for the transfer of legume-fixed nitrogen
from the soil to the companion plant: l.) excretion of nitrogenous compounds
from the nodules of the legume to the growing medium, 2) release of
nitrogenous material to the soil from droppings of grazing animals,
3) decomposition of aerial parts of legume plants, and 4) the decay of
root and nodule tissues.

Whitney (1975) reviewed nitrogen fixation by legume-grass mixtures

such as Desmodium intortum, D. canum and Centrosema pubescens with and
without pangola (Digitaria decumbens) and napier (Pennisetum purpureum)
grasses (Table 14).

Table 14 NITROGEN FIXED BY THREE LEGUMES IN PLASTIC-LINED

CINDER PLOTS
(Whitney, 1975)

Net kg N/ha/yr recovered in

Legume or mixture
tops roots total

Desmodium canum alone 45 49 94

D. canum + pangola1 35 12 47
D. canum + napier2 106 30 136
Centrosema pubescens alone 257 14 271
C. pubescens + pangola 118 14 132
C. pubescens + napier 108 15 123
Desmodium intortum alone 350 56 406
D. intortum + pangola 330 30 360
D. intortum + napier 327 51 378

1
Digitaria decumbens

2
Pennisetum purpureum

Net nitrogen-fixation, including secretion of nitrogen in the root mass,

was low for D. canum except for the mixture with napier grass.
Centrosema pubescens was intermediate in result, but nitrogen-fixation
with grass was also low. D. intortum was effective in fixing nitrogen
(supplying about 400 kg N/ha/year in pure stand and only slightly less
in the grass-legume mixture).

In another study (Table 15) nitrogen fixed and transferred between

Desmodium and pangola grass was determined. D. canum fixed nearly
100 kg N/ha/year, of which an estimated one third was transferred into
the pangola grass. Although D. intortum fixed 264 kg N/ha/year, it
transferred only slightly more nitrogen to the grassland than did
D. canum, amounting to one eighth of the total nitrogen fixed. D.
Intortum in pure stand fixed the most nitrogen.
Table 15 NITROGEN CONTRIBUTION BY TWO DESMODIUM FORAGE LEGUMES
IN ASSOCIATION WITH PANGOLA GRASS.
(after Whitney, 1975)

Apparent kg N/ha/yr
T r e a t m e n t
Legume fixed transferred
%
D. canum (uninoc.) + pangola1 26 80 26
D. canum (inoc.) + pangola1 27 97 32
D. intortum (90 cm rows) + pangola1 54 213 42
D. intortum (45 cm rows) + pangola 64 264 37
D. intortum (45 cm rows) alone 100 290 -

1
Digitaria decumbens

ii. Legumes as green manure crops

The economic feasibility of using legumes as green manure crops, as a
general procedure in tropical agriculture remains to be proven. In
contrast to temperate conditions, decomposition of the plant residues
is too fast, and the residual effect is generally short-lived (and not
much beyond one year). However, there are other beneficial short-term
effects such as improved water infiltration and water holding capacity,
and control of plant diseases and pests. Green manuring of maize with
Crotalaria juncea, Canavalia ensiformis, or Stizolobium deeringianum
corresponded to the addition of 60 kg N/ha/yr of fertilizer N, but the
economics of this did not compare favourably with adding higher levels
of mineral nitrogen fertilizer. Table 16 shows the superiority of
Crotalaria juncea over other legumes and sorghum as a green manure
for field beans, Phaseolus vulgaris, (Dobereiner and Campelo, 1975).

Table 16 EFFECT OF GREEN MANURE INCORPORATED IMMEDIATELY BEFORE

PLANTING ON THE YIELD OF FIELD BEANS (PHASEOLUS VULGARIS) IN
SAO PAULO STATE, BRAZIL.
(Dobereiner and Campelo, 1976)

Fresh weight Grain yield

Green manure incorporated kg/ha

Crotalaria juncea 57.797 800

Cajanus cajan 25.026 637
Lablab purpureus 31.656 591
Tephrosia candida 13.884 609
Sorghum vulgare 56.025 416
None 418

L.S.D. 165
 4. BLUE-GREEN ALGAE

4.1 INTRODUCTION
Recognition of blue-green algae as nitrogen fixers dates back to
Frank in 1889 when he noticed that soils containing blue-green algae fixed
nitrogen. Prantil also, reported (in 1889) that impure cultures of Nostoc
fixed nitrogen and other workers around the same period observed similar
phenomena. But this was followed by a period of doubt until Drews in 1928
published evidence that Anabaena variabilis, Anabaena sp, and Nostoc
punctiforme grew well in pure culture in a medium free of combined nitrogen
(Stewart, 1975).

4.2 NITROGEN-FIXING BLUE-GREEN ALGAE

With the developments of techniques such as 15N and acetylene
reduction assay, it became evident that there are three main groups of blue-
green algae: unicellular, filamentous heterocystous and filamentous non-
heterocystous, that include types which fix nitrogen (Stewart, 1977, Tables
17 and 18). In all these groups, the nitrogenase appears remarkably similar
and the main differences appear to be in the localization of the enzyme and
in the ways in which it is protected from inactivation by oxygen (Fig. 11).

4.3 CLASSIFICATION
According to the 8th edition of Bergey’s Manual of Determinative
Bacteriology, blue-green algae are classified in Division I which includes
the phototrophic procaryotes, “The cyanobacteria”. The division includes
3 classes:
Class I : Blue-green photobacteria
Class II : Red photobacteria
Class III: Green photobacteria.
(Class I and II represent part I and include the phototrophic bacteria).

All phototrophic procaryotic organisms use water as an electron donor

and hence produce oxygen in the light.

The characteristics of cyanobacteria are summarized by Stanier (1974)

as follows:

Cells are always enclosed by a rigid, multilayered wall with an inner

peptidoglycan layer. The wall may in turn be surrounded by a gelatinous
or fibrous sheath. Most cyanobacteria are mobile at some stage of
development; motility is always of the gliding type, dependent on
surface contact. The cytoplasmic region is traversed by an extensive
system of paired photosynthetic lamellae (thylakoids); the outer surfaces
bear characteristic granules (phycobilisomes) composed of aggregates of
the phycobiliprotein pigments.

Some cyanobacteria are unicellular, others consist of colonies of cells

(filaments), either simple or branched. Reproduction of unicellular forms
may occur by binary fission, by multiple fission or by the aerial release
of apical cells (exospores) from a sessile individual. Forms that consist
of filaments grow by repeated intercallary cell divisions and reproduce
either by random fragmentation of the filament or by terminal release of
short motile chains of cells (hormogonia). Certain filament formers can
produce specialized cells known as akinetes and heterocysts. Akinetes are
Table 17 HETEROCYSTOUS BLUE-GREEN ALGAE REPORTED TO
FIX NITROGEN IN PURE CULTURES
(Stewart, 1977)
Anabaena ambigua Fischerella major
A. azollae F. muscicola
A. cycadeae
A. cylindrica Hapalosiphon fontinalis
A. fertilissima
A. flos-aquae Mastigocladus laminosus
A. gelatinosa
A. humicola Nostoc calcicola
A. levanderii N. commune
A. naviculoides N. cycadeae
A. variabilis N. entophytum
N. muscorum
Anabaena sp. N. paludosum
N. punctiforme
Anabaenopsis circularis N. sphaericum
Anabaenopsis sp.
Scytonema arcangelii
Aulosira fertilissima S. hofmanni

Calothrix brevissima Stigonema dendroideum

C. elenkinii
C. parietina Tolypothrix tenuis
C. scopulorum
Westiellopsis prolifica
Chlorogloea fritschii

Cylindrospermum gorakhpurense
C. licheniforme
C. majus
C. sphaerica

Table 18 NON-HETEROCYSTOUS ALGAE REPORTED TO FIX NITROGEN

IN PURE CULTURES
(Stewart, 1977)

Gloeocapsa 795 Phormidium sp.

Gloeocapsa 6501 Plectonema boryanum
Lyngbya 6409 Plectonema 6306
Oscillatoria 6407 Plectonema 6402
Oscillatoria 6412 Rhaphidiopsis indica
Oscillatoria 6506 Trichodesmium (impure)
Oscillatoria 6602
 a.

Fig. 11 Some nitrogen-fixing blue-green algae.

From top, Gloeotrichia sp., Nostoc sp. and Anabaena sp.
(contributed by I. Watanabe)
 larger than the vegetative cells in the filament and represent a resting
stage, which germinates with a release of a hormogonium. Heterocysts are
non-reproductive cells, distinguishable from the adjoining vegetative cells
by the presence of refractile polar granules and of a thick outer wall:
they are believed to be physiologically specialized cells that serve as
sites of nitrogen-fixation.

The characteristic photopigments include chlorophyll as the only

Chlorophyllous pigment and phycobiliproteins (allophycocyanin, phycocyanic
and sometimes phycoerithrin). The cellular absorption spectrum has a peak
at approximately 680 nm attributable to chlorophyll(a) and a broad
absorption band with one or more peaks, attributable to phycobiliproteins
between 560 and 630 nm.

A detailed discussion on the status of the classical taxonomy of

blue-green algae is given by Desikachary (1973).

4.4 GENETICS OF BLUE-GREEN ALGAE

Stewart (1975) indicated that little progress had been made in
knowledge of the genetics of nitrogen-fixation by blue-green algae. A few
reports have dealt with the isolation of large numbers of mutants and some
eveidence has been presented of genetic recombination, and of transformation.

4.5 THE SITE OF THE NITROGENASE ENZYME IN BLUE-GREEN ALGAE

Nitrogen-fixing blue-green algae have three distinct cell types:
vegetative cells, spores (akinetes) and heterocysts. The cellular localization
of nitrogenase in heterocystous algae has been, and still is to some extent
the subject of much debate. Although in theory, heterocysts, akinetes and
vegetative cells could all produce a nitrogenase, the akinetes have been
eliminated because tests have shown that they do not reduce acetylene
(Fay, 1969).

Experimental evidence has been presented that heterocysts do not have

photosystem II and hence would not be able to assimilate CO . It has also
2
been shown that carbon supplied as 14CO is fixed in the vegetative cells and
2
transferred into heterocysts. The transport takes place intrafilamentously,
possibly via microplasmadesmata which transverse the membrane separating the
cells. Nitrogen fixed in the heterocysts is also thought to travel intra-
filamentously (Stewart et al, 1969).

The discoveries of aerobic nitrogen-fixation in two strains of the

unicellular algae Gloecapsa alpicola and in unialgal cultures of the
non-heterocystous alga Trichodesmium sp, and the anaerobic nitrogen-fixation
of the non-heterocystous filamentous alga Plectonema boryanum 594, suggest
that vegetative cells of heterocystous algae might at least fix nitrogen
under anaerobic conditions even if they cannot do so aerobically.

4.6 ISOLATION AND PURIFICATION OF BLUE-GREEN ALGAE

Under natural conditions, algae grow as mixed communities which include
species and genera. It is therefore obvious that in order to study individual
species, the organisms must be cultivated without other types or forms present

4.6.1 Unialgal Cultures

The term “unialgal culture” refers to one containing only one species
of alga in the presence of other organisms.
 Felfoldy and Zssusza (1959) used the serial dilution pour-plate
technique on agar plates containing inorganic salts after shaking to separate
individual cells. De (1939) obtained unialgal cultures by distributing the
algal cells over the surface of the solidified agar with the aid of a sprayer,
bent glass rod, and brush or platinum loop. Gerloff et al (1950) used a
micromanipulator as a means to isolate the desired algae.

4.6.2 Bacteria-free Cultures of Algae

As the term indicates, the culture should contain a single species of
alga, and be free from other bacteria.

Phototactic motion (creeping movement) is useful as a means of

eliminating the bacteria. During multiplication, the algae spread over the
sterile agar surface and the attaching bacteria are left behind.

Shalan (1974) used silica gel plates to obtain bacteria-free

Anabaena naviculoides and A. variabilis. Wieringa (1968) obtained bacteria-
free cultures of Anabaena variabilis, A. azolla, Nostoc muscorum, N. paludosum,
N. piscinale, Nodularia sp and Microchaete sp by using heat treatment.

Pure cultures of Nostoc linckia, N. muscorum and Anabaena variabilis

were obtained with the aid of a gamma cell producing 6.48 x 104 rad/h at the
irradiation site (Kraus, 1966). These pure cultures were obtained upon using
288 000 rad for 4.75 h.

Gerloff et al(1950) employed ultraviolet irradiation to obtain bacteria-

free cultures of Aphanizomenon flos-aquae and Diplocystis aeruginosa by
exposing the algal suspension in the hormogonial state to ultraviolet light
from a quartz-mercury vapour lamp for 20-30 minutes. Pure cultures were
obtained of Hapalosiphon fontinalis and Anabaena variabilis by UV-irradiation
for 15-20 minutes at a distance of 40 cm, but Calothrix elenkinii only
required UV-irradiation for 1-3 minutes at a distance of 12 cm (Taha, 1963).
The antibiotics streptomycin (0.05-0.4 mg/ml) and penicillin G (4-40 IU/ml)
were used to obtain pure cultures of Scenodesmus acuminatus and S. quadsicanda,
Navicula sp and Euglena sp after a few subcultures had been made.

Gupta et al (1956) succeeded in obtaining pure cultures of algae using

mercuric chloride. Chlorine water, 25 ppm or 2.5% for 2 minutes, was used to
purify cultures of Anabaena cylindrica and A. variabilis. An electric field
of 500 V and 1.0 mA for 48 hr gave bacteria-free cultures of Nostoc (Coler
et al, 1969), and Shalan (1974) secured the best results using silica gel and
sodium tellurite treatment.

4.6.3 Tests for Purity of Algal Cultures

Two sources of errors may occur when purifying algae:
a. the contaminating bacteria may not be able to grow in the medium
used;
or
b. some bacteria may be embedded in the mucilage of the alga and,
failing to pass out into the medium, will not produce visible
contamination.

In order to ascertain the purity of algal cultures they must be tested

on a set of culture media suitable for various possibly contaminating bacteria:
i. caseinate agar;
ii. soil extract sucrose nitrate broth and agar;
iii. albumate agar;
iv. litmus milk:
v. dextrose peptone broth (1% each of dextrose and difco proteose peptone);
vi. bacto-tryptophane broth (difco, 1% solution);
vii. 2% mannitol in tap water + 0.02% potassium monohydrogen phosphate (a
medium suitable for Azotobacter).

4.6.4 Methods for Testing the Ability of Blue-Green Algae to fix Nitrogen
In experiments designed to show the nitrogen fixation by a given alga,
two points should be carefully considered:

i. the alga must be in absolutely pure culture, because even with a

contaminant which is known to be otherwise incapable of fixing nitrogen,
it is possible that it may fix nitrogen in the presence of the organism
under examination;
ii. there must be no doubt whatsoever that any increase in fixed nitrogen in a
culture, is due to the uptake of free nitrogen.

It is important to make sure that nitrogen is not absorbed in the form

of ammonia or oxides of nitrogen from the atmosphere. Before being passed
over the culture, air must be purified from traces of combined nitrogen by
passing it through a solution of sodium bicarbonate and a 25% solution of
sulphuric acid; the wash bottles should be the type filled with glass bubbles.

Some methods which have been developed for testing include the following
15
- N Tracer. Williams and Burris (1952) developed a method for
testing Calothrix parietina (Appendix D.l);
- Microkjeldahl (Appendix A.2);
- Acetylene reduction (Appendix D.3);
- Detection of ammonia. Stewart and Rowell (1975) indicated that it
is possible to get the blue-green alga Anabaena cylindrica to
secrete ammonia into the medium. This method depends on two facts.

1) The main pathway of incorporation of newly fixed nitrogen by

nitrogen-fixing organisms involves the enzyme glutamine synthetase
and also (usually)the glutamate synthase.

2) The analogous methionine sulphoximine (MSO) inhibits the GS pathway

of ammonia incorporation and when this happens, the newly fixed
nitrogen, rather than being incorporated into general cell
metabolism is excreted into the medium and can be measured
calorimetrically.

MSO should be used at the rate of 1 N mol(use filter-sterilized M50,

e.g. 1 ml of 100 µ mol solution in 99 ml of the cultures). MSO
experiments should not exceed 12 to 24 hours. If left for 48 hours
or more the experiment may cease to function.

Extracellular ammonia can be detected by the method adopted by

Solorzano (1969) (Appendix D.2).
4.7 GROWTH RATE OF BLUE-GREEN ALGAE
The specific growth rate of blue-green algae depends on many factors
such as the nature of the organism, quality and quantity of light available,
carbon dioxide concentration, nutrient status, metabolites, etc. (Venkataraman,
1969).

Hoogenhout and Amesz (1965) tabulated the growth rates of photosynthetic

organisms. The number of doublings per day for some blue-green algae are
presented in Table 19.

Table 19 NUMBER OF DOUBLINGS PER DAY OF SOME BLUE-GREEN ALGAE

(Hoogenhout and Amesz, 1965)

Name of algae K Temp.°C Illumination Medium

Agmenellum quadriplaticum 8.00 39.0 ? S

Anabaena cylindrica 0.96 25.0 I S
A. variabilis 3.90 34.5 I S
Anacystis nidulans 11.50 41.0 I S
Cylindrospermum sphaerica 0.17 25.0 nI S
Gloeotrichia echimula 0.20 26-27 nI S
Microcystis aeruginosa 1.60 23.0 ? S
Nostoc muscorum 3.10 25.0 ? S
Nostoc muscorum 2.90 32.5 I S
Schizothrix calcicola 3.40 30.0 I S
Synechococcus lividus 8.50 52.0 ? S
Synechococcus sp 9.90 45.0 ? nS
Synechocystis sp 8.00 37.0 ? S
Tolypothrix tenuis 8.00 38.0 I S

I: illuminated at saturation S: synthetic medium

nI: non-saturating light intensities nS: non-synthetic medium
?: unknown K: number of doublings

4.8 ECOLOGY AND FACTORS AFFECTING NITROGEN-FIXING BLUE-GREEN ALGAE

4.8.1 Distribution
Blue-green algae occur in many habitats extending from the Antarctic
to the Tropics, but they are only in certain localized areas such as rice
fields, mesotrophic and eutrophic fresh waters and alkaline hot spring
regions, and in symbiosis with other plants. Blooms of Trichodesmium
occur in some tropical waters.

4.8.2 Nutrients for Blue-Green Algae

i. Carbon
Blue-green algae have a photosynthetic system which enables
them to utilize carbon dioxide as the sole carbon source in
the light.
Carbon dioxide bubbled into the culture of the algae at a
Rate of 0.5 to 5% of air passing through increases the growth rate.
However, higher levels of carbon dioxide frequently result in a drastic decrease in pH
even in a buffered medium.
Aeration with air containing 3% carbon dioxide favours the growth of Nostoc
spongiaeforme. While 5% carbon dioxide in air is toxic to cultures of Anabaena
cylindrica,it is satisfactory for very dense cultures of this species at saturating
light intensity.

It appears that glucose, sucrose and mannitol stimulate the growth of, and nitrogen-
fixation by Tolypothrix tenuis, Nostoc muscorum and Calothrix brevissima under
continuous illumination, while sodium acetate or citrate have no effect
or are inhibitory (El-Sayed, 1974).Shalan (1974) observed that in the presence of light,
maximum growth and nitrogen-fixation by Anabaena sp and Aulosira sp took place with
molasses, followed by glucose and mannitol at a concentration of 0.2%.

ii. Nitrogen
Nitrogen-fixing blue-green algae can readily assimilate inorganic and organic nitrogen,
and when combined nitrogen is available it may be assimilated prerentially thus
inhibiting fixation. This inhibitory effect depends however on the level and type of
combined nitrogen, the exposure periiod to it, the physiological status of the algae and
whether or not the experiments are carried out with whole cells or extracts.

iii. Phosphorous
De and Sulaiman (1950) reported that the addition of phosphates to rice fields,
whether in soluble form as potassium phosphate or in insoluble form as calcium
phosphate, stimulated nitrogen-fixation by blue-green algae. Similarly,De and Mandal
(1956) showed that superphosphate at the rate of 67 kg/ha of P O
2 5
incerased nitrogen-fixation only in soils relatively poor in this nutrient.

El-Nawawy et al (1972) demonstrated that the

nitrogen fixed by the)bdueogreentad that the growth and/or nitrogen fixed by the
blue-green alga T. tenuis were incresed with an increase in the P O
2 5
level up to 63 ppm, but were depressed with 123 ppm. Similarly, dry weight and/or
nitrogen fixed by N. linckia var. arvense were increased and reached a maximum at the
level of 40 ppm P O , after which a gradual decrease occurred (Taha et al,1974).
2 5

iv. Calcium

Calcium has been shown to be required for the growth of blue-green algae. It
plays a particularly important role in the maintenance of plasma membrane, salt
formation with colloids and cell wall materials. It has been shown that calcium
is required for Anabaena cylindrica whether the alga grows with molecular or combined
nitrogen and at least 20 ppm of calcium are needed for optimum growth and this element
cannot be replaced by strontium (Allen and Arnon,1955).
v. Trace elements
Cobalt is an essential element for nitrogen-fixation by
Nostoc muscorum and Calothrix sp although the requirement
is very small, (0.004 ppm is sufficient for growth).
Molybdenum is essential for the growth of seven species of
blue-green algae in medium without combined nitrogen. It
has been shown that the optimum concentration required for
Anabaena cylindrica in a nitrate medium is 0.1 ppm, whereas
more than 0.2 ppm is required to give optimum growth in
elemental nitrogen. The optimal concentration of molybdenum
for T. tenuis in the absence of combined nitrogen or in the
presence of N0 -N was found to be 0.01 ppm, but when ammonium
3
sulphate, urea or monosodium glutamate were supplied no
growth response to molybdenum was observed. Nostoc commune
responded to added molybdenum reaching a maximum at about
4 x 103 mg/l after which a decline in algal growth occurred
(Taha and El-Rafai, 1962).

Boron has been found essential for maximum growth of various

blue-green algae. Nostoc commune G when inoculated into a
boron-free medium cannot develop naturally and requires
boron for good growth. Cell counts in boron-free cultures
show relatively small numbers; chlorosis starts slowly
during the 3rd week and by the end of the 8th week the alga
becomes comnpletely white.

Manganese is needed as a trace element in the medium for

good growth of some blue-green algae. The quantities required
differ depending upon the type of the alga and the environmental
conditions under which it grows. It has been shown that the
presence of manganese in the medium stimulates intensely both
nitrogen-fixation and photosynthesis in the culture of
Anabaena ossillaroides and Hapalosiphon fontinalis f.globosus.

4.8.3 Light
Stewart (1975) has cited numerous studies on the relationship between
light and nitrogenase, both with free-living blue-green algae and with lichens.
Generally there is a correlation between nitrogen-fixation and acetylene
reduction in natural ecosystems. The relationship is also complicated by other
factors such as the effect of desiccation, while in aquatic systems light
intensities may be excessive at the water surface. For example the activity
of samples collected at 0.9m depth in Lake Mendota was found to be more than
that in the surface waters (Rusness and Burris, 1970).

Under controlled laboratory conditions it is still debateable as to

whether algae grow better when given a dark period each day or whether
maximum growth per unit of time is obtained when the cells are continuously
illuminated. Allen and Arnon (1955) showed that the rate of growth of
Anabaena cylindrica was essentially the same when the alga was grown under
11 hours or 13 hours of darkness or 24 hours of light each day. However,
Ukai et al (1958) found a light intensity of 8 000 lux and a temperature of
32°C suitable for optimum steady growth of Tolypothrix tenuis without
causing any sign of bleaching. Half saturation of growth was observed at
light intensities of 1 000 and 1 500 and 2 500 lux at 26, 32 and 38°C,
respectively. They also showed that the apparent half-saturation of nitrogen-
fixation was obtained at an intensity lower than the lowest examined i.e.
800 lux.
 In the case of the algae Hapalosiphon fontinalis, Anabaena variabilis
and Calothrix elenkinii it was found that they did not grow in the dark in
a nitrogen-deficient medium even in the presence of various alcohols, sugars
and organic acids. The best algal growth occurs under continuous illumination
and the nitrogen-fixing capacity increases with increasing light intensity
(Fogg and Than-Tun, 1960).

Granhall (1970) reported that the reduction of acetylene by the blue-

green alga Nostoc punctiforme isolated from Swedish soils, was at light
intensities below 100 lux and the highest saturation level for the process
was about 3 000 lux.

4.8.4 Temperature
Blue-green algae are able to withstand temperature extremes. Nitrogen-
fixation by hot spring algae was noted in Alaska and in Yellowstone National
Park where the upper limit of temperature was near to 60°C. Blue-green algae
dominant at 40-60°C include species of Mastigocladus (Stewart, 1975). In hot
springs in Japan, potential nitrogen-fixing species of Anabaena Calothrix
Cylindrospermum, Hapalosiphon, Mastigocladus, Nostoc and Scytonema all occur
at temperatures ranging from 30-50°C.

At the low temperature extreme, in the Antarctic, blue-green algae

are abundant, and the most common genera in this habitat are Nostoc,
Scytonema hofmanni, Anacystis and Coccochloris.

4.8.5 pH
Blue-green algae are found in most abundance in natural or slightly
alkaline habitats, and rarely in acid ones.

Several workers have determined the growth of nitrogen-fixing blue-

green algae as affected by hydrogen ion concentration. Nostoc muscorum G
can grow in a medium containing nitrate at pH 6.9-9.0. pH values of 7.0-7.5
were found to be optimum for Anabaena flos-aquae and A. scherematrevi; pH 8.0
for Aphanizomenon and pH 9.0 for Microcystis aeruginosa. HapaZosiphon
fontinalis, Anabaena variabilis and Calothrix elenkinii grew at pH 7.0-8.0.
Maximum growth of Nostoc linckia var.arvense was attained at pH 8.0

4.8.6 Desiccation
Desiccation is one of the more important factors regulating the
nitrogenase activity of blue-green algae. Nitrogen-fixation by Nostoc and
Collema in Antarctica is greatly affected by the snow-free seasons and
desiccation.

In the hot deserts of America, blue-green algae occur particularly in

any puddled areas, in intermittent streams and transitory pools, in lichen
symbiosis and under pebbles, stones and rocks. In temperate regions, in situ
rates of nitrogen-fixation by Nostoc in sand -d une ecosystems were found to
be much higher in areas shaded by long grass than in more open, desiccated
areas (Stewart, 1965).

4.8.7 Oxidation Reduction Potential

Blue-green algae grow well under reducing conditions at a redox of
170 to 600 mV. Singh (1961) reported that in Indian paddy soils the redox
potential of the soil stabilises on the reducing side at the time of puddling,
and then thick growths of blue-green algae develope.
4.8.8. Pesticides
Pesticides commonly used in agriculture may or may not affect the
growth of blue-green algae, depending on the type, concentration and time of
application of the compound. For example it has been found that Gamma BHC
stimulates algal population in rice fields by inhibiting the predators which
feed on algae.

On the other hand Hamdi et al (1970) evaluated the effect of the

herbicides molinate, propanil, trifluralin and 2,4-D. They found that dry
weight and nitrogen contents of the alga T. tenuis were reduced in the
presence of these when added either at the beginning or 10 days after the
start of growth (Table 20). Chlorophyll synthesis was stimulated by low levels
of molinate, trifluralin and 2,4-D in both treatments but was inhibited by
propanil.

Venkataraman and Rajyalakshimir (1971,1972) also examined the effect

of various pesticides. They found that while most of the strains of Anabaena
could tolerate 100 ppm of “Ceresan M” a few were sensitive to a concentration
as low as 0.1 ppm. T. tenuis and Aulosira fertilissima tolerated high
concentrations of “Ceresan M”, “Zineb”, 2,4-D and “Dalapon”. However,
A. fertilissima was found to be sensitive to substituted urea compounds such
as fluometaron, diuron and linuron, as well as the triazine herbicide
propazine.

Table 20 EFFECT OF HERBICIDES ON DRY WEIGHT, NITROGEN AND

CHLOROPHYLL CONTENT OF T. TENUIS
(Hamdi et al, 1970)

After inoculation on 10 day-old culture

Herbicides
Dry wt. Nitrogen Chloro- Dry wt. Nitrogen Chloro-
(ppm) (g) (mg) phyll (g) (mg) phyll
(mg) (mg)

Molinate
0.25 280 8.3 3.2 395 10.6 3.2
2.50 290 7.8 2.0 371 6.7 3.2
25.00 101 6.6 1.0 340 8.2 2.9

Trifluralin
0.25 307 8.5 3.3 221 10.3 2.7
2.50 243 7.7 2.1 289 9.2 3.5
25.00 96 8.6 0.6 309 7.9 2.5

2,4-D

0.25 316 10.4 8.6 276 9.5 2.8

2.50 375 9.5 2.7 280 8.3 2.9
25.00 375 9.2 3.1 270 9.1 2.5

Propanil
0.18 308 9.2 3.3 407 10.4 3.8
1.80 231 4.6 1.0 427 6.4 2.8
18.00 No growth 199 1.8 1.1
Control 448 13.0 448 13.0 2.0
4.8.9 Algal Pathogens
Blue-green algae are attacked by various organisms such as aphids,
protozoa, fungi, bacteria and viruses (Singh, 1978) and these manifest
themselves as pale or white spots, or rings, on algal lawns. The variability
in algal population from field to field and the sudden disappearance of algae
are generally attributable to attack by these organisms. The chytrids and
bacteria (Myxobacteria, Cytophaga) are found in many habitats and laboratory
work has shown that they can infect algal lawns to plague proportions. Daff,
Begg and Stewart (1970) isolated a virus pathogenic to Plectonema, Phormidium
and Lyngbya from a variety of fresh waters in Scotland. Blue-green algal
viruses from rice fields in India have been studied in detail (Singh and
Singh, 1967).

4.8.10 Salinity
Blue-green algae are widely distributed in marine ecosystems, although
they are seldom dominant. Hof and Fahmy in 1933 (Fogg et al, 1973) divided
the algae into two physiological groups: halotolerant and halophilic. For
example a halotolerant species is Calothrix scopulorum,while an example of a
halophilic species is Spirulina subsalo.

El-Nawawy et al (1968) reported that the fixation of atmospheric

nitrogen by the local strain of blue-green alga, Calothrix sp was slightly
affected by 1 747.5 ppm of sodium chloride, and severely depressed by
3 395.5 ppm. However, concentrations of 795 and 1 590 ppm had no influence
on the nitrogen-fixation of the alga. Growth of the alga was not affected
appreciably until the highest concentration of 4 191.0 ppm was used.

Khadr (1975) showed that growth of Nostoc calcicola increased as a

result of increasing the sodium chloride concentration up to 9 000 ppm after
which it began to decline. Growth of Anabaena variabilis was slightly
increased by 9000 ppm of sodium chloride but it was markedly decreased by 12 000
and 15 000 ppm. Higher concentrations were found to be lethal for this alga.
Growth of Anabaena oryzae and Nostoc muscorum significantly decreased at
21 000 ppm and the same concentration of sodium chloride was lethal to
Anabaena naviculoides.

Brock (1976) isolated the halophilic blue-green alga Aphanothece

halophytica from the Great Salt Lake in Utah,U.S.A.. The optimum salinity for
growth of this organism was found to be about 16% of sodium chloride, but it
could grow very slowly even in a saturated (30%) solution.

4.9 AMOUNTS OF NITROGEN FIXED BY BLUE-GREEN ALGAE

4.9.1 Under Laboratory Conditions

Amounts of nitrogen fixed under laboratory conditions vary according
to the species, type of media, period of incubation, method of determination,
etc. Table 21 summarizes some of the available data on the amounts of nitrogen
fixed in culture media.

4.9.2 Under Field Conditions

Various reports indicate that there is an increase in the nitrogen
content of the soil through algal inoculation. Studies using 15N tracer
methods showed that algae fixed about 22.5 kg of nitrogen per hectare and
that the crop absorbed 4.8 kg nitrogen (Nishigaki et al, 1953).
Table 21 AMOUNTS OF NITROGEN FIXED BY DIFFERENT BLUE-GREEN
ALGAE IN LIQUID CULTURES

Nitrogen fixed
Species
Period Quantity Reference
days

Anabaena naviculoides 60 3.6 mg/100 ml De 1939

A. oryzae 60 4.4 " " "
A. variabilis 60 5.7 " " "

Nostoc commune 60 70.6 mg/1 El Borollosy 1972

Tolypothrix tenuis 60 141.9 " " "
A.naviculoides 45 154.4 " El Sayed 1978
A. oryzae 45 270.2 " " "
N. calcicola 45 187.3 " " "
N. muscorum 45 175.5 " " "

A. naviculoides 55 83.0 " Khadr 1975

A. oryzae 55 117.0 " " "
A. variabilis 55 92.7 " " "
N. calcicola 55 99.3 " " "
N. muscorum 55 101.3 " " "
N. sphaericum 55 83.0 " " "

A. ambigua 45 5.7 mg/100 ml Singh 1942

A. fertilissima 45 6.7 " " "
Aulosira fertilissima 45 8.7 " " "
Cylindrospermum gorakhpurense 45 5.0 " " "

N. amplissimum 90 1.26-1.45 g/l Subramanyan and Sahay

N. sphaericum 90 1.2 -1.3 " " 1964
T. campylonemoides 90 1.6 -1.81 " " "
Westiella sp. 90 1.0 -1.1 " " "

A. variabilis 27 mg/100 ml Taha 1963

Calothrix elenkinii 57 " " "
Hapalosiphon fontinalis 22 " " "

Cylindrospermum sp. 60 78.6 mg/1 Taha et al 1973

N. linckia var arvense 60 91 " " "
N. paludosum 60 61.6 " " "
N. piscinale 60 87 " " "

Anabaenopsis sp. 60 34 mg/100 ml Watanabe et al 1951

Calothrix brevissima 60 52 " " "
Nostoc sp. 60 31 " " "
T. tenuis 60 96 " " "

Singh (1961) attributed a fixation of about 80 kg of nitrogen per

hectare in maize fields in Utter Pradesh to algae. Sankaram (1971) showed
a gradual increase in the organic matter content due to algal inoculation,
although the increase was small (Table 22).
Table 22 BUILD UP OF ORGANIC MATTER DUE TO ALGAL INOCULATION
(Sankaram, 1971)

Increase/decrease of organic matter

over uninoculated fields
1 9 6 4 1 9 6 5

Control 0.06 0.09

F Y M (20 kg N/ha) 0.02 0.08
Green manure (20 kg N/ha) 0.05 0.09
Ammonium sulphate (20 kg N/ha) 0.06 0.04
Urea (20 kg N/ha) 0.08 0.01

In West Bengal, De and Mandal (1956) estimated a fixation rate of

15-49 kg N/ha over a six week period. When the soils were fertilized with
phosphorus, the amount of nitrogen fixed was calculated to be 18-69 kg N/ha
during the same period. Watenabe et al, (1951) reported nitrogen-fixation
by Tolypothrix tenuis inoculated in rice fields in Japan to be about 22 kg
N/ha.

Granhall (1976) reported data on in situ nitrogen-fixation rates in

Swedish soils near Uppsala by a 0-2 cm layer of blue-green algae. The results
varied between 0.06 and 27.7 mg N/m2/hr in the light. The main nitrogen
fixing genera were Anabaena, Cylind.rospermum and Nostoc. Clay soils were
among the most suitable habitats for blue-green algae and the environmental
factors, e.g. moisture, temperature, pH and light conditions, were nearly
optimum during incubation. Heterotrophic fixation by bacteria, as judged
from samples kept in the dark for 24 hr, was sometimes observed and varied
between 0.09 and 1.2 mg N/m2/hr. Henriksson et al(1972) reported values from
0.01 to 4.5 mg N/m2/hr for aerobic algal fixation in other clay soils near
Uppsala. Stewart (1967) reported values of 2.5 mg N/m2/yr for alga in sand
dunes.

4.10 TRANSFER OF FIXED NITROGEN

Blue-green algae may liberate large quantities of ammonia extracellularly
under certain conditions. Many reports also indicate that a variety of organic
compounds are liberated (Stewart, 1975).

Studies on symbiotic systems have shown clearly that the nitrogen fixed
by blue-green algae is rapidly transferred to the host. Bergersen et al (1965)
have shown that nitrogen fixed in cycads appeared in all parts of the plants
within 48 hours. Nitrogen fixed in lichens was transferred to the mycosymbiont
15
within 3 hours. N appeared in the angiosperm in a Gunnera system within 1.5
hours of supplying 15N to the alga.

Pot and field experiments have shown that the nitrogen fixed by blue-
green algae becomes available to associated rice plants, which results in
increased crop yield.

/
4.11 PRODUCTION OF VITAMINS AND GROWTH SUBSTANCES BY BLUE-GREEN ALGAE
Many algae produce vitamin B and this process is promoted by the
12
addition of cobalt salt but is not much affected by carbon or nitrogen sources
(Venkataraman et al, 1964; Venkataraman and Neelakanthan, 1967).

In addition to vitamins, the algae produce auxin-like growth substances;

and considerable amounts of ascorbic acid (Vaidya et al, 1970). The algae
liberate these substances either by excretion or autolysis into the surrounding
medium, from which the crop plants can assimilate them.

4.12 MASS CULTIVATION OF BLUE-GREEN ALGAE

An extensive review of the knowledge on the engineering, biological
and operational aspects has been presented by Venkataraman (1969). The
following are summaries of the techniques used by various workers for the
production of blue-green algae biomass.

4.12.1 Japanese Technology (Watenabe et al, 1959)

i. Preliminary shake culture
The alga from agar slants was transferred to round flasks which were
constantly shaken at 32°C. The cultures were illuminated by incandescent
lamps set in the inner wall of the incubator. Two week old cultures
were used as inocula for the tank cultures.
ii. Tank culture
The tank was a 30 litre pyrex glass cylinder housed in a water bath
thermostatically controlled at 32°C. The water bath was provided with
three windows for illuminating the culture from outside. The light
intensity was controlled by adjusting the position of the light source.
The tank had a stirrer and a pipeline for aeration. The carbon dioxide
enriched air (3%) was bubbled at a rate of 350 ml/min into the culture
solution. A rubber tipped wiper was also provided to detach the alga
from the walls of the glass cylinder. Prior to inoculation, the chamber
and the culture medium were sterilized with steam for 15 min at 0.1 kg/mm2,
When the culture attained a density of about 2 g/1 (dry wt) about half
the culture was withdrawn and used as the inoculum for outdoor cultures.
The tank was then filled with fresh medium to the initial volume.
The average growth rate obtained with this tank culture was 0.2 g
dry wt/1/day.

iii. Closed circulation system

For outdoor culture, the culture chamber was made of a long flat
polyvinyl bag of 300 litre capacity, the two ends of which were
connected with each other through a 3 cm diameter and 8 m long tube.
The chamber was directly connected to the culture tank to facilitate
aseptic transfers from it to the outdoor growth chamber. The culture
was constantly stirred by a whirl pump at a rate of 20 1/min and 5% CO
2
enriched air was bubbled into the culture. The culture was cooled by
pouring tap water over the culture bag and the intensity of incident
light was controlled by a bamboo blind. Before starting the culture,
the entire system was sterilized with 1% hydrogen peroxide solution
which was later removed by steaming in sterilized water. The maximum
yield obtained with this outdoor device was 7.9 g dry wt/m2/day.
 The culture media used for the three types of culture described
above are presented in Table 23. For the tank and outdoor cultures
tap water was used instead of distilled water.

Table 23 CULTURE MEDIA USED TO PROPAGATE BLUE-GREEN ALGAE

(Watanabe et al, 1959). 1

g/l

Culture media Pre-culture Tank Outdoor

culture culture

K2HPO4 0.3 0.3 0.3

Mg SO4.7H2O 0.2 0.2 0.2
CaCl2 0.05 0.05 0.05
FeCl3 trace trace
KNO3 0.1 0.1
(NH4)2 MoO4 0.02
Na2CO3 0.2
EDTA 0.1
A5-micronutrients 1.0 ml

1
See Appendix D.8 for composition.

iv. Gravel culture

A special volcanic gravel was used commercially known as Kenuna-Tsuchi,
for growing Tolypothrix tenuis. Sieved gravel was washed several times
with distilled water, soaked in nitrogen-free medium and steam-sterilzed
at 1.1 kg/cm2 for 15 min. The gravel was then mixed with concentrated
algal suspension and the resulting inoculated gravel was placed in
polyvinyl tubes under weak illumination. After about 4 weeks incubation
the gravel cultures were transferred to vinyl bags or glass bottles and
kept at room temperature. These were used for field inoculation.

4.12.2 Indian Technology

Venkataraman (1961) considered the rate and cost of algal production
and developed simple techniques such as dry sand, foam and open air soil
cultures as described below.

i. Dry sand culture

Sieved quartz sand is washed well, boiled in distilled water for about
2 hours and dried at 120°C. The dried sand is soaked in nitrogen-free
medium and steam-sterilized at 1.4 kg/cm2 for 30 min. The sand is then
mixed with a concentrated algal suspension and gradually sun dried;
When dried, it is preserved in tubes or paper bags and stored. However,
an obvious advantage of sand culture is that the sand particles, being
heavy, sink into the mud, thus hampering the rapid growth of adhering alga.
ii. Foam culture
Synthetic sponge is cut into rectangular cubes of 2-4 cm, boiled in
water, soaked in nitrogen-free medium under suction and sterilized.
The sponge pieces are then spread on metallic boats, layered with
vinyl sheeting. The top of the boat is covered by a thick vinyl
sheet, supported by a number of arch-shaped metallic wires attached
to the side of the boat. The sponge pieces are either mixed with
algal suspension before spreading them on the boats or the algal
suspension is spread over them. The boats carrying the inoculated
sponge pieces are incubated under weak illumination. Within 3 to 4
days, the sponge pieces become completely covered with the algal
growth, and after a suitable period of incubation, they are removed
and stored in plastic bags. When these algal-covered sponge pieces
are inoculated into waterlogged soils, they float on the surface of
the water and the algae begin to grow rapidly and spread over the
surface of the water.

iii. Open air soil cultures

The merit of this system lies in the possibility it offers for farmers
to prepare their own inoculum. The basic principle is to grow the algae
using natural sunlight, under conditions simulating a rice field. A
thin layer of soil (2.5 cm) is spread in a rectangular tray of galvanized
iron measuring 0.6 x 0.9 m and flooded with 2.5 cm of standing water
(Fig. 12). After the soil settles down, the specific strain of alga
is inoculated into these trays with phosphate (0.2 g Na HPO /1) and
2 4
molybdenum (0.2 mg MoO /1). The trays are kept in the open air and
3
within 4 to 5 days, the entire surface of the water is covered by a
copious growth of the inoculated alga. The standing water is
allowed to dry and the dried algal mat cracks and peels off as flakes.
This dried material is stored in plastic bags and used for field
inoculation.

An average production of about 20 g/m2/day (dry wt) can be continuously

obtained in this way, with a ten day collection schedule. The
limitations of this method are essentially climatic, for during
the winter months, the rate of production slows down and during rainy
seasons the production may have to be temporarily suspended. However,
the rate of production during clear warm days and the inexpensive
operational costs outweigh some of these limitations. Such algal
nursery beds may be laid out in a farmer’s field, and enclosed by
earth embankments. Useful strains may be inoculated into these
beds just before the rainy season, or where water is available, the
beds may be kept waterlogged.
Prior to seeding, the beds may be given a dressing of lime, phosphate
and molybdenum to promote the growth of the inoculum. Even insoluble
phosphate, like rock phosphate, can be used. The algal scum (or mat)
can be scooped out, dried and used for broadcasting over the main field.
It may also be possible to direct the inflow of irrigation water over
the beds, so that the algal material may be distributed over the entire
field.

iv. Sewage propagation of blue-green algae

Venkataraman (1972) suggested the possibility of producing blue-green
algae in sewage oxidation ponds. The bloom of Oscillatoria grown in
such ponds at Ahmedabad was found to have a high manurial value for
rice-plants.
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4.12.3 Chinese Technology

The practice of algalization in China was observed on a FAO field tour
in 1977. At the Agricultural Research Station, Nanjing, an inoculum mixture
of Anabaena and Nostoc spp is prepared in flasks containing suitable media
under sterile conditions. The algae are then transferred into larger glass
bowls to grow under non-sterile conditions.

The fresh algae are subsequently used to inoculate a nursery plot

(5-7 m long, 1 m wide and 20 cm deep) at the rate of 150 g/m2. The water in
the nursery is 6-7 cm deep. After about 7 days, the algae in the plot attain
a density of 500-1 000 g/m2. The nursery is covered with transparent plastic
sheets to protect the plants from cold.

After preparing the main field for rice cultivation, it is flooded and
750 kg/ha of algae are spread as inoculum. The algae grow and attain 7.5 t/ha
and even up to 16 t/ha within 10-15 days at temperatures above 30°C.

4.12.4 Other Methods

Other methods have been developed to produce mass cultures of blue-green
algae for different research purposes, e.g. enzymology.
i Bath cultures
Haystead et al (1970) grew A. Cylindrica at 25°C in 10 litre batches
in aspirators illuminated.with four 40 watt “grolux” tubes at a
distance of 10 cm from the vessel. The culture was agitated with
a magnetic stirrer and sparged with the required gas mix at a rate of
200 1/h. The culture medium used was the nitrogen-free one of
Allen and Arnon (1955).

ii Continuous culture of blue-green algae

Bone (1971) grew Anabaena flos-aquae A-37 in continuous culture.
The medium used had the following composition per litre:
Na CO - 25 mg; K CO - 25 mg; Tris buffer - 100 mg;
2 3 2 3
Mg SO .7H O - 75 mg; CaCl .6H O - 60 mg; Fe SO .7H O - 60 mg;
4 2 2 2 4 2
Na EDTA - 120 mg;

H BO - 2.86 g; Mn SO .4H O - 1.81 g; Zn SO .7H O - 0.222 g;

3 3 4 2 4 2
Na MoO . 2H O - 0.391 g; Cu SO .5H O - 79 mg; CaCl .6H O - 39 mg;
2 4 2 4 2 2 2

Phosphate, the limiting nutrient, was added as K HPO and nitrate as KNO .
2 4 3

iii. Mass culture for various micro-algae

Soeder (1976) reviewed the technical production of micro-algae, other
than nitrogen-fixing blue-green algae, e.g. Chlorella and Scenodesmus
(green-algae) and Spirulina. Open air ponds for the cultivation of
micro-algae are either circular ponds or horizontal channels with
mechanical or hydropneumatic generation of turbulence by inclined planes.
Further experimentation is needed to refine these methods before they
can be generally adopted for the production of blue-green algae.

4.13 ALGALIZATION OR THE APPLICATION OF BLUE-GREEN ALGAE TO SOIL

4.13.1 Methods of Inoculating Blue-Green Algae

Singh (1961) broadcasted the algal stratum mixed with 1 kg of lime
onto paddy fields after the rice had been transplanted. Sankaram (1971)
mixed an algal suspension with washed sand and spread the mixture over the
field one week after transplanting rice.

Venkataraman (1972) mixed multistrain soil cultures in a bucket of

water containing molybdenum (0.5 kg sodium molybdate/ha) and sprinkled it
uniformly over the water in the rice field, one week after transplanting
the rice.

The multistrain inoculant contained T. tenuis, Cylindrospermum

muscicola, Aulosira fertilissima and Nostoc sp. The idea of using a mixture
of algae was to offset the ecological or edaphic changes to any one particular
strain in a given locality. All these four forms have been found to have good
competitive ability under field conditions. The only shortcoming is that
when several forms are used as inocula, the ultimate proportion of individual
strains in the soils tends to be unpredictable under field conditions; this
variation is not too important, because the establishment of any efficient
form will suffice.
 When direct sowing of rice seeds by broadcasting is adopted, seed
inoculation may be achieved as follows: the suspension of the soil culture
is mixed rapidly with the rice seeds and 2-3 kg of calcium carbonate per
10-20 kg of seed, until all seeds are evenly coated. The coated seeds are
then air dried in shade and sown.

As regards the optimum quantity of inoculum (1-6 kg/ha) and frequency

and time of application, the results are rather inconclusive. Of the
different methods of inoculation, soil application and seed inoculation seem
to be preferable (Venkataraman, 1972)

4.13.2 Algae on Soil Effect of Blue-Green and Rice Yield

In India many investigators have studied the response of rice to algal
inoculation. De and Sulaiman (1950) ran a 5 year pot culture experiment to
find out the effect of algal growth in soils on the yield of rice. In this
experiment, rice plants were grown in pots, some of which were kept in the
dark (without algae), while in others the soil surface and supernatant water
above were exposed to light (with algae present). The results showed that
during the first, second and third years, the yields with algae present and
absent were not very different; However, rice yields were much higher in the
fourth and fifth years. Upon determination of nitrogen at the end of the
experiment there was a considerable increase in nitrogen in the soils in
which algae grew abundantly, but in the soils in which algae were absent, a
loss of nitrogen was evident.

Singh (1961) indicated that after inoculation of rice (variety T9)

with Aulosira fertilissima there was an increase in yields of 368% in pots
and 114.8% under field conditions over the controls (Table 24).

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In a field experiment, Subrahmanyan et al (1965) found that

inoculation with blue-green algae increased the yield of rice over control
by 30%. An increase of 266% was obtained in pot experiments by inoculation
of A. fertilissima (Sundara Rao et al, 1963).
 Relwani and Subrahmanyan (1963) observed that partial soil sterilization
and blue-green algae with a nutrient mixture consisting of lime (1 000 kg/ha)
superphosphate (100 kg/ha) and molybdenum (0.28 kg sodium molybdate/ha)
contributed to an increase in yield of 45% under field conditions. This was
comparable with the yield obtained by the application of 20kg nitrogen/ha as
ammonium sulphate.

Jha et al (1965) reporting on field experiments in Sabour indicated

that algal inoculation increased yield by 40% and its effect in combination
with phosphate was similar to that of green manure plus phosphate (Table 25).

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Venkataraman and Goyal (1969) showed that combined application of

algae and nitrogen at all levels was better than the corresponding nitrogen
level alone and was comparable to the next higher level of nitrogen (Table 26).

Table 26 GRAIN YIELD OF PADDY (IR 8) DUE TO ALGAL INOCULATION

AT DIFFERENT LEVELS OF NITROGEN 1
(Venkataraman and Goyal, 1969)

1967 1968
Treatments
Grain yield (g) Grain yield (g)

l. Control 3.96 2.16

2. Algae 5.83 4.40
3. 20 kg N/ha 6.90 4.45
4. 40 kg N/ha 10.31 7.68
5. 60 kg N/ha 13.55 11.58
6. 80 kg N/ha 14.40 14.20
7. 100 kg N/ha 13.86 14.88
8. (3) + algae 7.30 7.26
9. (4) + algae 10.58 9.26
10. (5) + algae 16.15 13.40
11. (6) + algae 18.03 14.55
12. (7) + algae 24.88 19.55

C.D. at 5% 3.66 1.94

1
Pot culture experiment values are average of six replications.
 In a trial conducted in a farmer’s field with rice variety IRB, it
was observed that algal inoculation increased the yield by 20% over control
with no chemical nitrogen and by 12% over 100 kg nitrogen/ha alone (Venkataramar
and Goyal, 1968). In another cultivator’s field with no added nitrogen
fertilizer, the same authors observed an increase of about 18% in the grain
yield of ASD 5 rice variety compared with an application of a fertilizer
mixture of superphosphate and sodium molybdate (Table 27).

Table 27 GRAIN YIELD OF RICE VARIETY ASD 5 DUE TO ALGAL INOCULATION

(Venkataraman and Goyal, 1968)

Treatments Grain yield increase

kg/h %

l. Control 5 859.4
2. Fertilizer mixture
(112.1 kg superphosphate +
0.25 kg Na molybdate/h) 6 453.4 10.13
3. Fertilizer mixture + algae
(1 kg dry wt/h) 7 470.7 27.48 over 1
18.76 over 2

Sankaram (1971) indicated (in a cultivator’s field in Bihar) that

algal inoculation gave a comparable yield from rice variety BR 34 to that
obtained with an application of 45 kg N/ha (Table 28).
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Recent investigations have shown that, irrespective of varietal

differences among the high yielding fertilizer-responsive dwarf rices, all
respond to algal inoculation (10-15% increase in the yield) even in the
presence of high levels of nitrogen fertilizer (100-150 kg/ha). This results
in an increased output per unit input of added fertilizer (3-5 kg grain/kg N)
(Venkataraman, 1972).
 In Japan, large-scale tests of the effect of Tolypothrix tenuis on
rice yield were started by Watanabe in 1951 at eleven experimental farms and
continued for four years (Watanabe, 1956). As a result of applying this
blue-green alga, the yield of rice (averaged over 11 fields) was increased
by 2.7% in the first year and 8.4% in the second year. In the first year,
only one third of the alga multiplying in the rice field decomposed and was
absorbed by the rice plants; the rest remained as nitrogen fertilizer in the
soil. In one of the experiments, it was clear that the effect of inoculating
the rice field with this alga was almost similar to that of manuring with
71.68 kg/ha ammonium sulphate. Finally it was shown that 4 years after
inoculation with the alga, fields of rice could yield 128% more than
uninoculated controls.

In the U.S.S.R., an increase in crop yield of 13 to 20% has been

attributed to the application of Amorphonostoc punctiforme. An accumulation
of from 3 to 15 mg/100 g of soil was observed after inoculation of sodpodzolic
soil with blue-green algae (Shtina et al, 1968).

In China, Ley (1959) reported that inoculation with Anabaena azotica

increased rice yield by about 24% under field conditions. During a field
trip in China, (FAO 1977), it was observed that the practice of inoculating
rice with a mixture of Anabaena sp and Nostoc sp caused an increase of 10%
in rice yield.

In Egypt, El-Nawawy et al (1958) inoculated one gram (dry weight) of

T. tenuis into lysimeters (20 x 2 m) planted with rice to give a final
concentration of 494 g/ha. This was found to be sufficient to satisfy the
nitrogen requirements of the rice. Abou-el-Fadl et al (1964) studied the
response of rice to inoculation with T. tenuis together with ammonium sulphate,
compost, straw and superphosphate. In general, inoculation with T. tenuis
significantly increased soil nitrogen but had no effect on rice yield.
Apparently the addition of organic matter (compost or straw) stimulated
nitrogen-fixation but the ammonium sulphate was inhibitory. Furthermore the
addition of superphosphate together with ammonium sulphate or compost markedly
inhibited nitrogen-fixation.

Ibrahim et al (1971) also studied the effect on rice yield and soil
nitrogen of adding T. tenuis, phosphorus and nitrogen fertilizer. Addition
of the alga increased the grain, straw and nitrogen yield, as shown in
Table 29. Algal inoculation, without fertilizer, increased the yield of
rice grain and straw by 4.2% and 19.3% respectively. In the presence of
phosphorus, the increase was 7.0% and 56.6% with maximum yield being obtained
from alga plus 0.5 g phosphorus per pot.

El-Borollosy (1972) evaluated the response of rice to inoculation with

the following blue-green algae: N. linckia var. arvense, Cylindrospermum sp,
N. paludosum, N. piscinale, T. tenuis and N. commune (Fig. 13). Five rice
seeds were planted in washed sand in pot cultures. These pots were divided
into two series. The first was irrigated with nitrogen-free nutrient solution,
while the second was treated in the same manner but supplemented with nitrogen.
The highest total nitrogen levels in the plants and in the sand cultures, as
well as the highest fresh and dry weight of rice plants after 115 days were
observed inpots inoculated with T. tenuis, followed by the local isolate
N. linckia var. arvense, then in decreasing order: N. piscinale, Cylindrospermum
sp, N. commune and N. paludosum. The results are well illustrated in Fig. 13.
Table 29 EFFECT OF INOCULATION WITH ALGA T. TENUIS ON THE
YIELD OF RICE (g/pot)
(Ibrahim et al, 1971)

Grain Straw
Treatment
-alga +alga % increase -alga +alga %increase

Control 10.45 10.89 4.2 20.42 24.37 19.3

0 g N+P1 12.37 13.23 7.0 21.06 32.99 56.6
0.25 N+P 24.14 28.03 16.1 32.35 52.96 63.7
1.0 N+P 36.44 39.20 7.5 41.98 62.77 39.5
2.0 N+P 39.15 41.48 5.9 45.68 63.63 39.3
L.S.D. 5% 1.97 14.63

1
The content of P is stable at 0.5.

Field experiments on the inoculation of rice paddies by T. tenuis were

carried out over two successive years (Abou-el-Fadl et al, 1967). The
results (Table 30) showed that the average rice yield following beans was
much higher than that planted after wheat. When no phosphate was added, the
application of alga, or nitrogen, at the two levels gave approximately similar
results. With calcium superphosphate, the effect of 240 g alga was generally
less than that caused by 24 kg N, but the difference was insignificant when
rice followed beans. Moreover, the effect of 480 g alga together with
phosphorus was much smaller than without phosphorus.

Abou-el-Fadl, et al (1970) studied the response of rice to algal

inoculation as affected by crop rotation and the time of application of
nitrogen and phosphorus fertilizers. The data obtained (Table 31) indicate
that: i) in the first four experiments, the best rice yield was from
fertilization with 48 kg N and 36 kg P O per ha. Algal inoculation gave a
2 5
yield similar to fertilization with 24-48 kg N/ha. The algae + 36 kg P O
2 5
did not give a better yield than that without P O ; ii) with the application
2 5
of superphosphate two weeks before and ammonium sulphate four weeks after
transplanting, algal inoculation caused an increase of 23.1% in yield
(experiment 5); iii) in experiment 6 where 72 kg N/ha was added four weeks
after transplanting, algal inoculation increased rice yield by 22.3%;
iv) when the amount of ammonium sulphate was reduced to two thirds the recommended
dose (experiments 7 and 8), inoculation with alga gave a greater yield than
when maximum levels of ammonium sulphate alone were used. In general, the
greenhouse and field experiments indicated that blue-green algae served as a
useful nitrogen source for rice growth.

In a pot experiment Khadr (1975) showed that the nitrogen contents

of rice plants increased as a result of algal inoculation (Table 32).
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Table 30 EFFECT OF NITROGEN AND ALGAL INOCULATION ON RICE YIELD
IN THE ABSENCE OR PRESENCE OF PHOSPHATE
(After Abou-el-Fadl et al, 1967)

Preceding crop
Treatment Horse bean Wheat
per ha Yield Increase Yield Increase
kg/ha % kg/ha %

Without Ca-superphosphate
24 kg N 8 182 9.28 6 058 28.18
48 kg N 8 074 7.84 6 177 30.69
240 g alga 8 312 11.01 6 296 33.21
480 g alga 7 768 3.75 6 476 37.01

With Ca-superphosphate
(36 kg P O /ha)
2 5
24 kg N 8 910 18.99 7 020 48.51
48 kg N 9 108 21.64 6 627 40.21
240 g alga 8 539 14.04 6 130 29.70
480 g alga 7 196 - 6 004 25.20

For consistency, the original figures presented in ardebs and feddans have been
converted to metric units. The following conversion factors have been used:
1 ha = 2.4 feddan
1 ardeb = 150 kg (approx)

Field trials in Egypt (Hamissa, Khadr and Gad, 1978) using the blue-
green algae A. oryzae (local isolate) and A. fertilissima (imported culture),
with and without nitrogen fertilizer, showed that the yield of rice (seed
and straw) was significantly increased: 31.6% by the alga A. oryzae.
Nitrogen uptake by plants was also increased in the presence of the blue-green
alga A. oryzae by 25 to 42.5%. A. fertilissima, however, did not stimulate
the response of rice as much as the nitrogen uptake. Algalization combined
with 36 kg N/ha caused an increase in yield of 72%; nitrogen fertilizer gave
a 45.62% increase.

In another study, Khadr et al (1978) showed that T.Tenuis increased

the yield of rice and nitrogen uptake when it was grown in three different
Egyptian soils. The addition of nitrogen (36 kg/ha) and phosphorus
(36 kg P O /ha) increased the response to algalization. (Table 33).
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4.13.3 Effect of Blue-Green Algae on Crops other than Rice
In addition to the positive effect of algae on rice yield, algalization
has a beneficial effect on other crops as well. For example Venkataraman
(1972) reported that barley benefited, and the introduction of Azotobacter
and algae together increased the yield of oats by 34% (a greater increase than
that obtainable by each constituent separately).

Aiyer et al (1972) reported a significant increase in the vitamin C

content of tomato fruits through algalization. Singh (1961) reported an
increase of 202.7% in grass yield after soil inoculation with Cylindrospermum
licheniforme, but no significant effect on the yield of wheat from algal
inoculation was observed.

Dadhich et al (1969) observed a significant increase in yields and

nitrogen uptake by Capsicum annuum (Chili) and Lactuca sativa (lettuce) due
to inoculation with blue-green algae Calothrix anomala. In these cases, a
combined application of a nitrogenous fertilizer like urea, and alga, was
found to be more effective than an individual application of either urea or
alga. This result suggested that the beneficial effect of the alga was
largely due to the production of growth promoting substances.
 5. FREE-LIVING NITROGEN-FIXING BACTERIA

5.1 AZOTOBACTERIACEAE. TAXONOMY

According to Becking (1974a) the family Azotobacteriaceae comprises four
genera: Azotobacter, Azomonas, Beijerinckia and Derxia. Every genus has one
or several species as follows:
Genus AZOTOBACTER has four species:
A. chroococcum A. beijerinckii A. vinelandii A. paspali
Genus AZOMONAS has three species:
A. agilis A. insigne A. macrocytogenese
Genus BEIJERINCKIA has four species:
B. indica B. mobilis B. fluminensis B. derxii
Genus DERXIA has one species:
D. gummosa

Normally these organisms fix at least 10 mg of atmospheric nitrogen

per gram of carbohydrate consumed, but some Beijerinckia strains fix less
(6-9 mg) because of copious polysaccharide production. The efficiency of
nitrogen fixation increases appreciably at lower carbohydrate levels or lower
oxygen tension. Organic growth factors are not required for growth but for
nitrogen-fixation. Trace elements, in particular molybdenum (a specific
catalyst of nitrogen-fixation) are required.

5.2 GENUS AZOTOBACTER

The genus Azotobacter is one of the oldest known nitrogen fixers. It
comprises four species: A. chroococcum (Fig. 14), A. beijerinckii, A..vinelandii
and A. paspali. Azotobacter chroococcum inoculant is known as Azotobacterin.

Fairly recently it has been discovered that A. paspali contributes to

the nitrogen resources of Paspalum notatum.

5.3 AZOTOBACTERIN
5.3.1 Historical Background
In 1902 Gerlach and Vogel were the first to study the effect of
Azotobacter in soil, on the growth of several plants. They noted no increase
in the dry weight of oats, but an increase of 42% in dry weight and 35% in
nitrogen content of white mustard and a decrease of 83% and 78% in dry weight
and nitrogen content respectively of carrot.

In the Sugar Industry Experimental Station in Prague, Stocklasa (1910)

showed that azotobacter caused a 10% increase in the yield of sugar-beet roots,
and 35% in the yield of beet tops. With oats, the grain yield was increased
by 13% and straw by 16%. Potato was also increased by 31%.

These results, and others reviewed by Rubenchick (1960) led to the

production of an azotobacter preparation in 1937 in all Soviet Union Research
Institutes. It was called azotogen, but the name was later changed to
azotobacterin.
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5.3.2 Production of Azotobacterin

Since 1937, methods of azotobacterin production have undergone
fundamental changes.
 In one method the parent azotobacter culture was grown on an Ashby agar
medium for two days. The cells were harvested and introduced into ground peat
suitable for azotobacter growth; 40 mg of wet bacterial cells per kg of dry
peat were used. After careful mixing, the peat was left for several days at
20-24°C. At the end of this incubation, the peat was diluted with peat powder
and again incubated at the same temperature for several days, until the
bacterial titre reached 108 cells/g peat.

With the development of growing azotobacter in submerged cultures,

A. chroococcum, used in azotobacterin preparations, was also grown in fermenters
and liquid azotobacterin was used. However, the shortcomings of this preparation
are rapid loss of viability upon storage, and difficulties in transportation.

Accordingly, different methods and carriers were tested to obtain dry

preparations of azotobacter, e.g. silica gel, lyophilization, sawdust, talcum,
starch and chalk. None of these media were successful because they affected
the viability of the azotobacter.

Various other methods for the production of azotobacterin were then

developed in state and collective farms and research stations as well as in
industrial plants in the U.S.S.R. The following is an example of a method
popularized at the Microbiology Institute of the Academy of Sciences of the
Ukranian S.S.R.

Well decomposed marshland peat or powdered humus with neutral or weakly

alkaline pH is sieved. The growth medium comprises 1 ton of the substrate:
10 kg sugar or 20 kg molasses, 1-2 kg superphosphate, 20 kg chalk or 10 kg
slaked lime, and a starter of azotobacter cells (harvested from the agar
surface of 25 Roux bottles). The Azotobacterin is then produced in 2 stages.
First, half the substrate is mixed with the appropriate amount of nutrients and
the entire amount of starter; this is mixed well with shovels and then spread
out in a 20-40 cm layer and incubated at 20-25°C. Then, after 3 days incubation,
the rest of the substrate and the nutrients are added. The resultant mass is
kept for a further 3-5 days, with daily shovelling. The bacterial fertilizer
is then ready for use.

5.3.3 Methods of Application of Azotobacterin

The most widely used method of applying azotobacterin is by treating
seed or planting material immediately (i.e. same day)prior to sowing or
planting.

Azotobacter cells are grown on agar in Roux bottles and harvested with
water. The bacterial suspension is further diluted with water and used to
spray seeds, or in the case of potato tubers or seedlings for example, they are
soaked in the suspension.

Peat and soil azotobacterin preparations are also diluted with water,
and the resulting suspension is sprayed on seeds and seedlings. Peat
azotobacterin is sometimes used as a top dressing on sugarbeet.

The following rates per hectare are used for different azotobacterin
preparations:
i. A ar preparation: growth from 600 ml Roux bottles containing
4.6 x 1010 azotobacter cells is used to treat cereals. The growth
from 350 ml Roux bottles is used for treating potato tubers and
vegetable seedlings .
ii. Peat preparation: 3 kg are used to treat cereals and 6 kg to treat
potato tubers and vegetable seedlings. One gramme of preparation
should contain 5 x 107 azotobacter cells.

iii. Liquid preparations from submerged cultures: 200 ml for cereals

and 500 ml for potato tubers and vegetable seedlings.

5.3.4 Effect of Azotobacterin on Plant Yield

Rubenchick (1960) reviewed 1 095 experiments in the U.S.S.R. on yield
response to azotobacter. In 890 of the experiments (81%) there was an increase
in yield of cereal, vegetable and industrial crops. The 1949 report of the
Ministry of Agriculture of the U.S.S.R. (Table 34) shows that increases in crop
yields exceeded 10% in the majority of experiments.

Table 34 EFFECT OF AZOTOBACTERIN ON CROP YIELDS IN VARIOUS

REGIONS OF U.S.S.R.
(Rubenchick, 1960)

Regions Number of Increase in yield after

experiments using azotobacter, %

Spring wheat
Poltava 2 5.48
Vladimir 6 12.92
Potato
Gorki 1 10.21
Kirov 12 23.28
Vorshilovgrad l 23.09
Poltava 1 30.00
Vladimir 2 32.37
Leningrad 5 7.56
Cabbage
Vladimir 1 23.73
Kirov 1 18.48
Voroshilovgrad 3 20.76
Barley
Kirov 2 11.90
Leningrad 1 21.73
Oats
Kirov 2 24.48
Gorki 1 8.98
Rye
Kirov 1 14.29
Vorshilovgrad 1 16.66
Poltava 1 28.57
 Another survey was made by the All Union Institute for Agricultural
Microbiology of experiments carried out at a number of research institutes
from 1949 to 1955 and at collective farms from 1949 to 1951 (Table 35).
Again in the majority of the experiments, azotobacterin increased crop yields.

Table 35 EFFECT OF AZOTOBACTERIN ON CROP YIELDS ON COLLECTIVE FARMS

IN U.S.S.R. 1949 - 1951
(After Rubenchick, 1960)

Plant Number of Positive

experiments experiments Control Crop Increment
% (kg/ha) (kg/ha)

Spring wheat 38 89.0 540 90

Winter wheat 5 100.0 1 040 180
Winter rye 4 100.0 660 85
Oats 26 92.0 755 110
Barley 15 86.0 905 85
Millet 3 66.6 400 70
Buck wheat 1 100.0 250 50
Corn (maize) 8 100.0 1 260 220
Foxtail millet 2 100.0 1 015 390
Seed flax 4 75.0 175 35
Sugar beet 10 100.0 11 520 1 145
Cabbage 3 100.0 10 900 3 265
Cotton 27 81.0 1 280 140
Potato 35 85.7 6 015 1 220

Original figures in centner/ha converted to kg/ha

(one centner = 50 kg).

Experiments on azotobacterin in Czechoslovakia in 1954 (Czechoslovakian

Academy of Agricultural Sciences) showed that yield increases for sugar beet,
maize, carrot and cabbage were 39%, 15.4%, 19.2% and 2.9% respectively
(Rubenchick, 1960). Studies in Romania in 1954, showed an increase of 25 to
50% in sunflower yield.

Results of studies on the response of various crops to azotobacterin

outside the U.S.S.R. have been inconsistent. Increases of 11% in carrot yield,
6.25% for potatoes and 13% in the green matter of mustard have been reported
in Germany. In other instances, e.g. in Switzerland, France, Denmark, Finland
and the U.S.A. results have been negative.
 Brown et al (1962, 1964) studied the effect of inoculation with
Azotobacter chroococcum on plant growth and yield using three methods to
introduce the bacterium into the soil and rhizosphere i.e. cultures applied to
seeds, roots and soil. A wide range of crops was tested, e.g. spring and
winter wheat, barley, lettuce, radish, cabbage, cress, carrot, spinach, sugar
beet, and tomatoes. The results were variable and inconclusive.

Careful investigations using chemical and isotopic techniques have snown

that heavy inoculation with azotobacter cultures fails to effect the incorpora-
tion of nitrogen into soil. The inoculated bacteria frequently die (Alexander,
1977). Nevertheless, plant responses are sometimes evident and thus explanations
must be sought other than in nitrogen metabolism.

A more plausible alternative hypothesis is that species of Azotobacter

produce compounds that act as plant growth regulators. Indeed azotobacters
synthesize stimulatory compounds such as gibberellins, cytokinins and indole
acetic acid (Vancura, 1961; Brown, Jackson and Burlingham, 1968).

Another hypothesis is based on the control of disease by azotobacter

inoculation. Brown et al (1968) reviewed cases where azotobacter inoculations
reduced disease, e.g. flax wilt, lodging of spring cereals, potato scab and
potato blight. They indicated that it is difficult to assess the value of
these reports and unlikely that all the effects are explicable by azotobacter
being antagonistic towards all pathogens, although azotobacter has indeed been
shown to antagonize certain bacteria and fungi, e.g. Candida albicans, Monilia
and Alternaria. Indirect effects cannot be explained either, i.e. stimulation
by azotobacter of some organisms antagonizing pathogens or synthesizing
biologically active substances that stimulate plant development, thus enabling
seedlings to grow away from the disease.

Patel (1969) investigated the effect of azotobacter inoculation on

wheat and tomato rhizosphere microflora. Azotobacter became established in
the rhizosphere of wheat and tomato plants and stimulated their growth. All
the bacterial groups examined were more abundant in the rhizosphere than in
the soil. Inoculation with azotobacter delayed the colonization of roots by
bacteria, actinomycetes and fungi in the rhizosphere, but had no effect on other
organisms. Inoculation did not affect the dominant root surface fungi and
minor changes were not consistent.

Ridge and Rovira (1968) conducted an extensive series of glasshouse and

field studies in South Australia to evaluate the inoculation of wheat with
Azotobacter, Bacillus polymyxa and Clostridium pasteurianum.

The results of trials carried out in 1963 and 1964 are summarized in
Table 36, giving yield relative to 100 for uninoculated plots. The Table
shows that there was a much greater tendency for inoculation to increase grain
yield than to decrease it. If ± 5% is accepted as a minimum meaningfull yield
difference (though not necessarily statistically significant), then, of the 71
comparisons, there were 28 increases and four decreases following inoculation.

5.4 AZOTOBACTER PASPALI

This species was named by Dobereiner (1966). It associates with the
grass Paspalum notatum, a subtropical grass widely distributed in South America.
This organism occurred abundantly in all 75 root surface soil samples from
P. notatum and in 3 out of 4 samples from the root surface of P. plicatum.
Root surface soil samples from 81 other pasture plants, including more than
38 species of gramineae and 8 species of leguminosae, were all negative.
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5.4.1 Potentiality of Nitrogen-Fixation

Dobereiner, Day and Dart (1972a) reviewed the characteristics of
A. paspali. In the field, A.paspali becomes permanently established in the
rhizosphere of four tetraploid cultivars of Paspalum notatum, but not in the
rhizosphere of other, mostly diploid, cultivars, or other Paspalum species,
even when artificially inoculated.
5.4.2 Location of Nitrogenase Activity
By dissecting paspalum roots into different components and measuring
nitrogenase activity by acetylene reduction, Dobereiner et al (1972a) found
that high activity is associated with the presence of roots. Thin sections
showed that P. notatum “batatais” roots with high nitrogenase activity have
a prominent mucigel layer.

Colonies of bacteria were observed on the root surface associated with

mucigel which might provide the right condition for fixing nitrogen and also
protect cells from being washed off the roots.

5.4.3 Amount of Nitrogen Fixed in the Field

Dobereiner et al (1972a) extrapolated that rates 15, 44 and 93 kg N/ha/
year could be obtained in different locations in Brazil. This amount of
nitrogen-fixation would suffice for the growth requirements of P. notatum in
the field. It is also consistent with its ability to establish a good cover
on poor sandy soils without any fertilizers.

5.5 BEIJERINCKIA
Azotobacter indicum was described as a new azotobacter species by
Starkay and De (1939) when isolated from two Asiatic soils of low pH, e.g.
4.9 and 5.2 (Becking, 1961). Later, Derx (1950) urged that this organism be
set in a new genus named Beijerinckia.

Cells are single, straight or slightly curved rods or pear-shaped,

with distinctly rounded ends. Characteristically large, highly refractile
lipoid (poly B-hydroxy butyrate) bodies occur at each end of the cell.

In liquid media no surface pellicle is formed, but the whole medium

becomes a homogenous, highly viscous, semitransparent mass. In some species
(e.g. B. fluminensis) the whole medium becomes opalescent and turbid, and
adhering slime is not produced.

On agar medium, copious slime is produced and giant colonies develop

with a smooth, folded or pellicated surface. The slime is extremely tough,
tenacious or elastic and makes it difficult to remove part of the colony with
a loop (Fig. 15).

Atmospheric nitrogen is fixed in a nitrogen-deficient medium. Molybdenum

is required for nitrogen-fixation.

Cells are highly acid and alkaline tolerant, and will grow between
pH 3.0 and 10.0.

5.5.1 Taxonomy
Four species have been recognized in the genus Beijerinckia: B. indica,
B. mobilis, B. fluminensis and B. derxii (Becking, 1974a).

5.5.2 Geographic Distribution

One of the main features of this genus is its apparent restricted
geographic distribution. Although the organism has been isolated from soils
outside the tropics, its main distribution with respect to cell numbers per
quantity of soil is in tropical soils and to a lesser degree in subtropical
soils(Becking, 1977).
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5.5.3 Soil pH
Becking (1961) showed that soil samples of pH 5.0-5.4, 5.5-5.9 and
6.0-6.4 contained the greatest proportion of Beijerinckia positive samples.
In growth experiments in culture medium, maximum nitrogen-fixation was
obtained at pH 4.5. In some strains, the optimum pH was 4.0 and a considerable
decrease in nitrogen-fixation was observed at higher and lower pH values.
However, some reports indicate that Beijerinckia has been isolated from soils
with pH 8.27 and 7.25.

5.5.4 Vegetation
Vegetation seems to exert an influence on the distribution and
occurrence of Beijerinckia in soil. Derx (1950) noted the particular abundance
of Beijerinckia in hydromorphic paddy soils. It also tends to be more abundant
in the rhizosphere of the rice plant than in tree soils.

Dobereiner (1961) found that in sugarcane fields, Beijerinckia was

particularly abundant along the root surface, suggesting that the root
secretions in the rhizosphere promote the development of this organism in the
vicinity of the roots. However, according to Hegazi et al (1979) Beijerinckia
was not isolated from the rhizosphere soil or root surfaces in sugarcane fields
in Egypt, where the soil pH was between 6.8-7.2.

5.5.5 Significance in Soil Improvement

Evidence has been presented by 15N studies that the roots of sugarcane
can fix 0.2 to 0.38 and 0.44 to 23.8 g of nitrogen/ha/day based on the values
of 15N uptake by non-ammended intact systems and disturbed systems, respect-
ively (Dobereiner, 1961; Ruschel et al, 1977). A similar calculation of
fixed nitrogen taken up by sugarcane leaves in intact systems gives values of
5.7 to 16.9 g of nitrogen/ha.

On the other hand, 15N fixed in the soil, as tested in a disturbed

system, amounted to 250 g nitrogen/ha/day, assuming a soil density of 1.3 and
a 20 cm thick layer where roots are more effective, and a linear rate of
growth. Assuming at least 200 days in the life of the crop, this could amount
to 3.38 kg for plant fixation and 50 kg for rhizosphere nitrogen-fixation.
Examination of the microbial population in this system revealed the presence
of Azotobacter, Beijerinckia, Derxia, Caulobacter, Clostridium and Vibro
(Ruschel and Vose, 1977).

5.5.6 Efficiency of Nitrogen-Fixation

The efficiency of Beijerinckia to fix nitrogen in pure cultures, was
found to be about 16 mg N/g glucose consumed. Beijerinckia grew slowly and
after 20 days only 50% of the glucose had been consumed (Becking, 1978c).

5.6 AZOSPIRILLUM
In 1922, Beijerinck reported the occurrence of nitrogen-fixing
Spirillum in an enrichment culture of A. chroococcum. At first he named it
Azotobacter spirillum, but afterwards, in 1925, he gave it the name
Spirillum lipoferum. Becking (1963) obtained isolates able to assimilate
15
N which required 0.01-0.005% yeast extract to grow below the surface of
semi-solid malate media. Later on, the organism was found to be common in
free as well as rhizosphere soils in different climatic conditions, as
indicated in Table 37. In Egypt, the occurrence of S. lipoferum was reported
in association with several plants (Table 38).
Table 37 OCCURRENCE OF NITROGEN-FIXING SPIRILLUM IN ROOTS AND SOILS
UNDER VARIOUS CLIMATIC CONDITIONS
(Dobereiner, 1975)

a b
Roots Soils
Country Latitude Number Number Number Number
of positive of positive
samples samples
examined examined

U.S.A. 40-48° 72 11 54 5
Brazil 28-30° 52 14 - -
0-23° 584 293 69 35
Senegal 15° 24 10 24 12
Gambia 14° 6 5 6 6
Liberia 6° 8 4 8 8
Nigeria 6° 20 10 18 6
Kenya (1 700 m) 2° 18 0.0 18 4

(a) Washed roots (0.5 cm) of grasses and legumes.

(b) Enrichment cultures in semisolid malate medium, which showed nitrogenase activity
above 10 nano mole CH ethylene/hr/culture (3ml) after 40-48 hr incubation at 33°C
4
and contained predominantly Spirillum lipoferum populations, were considered positive.
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5.6.1 Isolation of Spirillum lipoferum

S. lipoferum can be isolated from field grown root pieces with highly
active nitrogenase activity as estimated by the acetylene reduction method
(Day and Dobereiner, 1975).
 These Spirillum enriched cultures form a fine white pellicle 2-4 mm
below the surface of the medium (Fig. 16). The most active enrichment
cultures, selected by acetylene reduction, were streaked onto plates of the
same basic composition, but with calcium malate and 5 ml of 10% fresh yeast
infusion per litre added. After one week, typical irregular dry white
colonies, often with greenish centres, were selected, replicated into semisolid
calcium malate medium with 15 ml (0.05%) yeast extract/1, and after 24 hours
again checked for acetylene reduction. Purity of the most active of these
isolates was checked again on agar plates before they were stored under oil
in the semisolid calcium malate yeast medium.

The most characteristic forms were observed in the nitrogen-free

sodium-malate medium. They are curved rods of varying sizes with a half to
several spiral forms and with certain refractive lipid bodies. The cells are
extremely active and the mode of motility is very characteristic (Figures 17
and 18).

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5.6.2 Taxonomy
The taxonomic position of S. lipoferum is not yet agreed upon.
Sampaio et al (1978) indicated that there are three groups of isolates of
this species based on the physiological characteristics, especially nitrate
reduction, as follows.
Group I (includes type strain ATCC 29145): reduces nitrate and
produces gas from ammonium nitrate. Does not require biotin for
nitrogen-fixation or growth; does not use glucose; catalase positive;
relatively resistant to streptomycin, tetracycline, gentamycine and
chloramphenicol.

Group II Does not require nitrate; requires biotin for growth on

nitrogen and NH +; grows on glucose; catalase negative; more
4
sensitive to above-mentioned antibiotics.

Group III Identical to group I, except in the ability to reduce

nitrate further and that it shows higher sensitivity to tetracycline.

Krieg (1977) suggested the generic name Azospirillum and named two
species: A. lipoferum and A. brasiliensis.

5.6.3 Location of A. lipoferum

Accumulating evidence shows that A. lipoferum is present on the surface
as well as inside the roots of maize, Digitaria and other grasses (Dobereiner,
1978; Burris, 1977).

5.6.4 Nitrogen-Fixation Potential

Studies with 15N have shown that the organism is capable of fixing
nitrogen by itself (Burris, 1977).

The potential of the spirillum to associate with roots to fix nitrogen

was studied on an excised root system pre-incubated before the acetylene test,
and on an intact plant (Burris, 1977). Higher rates of nitrogen-fixation
(C H reduction) were usually obtained with excised roots rather than from
2 2
intact plants.

5.6.5 Nitrogen-Fixation Rates

The nitrogenase activity of washed roots of field grown tropical
grasses are summarized in Table 39 (Day, 1975). A large variation exists
between sites, species, and sampling time. The maximum values for
Pennisetum purpureum, if transformed into fixed nitrogen indicate fixations
of up to 1 kg nitrogen/ha/day. Activity was associated mainly with roots
and little was noted in the soil.

Dobereiner (1978) reported the nitrogenase activity of S. lipoferum

enrichment cultures from roots of different crops (Table 40).

5.6.6 Temperature
The optimum temperature for nitrogen-dependent growth is high, between
32 and 40°C and similar to that of organisms from tropical environments, e.g.
Derxia and A. paspali. Day (1975) found that below 24°C growth was very slow
and there was none below l8°C. At 42°C the nitrogenase became inactive and
little growth occurred.
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5.6.7 pH
Nitrogenase activity is dependent upon pH, the optimum being between
pH 6.8 and 7.8; there is little growth and nitrogen-fixation below pH 5.5 or above 9.0.

5.6.8 Oxygen
Control of the partial pressure of oxygen is critical in growing S. lipoferum
under conditions requiring nitrogen-fixation. If ammonia is supplied as a source
of nitrogen, the organism grows as a typical aerobic one. When the organism
is fixing nitrogen, it operates as a micro-aerophilic organism (Burris, 1977).

5.6.9 Response to Inoculation

Smith et al (1975) used a liquid inoculant of S. lipoferum for several
crops. The cultures were grown in 10 litre fermenter bottles using nitrogen-
free medium with 0.5% malate as the energy and carbon source. The mixture
was aerated with a mixture of 5% oxygen and 95% nitrogen. A temperature of
40°C gave faster growth than 30°C and was used when rapid growth was desired.
For inoculation, an aqueous solution of the culture was added to the rows to
give about 8 x 106 cells per metre of row and watered into the soil by sprinkler
irrigation immediately after application. Table 41 shows the rates of
nitrogen-fixation in the roots of various crops.

Table 41 FIELD WORK ON THE INOCULATION OF VARIOUS CROPS WITH S.

LIPOFERUM
(Smith et al, 1975)
Acetylene reduction (n mole g-1 dry root h-1)

No. of Range Lines above Lines above

lines 50 100
nana mole % nana mole %

Zea mays 63 0- 377 28 21

Sorghum sp 51 0-1974 88 69
Pennisetum americanum 5 0- 522 60 40
Chloris gayana 36 0- 180 17 5
Cenchrus ciliaris 3 0- 163 33 33
Panicum maximum 7 0- 115 29 14
Digitaria sp 21 0- 32 0 0
Paspalum notatum 3 0- 16 0 0
Hemarthria altissima 15 0- 16 0 0

Smith et al (1978) also reviewed several years work on the response of

various crops to inoculation with S. lipoferum. During the first year (1974)
inoculated Panicum maximum and Digitaria decumbens produced 80 and 61% more
protein respectively than uninoculated controls. In 1975, inoculation
produced significantly higher protein and dry matter yields in Pennisetum
americanum and P. maximum (Table 42). Projected yields using regression
analysis, of both crops indicated that about 40 kg N/ha/year were replaced
by inoculation. Although overall production was lower during 1976, yield
responses due to inoculation were similar to those of 1975 in Panicum maximum.
Response to inoculation was enhanced at higher rates of nitrogen fertilizer
application e.g. 80 and 120 kg N. Changes in population of S. lipoferum were
monitored using fluorescent immunological analysis 10 months after inoculation.
Inoculated plots gave significantly higher counts than the control plots but
the latter had a considerable number of bacteria. The bacterial populations
in uninoculated plots were relatively constant over the fertilizer treatments,
whereas in inoculated plots higher counts were obtained as more nitrogen was
applied.
Table 42 DRY MATTER FORAGE YIELDS INOCULATED WITH S. LIPOFERUM
COMPARED TO UNINOCULATED
(Smith et al, 1975)

Fertilizer N Dry matter yield Increase from

rate
kg/ha inoculated uninoculated inoculation
kg/ha

Pennisetum americanum (pearl millet)

0 4 750 5 300 -550
20 5 820 6 070 -250
40 7 350 6 040 1 310*
80 9 140 7 880 1 260*

Panicum maximum (guinea grass)

0 9 960 10 750 -790
30 15 550 13 460 2 090*
60 17 610 15 560 2 050*
120 20 020 19 470 550

Cenchrus ciliaris (buffle grass)

0 1 640 2 030 -390
20 2 938 2 642 292
40 3 097 2 544 548
80 3 625 2 974 651

* Significant at 5% level

Rinaudo et al (1975) reported that inoculating variety IR 8 rice

grown in a mixture of sand and alluvial soil (9 parts of sand : 1 part of
soil) with Azotobacter chroococcum and Beijerinckia camargensis did not
alter significantly the nitrogenase activity, but inoculation with
Spirillum lipoferum increased the activity significantly (Fig. 19).

In greenhouse experiments, Burris (1977) reported very little response

of maize inoculated with Spirillum lipoferum in terms of yield or total
nitrogen in the plants. Furthermore,in field trials in sandy soil in the
Wisconsin River valley, a slightly higher percentage of the uninoculated
plants gave a better total yield than the inoculated plants, but the
differences were well within the statistical variations of the experiment.

In a different heavier soil near Madison, Wisconsin, Burris (1977)

ran a field trial using 19 different varieties or species of plants
(Table 43); 14 of them showed a positive response and the other 5 a negative
response to inoculation with S. lipoferum.

Again the results are not statistically significant, but they were
definately in a positive direction toward benefit from S. lipoferum
inoculation. Use of these data to calculate yields and increased nitrogen on
a per hectare basis indicates a possible increase in total nitrogen of
2 to 5 kg/ha during a growing season.
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Table 43 FIELD TRIALS ON 19 VARIETIES OR SPECIES OF MAIZE AND OTHER

PLANTS WITH AND WITHOUT INOCULATION WITH S. LIPOFERUM
(After Burris, 1977)

Inoculated Uninoculated
Cultivar
g % N g %N

W 59 E 163 0.99 169 1.02

W 153 R 225 1.25 246 1.16
W 64 A 240 1.19 243 1.23
Wis 900 376 1.12 422 1.07
Field corn 519 1.09 318 1.07
Sorghum 385 0.37 478 0.40
Sorghum (Sudan) 592 0.56 356 0.45
W 729 D 223 1.23 223 1.10
W 182 E 205 1.13 178 1.08
W 629 A 113 1.39 103 1.45
W 513 205 1.28 145 1.25
W 61 235 1.19 218 1.13
W 629 237 1.15 223 1.10
W 438 289 1.11 266 1.12
A 654 211 1.26 223 1.19
Gold Rush 372 1.18 302 1.12
Golden Cross 439 1.15 390 0.96
Golden Beauty 329 1.10 286 1.12
White Cloud 320 1.06 262 1.06
 6. AZOLLA

6.1 INTRODUCTION
Azolla is a symbiotic algal association that grows on the surface of
water (Fig. 20). It has long been grown in lowland rice fields, both as green
manure to enrich the field and as a feed for domestic animals.

Mg' SO VuOUts 1,61-11 LIoucfuE ou muc61.

(coup4pncaq 0 I. muc9u1p6)

Unlike chemical fertilizers and other nitrogen-fixing plants, azolla

does not contaminate the environment nor does it consume the photosynthates
of the rice plants. In China acclimatization has made it possible for it to
live through the high temperatures of summer and the cold temperatures of
winter and now a total of more than 1.3 million ha of rice are cultivated
there with azolla.

6.2 TAXONOMY
The genus Azolla belongs to the family Azollaceae (Salvinaceae) of the
order Salvinales of the Division Pterodophyta (Lumpkin and Plucknett, 1978).
The six existing forms (Becking, 1979) are usually divided into two sections:
Section I Azolla (or Euzolla). This section contains four species:
- A. filiculoides Lam., found throughout the Americas and widely
distributed elsewhere,
- A. caroliniana Willd., found in eastern U.S.A., Mexico and the
West Indies,
- A. mexicana Presl., northern South America to British Columbia
and eastward to Illinois,
- A. microphylla, western and northern South America to southern
North America and the West Indies.
 Section II Rhizosperma. This section covers only two species:
- A. pinnata R. Br., widespread in the eastern hemisphere,
tropical and southern Africa, southeast Asia, Japan and
Australia,
- A. nilotica Decaisne. A large species occurring on the Nile
in Africa.

Tuan and Thuyet (1978) reported that Van Liem (in 1962) described three
varieties of azolla: green, purple and red (Table 44). It has been generally
accepted by farmers and research workers that green azolla is the most resistant
to high temperature, the red to low temperature, the purple one to acidity and
the red is also resistant to salinity.

Table 44 CHARACTERISTICS OF AZOLLA VARIETIES COMMON IN VIETNAM

(Tuan and Thuyet, 1979)

Azolla varieties
Characteristics
Green Purple Red

Size of leaf clusters medium great very great

Leaf arrangement dense dense sparse
Colour of ventral lobes white pinkish white pinkish
Colour of dorsal lobes
- favourable conditions light green dark green dark green
- unfavourable conditions orange purple dark red

6.3 AZOLLA-AZOLLA ANABAENA ASSOCIATION

In 1873 Strasburger published a monograph on Azolla in which he
indicated that there was almost always a blue-green alga belonging to the
Nostocaceae present in cavities of the leaves (Moore, 1969).
i. The fern
Azolla pinnata consists of a floating, branching rhizome, with small,
alternate overlapping leaves and simple roots which hang down into
the water (Fig. 21). Each leaf is bilobed. The upper thick green
lobe contains the green pigment chlorophyll and the lower thin lobe
is pink, without chlorophyll. The anthocyanin pigment is also found
in the upper lobe under certain conditions which gives it a reddish
brown colour. The lower surface of the lower lobe remains in contact
with the water.
Roots occur at branch nodes on the ventral surface of the stem. They
have root hairs and a sheathed root cap that falls off with age. The
roots contribute more to total fresh weight in plants lacking the symbiont
than to those containing it (Peters, 1976).
In nature (but also in the greenhouse), and under a combination of
appreciable light intensity and low temperature, azolla populations
may develop a brick-red colour. The nature of the anthocyanin compound
formed has recently been identified, by chromatographic analyses, to
be two pigments. The main pigment is a luteolinidin glycoside and
there is pigeninidin glycoside in smaller quantities ( Peters et al, 1977 ).
 k!K sl luottu bz.aatitq LGui
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ii. The symbiont

Examination of the Azolla-Anabaena association shows that the algal
symbiont grows in association with the fern at two different topographic
sites, i.e. in leaf cavities of the aerial upper lobe of the bilobed
azolla leaves and in close connection with the apical meristem of the
plants (Becking, 1978 a).
Anabaena azollae Strasburger is the only species mentioned in
symbiotic association with Azolla (Fig. 22)
Pockets of the symbiont, with approximately the same size and shape
as found in the individual leaf chambers, have been isolated by
digestion of the host tissues with cellulytic enzymes. These pockets
contain the symbiont and associated hair cells within the envelope.
When the pockets are ruptured to release the symbiont, the hair
cells remain associated with the envelope material (Peters et al, 1979).
These authors postulated that the hair cells and envelope may be
associated with the exchange of metabolites between the host tissues
and symbiont present in mature cavities.
Many workers have isolated the algal symbiont from A. pinnata
(Venkataraman, 1962), A. mexicana (Holst, 1976), A. filiculoides
(Ashton and Walmsley, 1976) and A. caroliniana (Wieringa, 1968;
Newton and Herman, 1977 ) . However, as no successful re-establishment
of the symbiotic state between any of these isolates and the algal-free
fern plants has been reported ( Peters et al, 1979 ) , caution must be
maintained when referring to any of the isolates as the algal symbiont
until this association can be reproduced.
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6.4 REPRODUCTION
Propagation of azolla is always through vegetative reproduction.
However, azolla is heterosporous and produces both mega- and micro- sporangia
within the sporocarps which are formed by the lower lobe initial of the first
leaf of a branch. Details of this sexual cycle have been reported by
several workers (Becking, 1978 a; Peters, 1978). Significantly, the symbiont
maintains its association with the host plant during the sexual cycle, an
outline of which is given in Fig. 23.

6.5 NITROGEN-FIXATION
Although azolla can utilize nitrogen present in its aquatic environment,
the algal symbiont is capable of meeting the entire nitrogen requirement of
the association. Nitrogen-fixation by Azolla-Anabaena symbiosis has been
demonstrated indirectly by the use of nitrogen-free nutrient solution,
acetylene reduction-gas chromatography and assay of H production, and
2
directly by the use of 15N tracer method (Lumpkin and Plucknett, 1978).

Nitrogen can be supplied to the association by nitrogen-fixation or by

absorption from an aqueous medium, or by any combination of the two without
the loss of nitrogenase activity (Peters et al, 1976). Nitrogenase activity
by A. azollae is protected by the fern from combined nitrogen in the medium.
Peters and Wayne (1974) found that even after six to seven months of growth
in a medium containing nitrogen, there was still appreciable nitrogenase
activity in the azolla fronds.
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Talley et al (1977) measured the nitrogenase activity of A. mexicana

and A. filiculoides in a greenhouse and under controlled environment conditions
(26°C and 300 microeinsteins/m2/sec). The results were respectively 800 + 25
and 773 + 38 n mole acetylene reduced per g dry wt/min. Midday acetylene
reduction rates obtained in the field for fully developed covers of these two
ferns have peak values of about half those found in the laboratory.

Ashton (1974) found that acetylene reduction for A. filiculoides was

960 n mole/g/dry wt/min at about 50% full sunlight and at 25°C. Rates at full
sunlight were only 60% of the optimum, implying an upper limit for acetylene
reduction in full sunlight of about 575 n mole/g dry wt/min.

Becking (1978a) showed that A. pinnata had a doubling time of 2.6 days,
a growth rate of 0.308 g/g per day and nitrogen-fixation of 9.86 mg N/g dry
weight per day.

Watanabe et al (1977) has reported that under the most favourable

conditions the doubling time of A. pinnata is 3 days, and it may fix 7.8 mg
N/g dry weight per day.
6.5.1 Effect of Combined Nitrogen
The symbiont retains significant nitrogenase activity when azolla is
grown with combined nitrogen. When azolla was grown for 85 days with nitrate
or urea as a nitrogen source the rate of acetylene reduction was decreased by
approximately 30% compared to the control grown on nitrogen (Table 45).
Prolonged growth on nitrate or urea (6-7 months) resulted in a 90% decrease
in the rate of acetylene reduction (Peters and Mayne, 1974). Ammonium chloride
was inhibitory to acetylene reduction and it did not support the growth of the
fronds as much as other sources.

Table 45 EFFECT OF COMBINED NITROGEN SOURCES ON ACETYLENE REDUCTION

(Peters and Mayne, 1974)

Rate of C H produced
2 4
Nitrogen source nana mole/mg total SD Number
chlorophyl/min

N 27.1 5.3 7
2
Nitrate 18.5 3.8 6
Urea 18.4 2.4 7
Ammonium 10.3 0.2 10
N dark control 0.09 0.08 4
2

Chu (1978)1 indicated that when the nitrogen concentration (as ammonium
nitrate) is increased nitrogen is reduced. Under a weak light of 1500 lux and
with 1000 ppm ammonium nitrate, activity of nitrogenase is completely checked,
but it retains some activity with 10 000 lux of light (Table 46). With a
weak light of 1500 lux and 100 ppm ammonium nitrate azolla grows better.
When the concentration is increased, the rate of growth decreases. However,
under strong light ( 10 000 lux) and 10 ppm ammonium nitrate, the yield
increases slightly. If the concentration is increased, the yield decreases.

Table 46 EFFECT OF VARIOUS CONCENTRATIONS OF NITROGEN ON THE YIELD

AND NITROGEN FIXATION CAPACITY OF AZOLLA
(Chu, 1978)2
Capacity of acetylene
Nitrogen conc. Yield ratio of azolla reduction C H
2 4
ppm NH NO (after 5 days) % nana moles/mg
4 3
fresh wt/day

Weak light 0 100.0 0.8710

1 500 lux 10 100.0 0.6105
100 116.7 0.3333
500 90.0 0.165
1000 90.0 0
Strong light 0 100.0 4.534
10 000 lux 10 109.8 3.432
100 74.1 2.228
500 68.5 1.452
1000 62.9 0.858

1
Personal communication
2
This Table is extracted from the working paper by Dr Chu for the IRRI Symposium on
Nitrogen and Rice.
Note. The base culture solution was Checkiang Agr. 6302 free from N; light was from tungsten iodic lamp, 28°C.
6.5.2 Nitrogen-Fixation Efficiency

The azolla association is capable of significant light-dependent nitro-

genase H evolution (Newton, 1976; Peters, 1978). In an argon atmosphere,
2
hydrogen production by the association and by the isolated alga was measured
to be 21.7 nana mole H /mg/chlorophyll (a)/min, and 88.6 nana mole/H /chloro-
2 2
phyll (a)/g. (Peters et al, 1976). Peters (1978) indicated that when azolla
was grown on N , efficiency values were 0.94-0.99 while those obtained when
2
the frond was grown on nitrate, were 0.60-0.84. Newton (1976) reported
efficiency values of 1 which indicates no H production in air took place and
2
0.89 when azolla was grown with nitrate. These results suggest that azolla
is a candidate for biosolar H production when grown on nitrate (Peters, 1978).
2

6.5.3 Nitrogen-Fixation and Conversion Factors

The ratio of C H reduced to that of nitogen fixed was found to decrease

2 2
as N pressure increased to 0.8 atm. Ratios of C H fixed at partial pressure
2 2 2
of N of 0.3, 0.6 and 0.8 atm were 3.2, 2.0 and 1.7 for the frond, but they
2
were 4.4, 3.0 and 2.5 for the blue-green algae, respectively (Peters, 1978).

6.5.4 Release of Fixed Nitrogen by the Algae

Anabaena azolla, when isolated from the azolla’s cavities and incubated
under N , releases ammonia into the incubation medium (Peters, 1976). No
2
ammonia is released during incubation under Ar or N + 2% Co. 15N experiments
showed that, in general, 50% or less of the nitrogen fixed is incorporated
into the symbiont. Newton (1976) indicated that fixed nitrogen constituted
up to 50% of the total nitrogen in the free nitrogen pools of the azolla
association when it was grown in the absence of combined nitrogen, and that
there were substantial levels of glutamine, glutamate and cystathionine.

Some azolla species secrete nitrogenous compounds into their aquatic

environment. Shen et al (1963) reported that a Chinese variety of azolla
(Whole River Bed) released 12-14% of its fixed nitrogen into the water. Brill
(Peters, 1977) indicated that a specimen of A. mexicana excreted about 20%
of its fixed nitrogen ammonia.

Because of the ability of azolla to fix nitrogen and to pump out ammonia,
it is hoped that this system can be employed as a living biosolar-driven system
for ammonia production in the future.

6.5.5 Photosynthesis of Azolla

Both the azolla plant and the algal symbiont removed from the plant are
photosynthetically competent. The azolla contains chlorophyll(a) and (b) while
the alga contains chlorophyll(a) and phycobilins. Both organisms contain
carotenoids. On the basis of three separate methods of determination, the
algal partner has been estimated to contribute 15 to 20% of the association’s
total chlorophyll (Peters et al, 1979).

6.5.6 Growth of Azolla

Talley et al (1977) studied the growth rates of A. filiculoides and

A. mexicana that are indigenous species in the Sacramento Valley of Central
California. A. filiculoides is ubiquitous in agricultural drainage channels
and other calm water surfaces. Healthy ferns growing in open water have a
biomass doubling time of less than 7 days during the summer. When fully
developed A. filiculoides may possess a biomass of over 180 g/dry weight/m2
and contain 58 kg N/ha. A. filiculoides is frost hardy and can withstand
temperatures as low as 5°C without suffering apparent harm.
 A. mexicana grows rapidly during the summer (with a doubling time of
2-8 days) but the fern is not resistant to frost. Maximum biomass development
which has been observed in the field is 98 g dry weight/m2, representing a
nitrogen cover of 29 kg/ha.

Azolla populations grown with rice during the summer have rapid initial
growth and reach peak biomass 28 days after inoculation. The subsequent
decline in biomass is rapid, especially in A. mexicana, implying a significant
nitrogen loss from the biomass to the aquatic environment.

Espinas and Watanabe (1976) determined the growth rate of Azolla pinnata.
The results (Table 47) showed that azolla doubled its weight in 3-5 days during
the first week. In two weeks, approximately 30-40 kg N/ha had accumulated in
the azolla tissue.

Table 47 NITROGEN INCREASE AND GROWTH RATE OF AZOLLA

(Espinas and Watanabe, 1976)

Nitrogen increase, weeks Growth rate,

Date inoculated kg/ha weeks, kg/ha
0-1 1-2 0-2 0-1 1-2

1 200 cm2 tray

11/7 12.9 23.1 36.0 3.7 5.6
12/2 9.8 17.8 27.7 4.6 5.9
1 884 cm2 tray
11/7 14.5 16.3 30.3 2.9 7.4
12/2 10.4 12.2 22.4 3.6 8.3

6.6 TOTAL NITROGEN INPUT ESTIMATES OF AZOLLA

Estimates of the total nitrogen input of azoZZa in paddy soils are
rather variable. Some examples are given below.

Becking (1972) calculated, upon C H reduction assays of A. pinnata,

2 2
a maximal input of 335-670 kg N/ha/year. Saubert (1949) reported a biomass
of 310 kg N/ha/year. Moore (1969) estimated a potential fixation value of
100-160 kg N/ha in three to four months. Olsen (1972) found an input of
95 kg N/ha with A. filiculoides over 3 months (June-August) which would give
380 kg N/ha/year if growth and nitrogen fixation should proceed throughout the
year. Watanabe (1977) measured an accumulation of 330 kg N/ha in 220 days.
Talley et al (1977) measured (in the Sacramento Valley of Central California),
nitrogen accumulation of over 50 kg N/ha within 35 days with A. filiculoides,
which means an annual increase of 500 kg N/ha. High figures are cited from
Vietnam, e.g. 1 000 kg N/ha, and China, e.g. 92.7-151.8 kg N/ha in 1.5 months
(Shen et al, 1963).

These estimates are based in most cases on unlimited continuous growth

of azolla in areas of open water with optimal conditions, whereas, in
agricultural practice, the growth of azolla is discontinuous. Correcting the
values by taking into account the cover and reduced nitrogen fixation at night,
a figure of 103-162 kg N/ha would be acceptable (Becking, 1976).
. 6.7 ENVIRONMENTAL FACTORS AFFECTING AZOLLA
6.7.1 Water
Water is a fundamental requirement for the existence of azolla. At a
relative humidity of less than 60% azolla becomes dry and fragile; and when
it is completely dry it dies. Although azolla can grow on a wet mud surface
or wetted peat litter, it prefers to grow in a free floating state on a calm
water surface, and may be found on ponds, canals, lakes and also on some
slow-moving rivers. It survives only in the presence of sporocarps (Becking,
1979).

6.7.2 Light
While some reports indicate that high intensity of light enhances the
growth of azolla, others show that the optimal light intensity for growth is
15-18 kilo lux and that at higher intensities growth and photosynthesis are
inhibited. At pH 5, high intensity of light enhances growth but at pH 6 and
7, it inhibits growth (Tuan and Thuyet, 1979).

Ashton (1974) observed that both the growth rate and nitrogenase
activity of A. filiculoides change with increasing light. In South Africa,
it was found that growth increased with increasing light intensity, up to a
maximum of 50% sunlight (i.e., about 49 000 lux). Further increases in light
intensity decreased the growth rate. In 25% sunlight the growth rate was
higher than in full sunlight. The nitrogenase activity followed the same
pattern i.e. highest at 50% sunlight and lower at both 25 and 75% sunlight.

Peters (1976) showed that nitrogen (C H ) fixation was already

2 2
saturated at the low light intensity of 4 840 lux in the intact association
of azolla,but only at 2 156 lux in the isolated alga. The low light
requirement of the symbiont can be explained as an ecological adaptation
because of the lower light intensities that reach the symbiont as a result of
the screening effect of host pigments in the intact association.

Brotonegoro and Abdulkadir (1976) studied the effect of prolonged

shading on the activity of A. pinnata. The exposure of plants shaded for 5
hours to light intensities of 16-57% of full sunlight did not affect the
nitrogenase activity of the association, but a shading of 16% of full
sunlight for 10 hours reduced the nitrogenase activity of the intact plants
to 30% of that of the control plants.

A 12 hour dark period caused the nitrogen (C H ) reduction rate to

2 2
remain constant at 25-50% of that in light. This demonstrated that, in the
dark, the intact association can supply the algal symbiont with endogenous
reductant accumulated during the previous light period (Becking, 1978a).
Talley et al (1977) showed that A. mexicana has a greater tolerance to high
light intensity than A. filiculoides.

6.7.3 Temperature
Several workers have reported various ranges in temperatures, but the
optimum temperatures for azolla spp seem to be between 18-28°C (Tuan and
Thuyet, 1979).

The optimum ranges of temperature for A. caroliniana, A. filiculoides

(Ashton and Walmsely, 1976) and A. pinnata (Watanabe et al, 1977) appear to
be between 25 and 30°C. Although A. mexicana will not tolerate frost,
A. filiculoides will survive it (Talley et al, 1977).
6.7.4 pH
Several workers have indicated that the optimal pH for azolla growth
varies from 4.5 to 7.5. They have shown that the response of azolla to pH
depends on many factors such as temperature, light intensity and the presence
of soil and soluble iron (Tuan and Thuyet, 1979) (Table 48).

Table 48 INFLUENCE OF pH ON AZOLLA GROWTH IN A RICE FIELD WATER

SOLUTION UNDER DIFFERENT CONDITIONS (RELATIVE DRY WEIGHT)
(Tuan and Thuyet, 1979)

Relative dry weight %

Normal High Low light With High With

pH condition temperature intensity iron temperature soil

5 100 100 100 100 100 100

6 87 61 93 110 92 121
7 82 54 86 108 88 119

Singh (1977) indicated that using aoils of different pH revealed that

the fern grew better in the slightly acid soils of Cuttack (ph 5.9) but
grew badly in Hyderabad soil of pH 8.1 (Table 49). The very acid soils of
Pokkali (pH 2.9) and Kattampalli (pH 3.6) did not support growth and the
plants died within 2 and 10 days in the two soils, respectively. The pH of
standing water on Cuttack, Hyderabad, Kattampalli, and Pokkali soils, was
6.6, 8.1, 3.4 and 3.2 respectively, after 15 days.

Table 49 GROWTH OF AZOLLA IN SOILS OF VARIED pH

(Singh, 1977)

pH after 15 days
Soil Initial of floodin Inoculum Growth after
collections pH fresh wt, g 15 day
Standing fresh wt.g
Soil water

Cuttack (CRRI) 5.9 5.9 6.6 2 15.1

Hyderabad 8.1 8.0 8.1 2 6.5
Kattampalli 3.6 5.6 3.4 2 0
Pokkali 2.9 3.0 3.2 2 0

6.7.5 Season and Soil Conditions

The growth of azolla on alluvial neutral soil, rich in phosphorus, is
better than on acid soils poor in phosphorus. But on acid soils, the yield
in summer is higher than that in other seasons (Table 50) (Tuan and Thuyet,
1979).
Table 50 AZOLLA GROWTH IN DIFFERENT SEASONS AND TYPES OF SOILS
(Tuan and Thuyet, 1979)

Seasons and Alluvial pH 7.0 Alluvial pH 5.5 Degraded light pH 6.1

Temperature P O 0.09% P O 0.57% P O 0.045%
2 5 2 5 2 5
range
Fresh wt Growth Fresh wt Growth Fresh wt Growth
t/ha/5 days rate t/ha/5 days rate t/ha/5 days rate

Spring
20-25°C 11.19 0.245 8.53 0.141 9.13 0.165
Summer
27-29°C 10.63 0.225 9.70 0.190 9.66 0.186
Autumn
26-29°C 9.36 0.170 9.22 0.168 7.78 0.111
Winter
17-22°C 7.19 0.090 9.34 0.173 9.51 0.180

6.7.6 Inoculum Density

Tuan and Thuyet (1979) indicated that as a general rule the optimum
inoculum density is 500 g/m2 For specific cases the following ranges of
densities were regarded as appropriate.
i. in paddy fields for the production of green manure: 250-500 g/m2
ii. in the inoculum production plots or for the production of compost: 500-750 g/m2
iii. in the production of animal feed, or where land is limited: 750-1 000 g/m2

However, it appears that densities can vary considerably in different

countries. In Vietnam, Thuyet and Tuan (1973) recommended a planting rate of
500 g/m2 for A. pinnata which can increase to a density of 1 000-1 600 g/m2
in winter and 1 000-1 400 g/m2 in summer. In India Singh (1977) recommended
rates ranging from 400-1 000 g/m2. In the U.S.A. an inoculum of 500 g/m2 of
A. mexicana and A. filiculoides is recommended (Talley et al, 1977).

6.7.7 Nutrition
i. Phosphorus
Rains and Talley (1978) indicated results of experiments with
A. filiculoides and A. mexicana to ascertain their requirement of
phosphorus. Azolla grown in a phosphorus-deficient solution
decreased or ceased growth, became red in colour and developed
curled roots (Watanabe and Espinas, 1976). A. filiculoides grown
as a fallow crop in the spring might require as much as 60 kg
P O /ha, either as a single application or as a split treatment
2 5
of 30 kg P O /ha each time. Maximal production of A. mexicana
2 5
was obtained with a split application of 15 kg P O /ha in equal
2 5
doses at 1, 10 and 21 days after inoculation. The higher yield
thus obtained seemed to result from less stimulation of competing
green or blue-green algae. At higher levels of phosphorus
fertilization, competing organisms proliferated and may have inhibited
azolla development (Rains and Talley, 1978).
ii. Iron
The addition of 1.5 kg Fe/ha was sufficient to eliminate the deficiency
in paddy water (Rains and Talley, 1978). The critical concentration
of iron for azolla growth was found to be 20µg Fe/l.
The availability of iron as a nutrient to plants is affected by pH.
When the pH is high ( alkaline ) iron is precipitated as hydroxide and
is therefore less available to plants. Unavailability of iron causes
yellowing of the ferns (Espinas and Watanabe, 1976).

Iron is usually present in culture medium at levels of 0.01 to 3.0 mg/l.

It can be added in the form of concentrated iron citrate or EDTA (or
versenate) to give a final concentration of 2-5 ppm iron, (Becking, 1979).
In the field, 9.1 kg Fe/ha are enough for a 5 month irrigation period
for rice with a water flow rate of 1.4 1/sec (Becking, 1972)

iii. Other mineral nutrients

Azolla plants require all the macro-nutrients (phosphorus, potassium,
calcium, magnesium, iron ) and micro-nutrients ( molybdenum, cobalt ) .
Vanadium and tungsten are reported to be a suitable substitute for
molybdenum ( Moore , 1969 ) .

Espinas and Watanabe ( 1976 ) compared the growth of azolla in

phosphorus, potassium, calcium and magnesium deficient media,
a nitrogen ammended medium and in a complete nitrogen-free
medium as control (Table 51 ) . Fresh weight, nitrogen content and
acetylene reduction activity measured when harvesting the azolla
in the P and Ca deficient media gave far lower values than those
in the control, K and Mg deficient media and the N-ammended medium.
Fresh weight and nitrogen content data for azolla in a potassium
deficient medium are still lower than those in the control medium but
acetylene reduction activity is twice as much or more than that in
the control medium. This means that the nitrogen-fixing ability of
potassium deficient azolla is kept active.

Table 51 FRESH WEIGHT, TOTAL NITROGEN AND ACETYLENE REDUCTION

ACTIVITY OF AZOLLA IN P, K, C AND Mg DEFICIENT MEDIA AND
N-AMMENDED MEDIUM
(Espinas and Watanabe, 1976)

Treatment Fresh weight Total N C H : C H assay

2 2 2 4
g/plate kg/ha 1 nana mole/g/ha

-N 35.40 ± 2.35 44.58 ± 0.21 340 ± 200

-N -P 8.15 ± 0.65 7.36 ± 0.22 12 ± 0
-N -K 11.60 ± 2.00 10.95 ± 1.95 830 ± 230
-N -Ca 3.41 ± 0.34 2.20 ± 0.11 0
-N -Mg 30.30 ± 1.70 34.28 ± 0.73 441 ± 539
+N 12.50 ± 0.90 19.31 ± 1.86 259 ± 119

1
Plate size was 314 cm2
2
The contaminated green algae were not completely removed
3
Average ± standard deviation
6.7.9 Pests of Azolla
Azolla is frequently attacked by lepidopterous and dipterous insects,
which are common in the summer and in rainy seasons (Singh, 1977; 1978), but
insect occurence is low when the morning water temperature remains below 20°C.
The larvae of insects, mostly Chironomus, Pyralis and Nymphulus (Thuyet and
Tuan, 1973) roll the leaves of plants together and feed on them, causing
brown patches in the azolla cover (Singh, 1979). Other pests reported from
different countries are reviewed by Lumpkin and Plucknett (1978) and among
them are mites, weevils, aphides and moulds.

Controlling pests is most important, otherwise the entire crop of

azolla can be damaged in a few days. These insects can easily be controlled
by carbofuran at the rate of 2-5 kg/ha. A smaller quantity (100-500 g) of
carbofuran mixed with the inoculum for one hectare can protect the crop and
encourage fern growth. Singh (1979) found that application of the insecticides
BHC, sevidol and carbofuran did not adversely affect azolla or its symbiont.
Chinese literature recommends the use of BHC and DDT (Lumpkin and Plucknett,
1978).

6.7.10 Pesticides
Becking (1979) tested 8 compounds in concentrations usually applied in
agricultural practice: carbofuran (100 mg/1), dimecron (80 mg/1), diazinon
(40 mg/1), thiodin (40 mg/1), 2,4-D (amine) (67 mg/1), DDT (50 mg/1),
dithiocarbamate (33 mg/1) and MCPA (67 mg/1). Nitrogenase activity was
assayed with the C H reduction test in 70 ml serum bottles, and incubated at
2 2
28°C at a light intensity of 12 000 lux. In a 48 hour incubation period, no
significant decrease of the C H reduction rate was observed, possibly because
2 2
the algal symbiont living in the leaf cavity of the aerial leaf was not in
direct contact with either the nutrient solution containing chemical, nor the
vascular system of the plant, so that the substance could not reach the
symbiont suring short-term exposure.

Cohn and Renlund (1953) showed that A. caroliniana present in a canal

could be controlled and eliminated by spraying with 1 000 to 10 000 ppm of
2,4-D. Diquat and paraquat was effective at l.0 ppm (Blackburn and Welden
1965). Paraquat was also effective at a concentration of 0.56-2.24 kg/ha
(Matthews, 1963). Diesel oil was also toxic to azolla when applied as a
foliar 1:1 spray with water (Oosthuizen and Walters, 1961).

6.8 CONTROL OF AZOLLA PESTS

Chu (1978) summarized methods recommended in China to control azolla
pests as follows:
i. intermittent drainage of rice fields is good for azolla and rice,
and favours the natural enemies of azoZZa pests, such as ants
and frogs;

ii. soil and azolla can be disinfected by using 4.8 kg/ha (8 catties)
of carbofuran at the last harrowing; 50% fenthion or 1 500 dilutions
of 50% fenthion may be used to soak mother azolla for 5 min;
iii. insecticides: it is important to know the characteristics of pests
before deciding on the kind and quantity of insecticide to use.
It is better to use different insecticides in rotation or mixtures
of them, or to use them in combination with fertilizers. A list
of suggested insecticides is given in Table 52.
Table 52 SOME PRINCIPAL INSECTICIDES FOR CONTROL OF AZOLLA PESTS
(Chu, 1979)

Name of Concentration Method of

insecticide ppm application Pests Effect

90% trichlorfon 1 000 Spraying Pyralis sp Quite good

Bagous sp “ “
80% dichlorvos 3 000 “ Pyralis sp “ “
Bagous sp “ “
Nymphula1 No effect
50% cartap 1 000-l 500 “ Pyralis sp Quite good
Nymphula 1 “ “
1
50% xenitrothion 800-2 000 “ Nymphula “ “
2
Polypedilum
50% malathion 2 000 “ Nymphula2 “ “
Pyralis sp “ “
25% phosmet 2 000 “ Pyralis sp “ “
50% fenthion 800 “ Polypedilum2 “ “
carbofuran 30-l00 Spraying or Pyralis sp Fairly good
4-5 kg/ha Nymphula sp “ “
mixed with soil Polypedilum sp “ “
Tea seed cake 30 Splashing Radix swinhoer3

1
Nymphula tarbata Butler
2
Polypedilum juinoense Hauber
3
Radix swinhoer H. Adams

6.9 FUNGAL DISEASES

Rymnaea causes grey spots on the surface of the leaves. The disease
is widespread at temperatures higher than the optimum. To control the disease
spraying with diluted defusit (1 part defusit to 250 parts of water) is
recommended after 7-10 days, using 1 125 l/ha of diluted mixture. A mixture
of equal proportions of trichlorfon (diptrex) and malathion (both at 0.1 to
0.2%) is also used to control the disease. The disease known as “damping off”
occurs at high temperatures and high plant densities and is controllable by
thinning and ploughing-under the crop (FAO, 1977).

6.10 ROLE OF AZOLLA IN AGRICULTURE

Azolla plays a significant role in agriculture in certain parts of the
world, where it is used as green manure and as animal feed, or grown with
rice. Another potential use of azolla is to improve quality of water.
However, in other parts of the world, it is still regarded as a nuisance and
a weed.

6.10.1 Azolla as Green Manure

Singh (1979) has outlined the method used by farmers in India to grow
azolla as a green manure. Fresh azolla should be spread on the surface of
standing water (5-10 cm deep) at a rate of 1 000 kg fresh weight/ha and
8-20 kg P O /ha should be applied. Mixing 100-500 gm/ha of carbofuran with
2 5
the inoculum protects the azolla.
 Between 10-20 days, when azolla covers the whole field, the plant
should be incorporated into the soil with a rotary hoe or other implement, and
the rice seedlings should then be transplanted into it. Azolla dies gradually
if left unincorporated on the soil surface.

If there is not sufficient water to irrigate a large area, small plots

of fallow land can be used for azolla cultivation and the plants can then be
gathered weekly, leaving some inoculum for further growth. About 5-10% of
the land will produce enough organic matter to fertilize the entire area in
2-3 months.

Fresh or decomposed azolla may be used as a top dressing at a later

stage of plant growth.

In China, azolla is grown on harvested rice fields or fallow land

between the first and second crop. When it is fully grown on the surface
water, it is turned into the soil with a small tractor with rotavator. The
remnants are left to propagate further for use as manure. The process is
repeated several times.

Soil mixed with azolla can be used directly in the fields where it
grows or can be dug out to use as fertilizer elsewhere. It is estimated
that in this way 66m3 of compost material per mou (1 000 m3/ha) can be obtained.
The results of experiments have shown that 10 tonnes of comnpost mazerial
generally increase rice yield by 470 kg/ha.

In the United States, Rains and Talley (1978) cultivated Azolla

filiculoides in 22 m2 plots at a rate of 50 gm/m2. The azolla inoculated
covered less than 10% of the plot area and contained 1.5 kg/ha. Within 18 days,
it covered the entire plot and the nitrogen had increased. After 35 days in
inoculated paddies, the azolla cover attained a biomass of 1 700 kg (dry weight)
/ha containing 52 kg N/ha. The azolla was mature at 35 days and further
experiments revealed a steady decline in nitrogen content and a steep decline
in acetylene reduction capacity. It was also shown that 40 kg N/ha added as
azolla produced almost the same yield response from rice as 50 kg N/ha added
as ammonium sulphate.

6.10.2 Azolla Grown in the Field

Subudhi and Singh (1978b) reported a nitrogen-fixing rate of 7.5
mg N/day/g dry weight of azolla in 10 days incubation.

When 0.1-0.4 kg inoculum per m2 was sown in the field (Singh, 1978)
8-15 t green matter per ha was obtained in 7-20 days and 333 t/ha, which
contained 840 kg N (Table 53) and 20 t dried compost, was produced annually
in breeding plots. A thick layer of azolla ensured about 30-40 kg/N/ha
because it contained 4-5% N on a dry weight basis (0.2 to 0.3% on a fresh
weight basis).

Shen et al (1963) compared the nitrogen-fixation ability of “red azolla”

with alfalfa (Medicago sativa) and soybean (Glycine max). They reported that
1.5 months of azolla cultivation increased the nitrogen content of the soil to
a level equal to that produced by a crop of soybeans, but to only 40% of the
level produced by alfalfa. Their calculations were based on alfalfa, soybean
and “red azolla” nitrogen contents of 2.87, 2.90 and 3.50%, respectively
(dry weight basis).

The yield of azolla varies according to the locality and species of

azolla. Table 54 summarizes data obtained by several workers.
--,ge
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The data were collected from 32 plots with a total of 256 m2 in the field and from
12 tanks with a total of 82.8 m2. The azolla crop was collected four times a month
over 20 months (June to January, 1978).

Table 54 YIELD OF AZOLLA SPECIES IN DIFFERENT LOCALITIES

Species Yield/t/ha
Fresh Dry weight N/kg/ha Reference

A. pinnata 37.8 2.78 Gopal, 1967

(India)
A. mexicana 0.98 24-45 Talley et al, 1977
(U.S.A)
A. filiculoides 1.8-2.57 58-105 “ “ “ “
(U.S.A)
A. filiculoides 3 90 Olsen, 1972
(Denmark)
A. pinnata 8-10 25-50 Thuyet and Tuan, 1973
(Asia)

6.10.3 Crop Response to Azolla

Singh (1979) showed that a layer of azolla covering a one hectare
rice field contains about 10 t of green matter and provides about 25-35 Kg N/ha.
This amount can be doubled by growing a second azolla layer after the first
one has been incorporated or gathered. Pot (Fig. 24) and. field experiments
conducted in both seasons (Rhabi and Kharif) over 3 years with high yielding
rice varieties (IR 8, Vani, Supriya, Ratna, Jaya, Kalinga-2, Pusa 2-21) and
cultivars (CR 1005, CR 181-5, CR 188-10, SG-1) revealed that the use of
azolla increased plant growth, number of tillers, grain and straw significantly
(Table 55).
 kW Sy cLob Lcebougo to %moire
TuocnjuCTOU
(Ltow !GIs co Litpc)
volin lucoLboL2ccq'
12q¡ro LyoutTutt trucl
coutuoi (uo lzuliq' uo utpuni.4)
(cAnuLtpniGq P. blr vap)

Although green manuring with 10 t of azolla per ha is as efficient as

a basal application of 30 kg/N, a split application of N fertilizer was found
to be slightly superior. Grain yield increased by as much as 54% when 10-12
tonnes of azolla were incorporated in the soil or allowed to decompose without
incorporation.

Table 55 EFFECT OF AZOLLA ON YIELD OF IR 8 AND SUPRIYA

RICE VARIETIES DURING RABI 1977
(Singh, 1978)

No. of panicles Wt. of panicles Grain yield Straw yield

per m2 g/m2 kg/ha kg/ha

IR 8 Supriya IR 8 Supriya IR 8 Supriya IR 8 Supriya

Control 339 434 745 548 4 722 3 489 3 607 2 571

Azolla, t/ha 430 545 880 825 5 918 5 125 4 643 3 786
Increase % 27 26 18 51 25 47 29 47

6.10.4 Availability of Azolla Nitrogen

The mineralization of azolla nitrogen and its incorporation with
flooded soil was faster at room temperature when incubated at 24°C (± 2°C).
About 56% of N as ammonia was released in 3 weeks of incubation at 24°C,
whereas 80% was released at room temperature (Singh, 1979). Watanabe et al,
(1979) reported that 62-75% of the azolla N available to the rice crop was
slightly less than the N from ammoniacal fertilizer.
6.10.5 Azolla Cultivated under Rice Plants
The beneficial effects of azolla grown under rice plants depend on
several factors as follows (Chu, 1979).

i. The variety of the fern: well.-grown vigorous plants usually contain

3.4% dry matter and 25-32% organic carbon and have a C : N ratio
of 10. Such green manure decomposes quickly in the rice field to
supply available nitrogen to the growing rice plants.

ii. The variety of the rice: rice varieties with a long growing season
generally give better yields than those with short ones because the
longer growth period permits greater nutrient absorption.

iii. The type of soil: the addition of green manure to soils of high
fertility usually increases crop yield by 9.9-27% but in soils of low
fertility a 13.3-42.7% increase in rice yield can be expected.

iv. Cultural practice: turning azolla under by hand or machine gives

better results than natural decompostion.

In the U.S.A. it appears feasible to grow azolla with rice provided

proper management is applied (Talley et al, 1977). The density of the rice
crop and the timing of the cultivation of azolla with rice are important
factors. In experiments rice yields from dual cultivation of A. filiculoides
and rice were 25% higher than those from unfertilized controls. The yield
response was equivalent to the addition of 10 kg/ha as ammonium sulphate.
With A. mexicana, rice yields were about 3 times those of controls without
azolla when 40 and 80 kg N/ha were applied as ammonium sulphate.

6.10.6 Place of Azolla in Rice Rotation in Vietnam

In Vietnam, azolla is considered as a winter crop like potatoes,
vegetables, maize and soybean. The place of azolla in the rotation is
summarized by Tuan and Thuyet (1979) as follows:

i. spring rice (February-June) - early summer rice (July-late October)

- maize, soybean, or multiplication of azolla.
ii. spring rice ( February-June ) - medium summer rice ( July-November )
- potatoes or vegetables.
iii spring rice (February-June) - late summer rice (July-November) -
azolla as winter crop.

6.10.7 Double Narrow Row Method

Chu (1979) has described a double (narrow) row method of rice
cultivation with azolla between the double rows of rice plants. This enables
the azolla to grow for most of the year and hence increases the rice yield.

The spacing between the double rows of rice plants is 53-66 cm, and
between the narrow rows of rice plants the spacing is 13 cm. The distance
between the clusters (hills) of rice in the rows is 6.5 cm. The azolla is
grown in the wider space between the double rows of rice plants (Fig. 25).

Table 56 shows that with this system an area of 2 ha produced an

average rice yield (i.e. with two crops) equivalent to 15.65 tonnes/ha. The
average yield of azolla was 133 tonnes/ha. The total nitrogen fixed (estimated
by the Kjeldahl method) was 602-618 kg/ha and 733-829 kg/ha within 10-12 months
for A. pocheng and A. filiculoides respectively.
 Note that the above weights and measures expressed in metric units
are approximate and have been converted from the original Chinese units using
the following relationships: 1 Chinese catty = 0.6 kg
1 “ inch = 3.3 cm
1 “ mou = 0.066 ha

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II:Iasi-1H E1.0 ./q9Z et Ell e.el EL! 2.6 1.01

AntJ Rum,/ bnS

not/pubolq 1/1qA E - A.21 a 5.8 0.0
.ThtsgilL1 31.0 lq12 C es
lud2 ma
no[J,ubol4 1i10 85
ob62t18 3.0 ,ciaa s
-
b. O.1 it! 1.r i.e
egal,vA
IsloT ee.1 A81 tynb EE1 1.31

adT DITIoa elznu vtt1 no'd b9/1,vroD moil Id, InntRIlo ,aan1dD
allnu an :awo110 1 ,aln110 uom = 000.0 ad
,a,n/d, 'Wisp = 0.0 0
1 ,nnol = 1 000 soi
6.10.8 Recommendations for Azolla Cultivation
Chu (1979) recommends the double row method of azolla cultivation with
rice outlined above (section 6.10.7).

The rice plants should be of the compact type, with straight narrow
leaves, hard stems, big ears, plenty of grains and medium tillering capacity.
(The same spacings as indicated in section 6.10.7 should be used).

The parent azolla should be grown in shallow water first until nearly
harvest time for the rice, when the water depth should be increased. When
the rice plants are fully grown use additional irrigation water, or drainage,
as necessary to ensure optimum growth of the azolla.

Pests and diseases must also be controlled as necessary.

6.10.9 Nutrients for Azolla During Summer

Chu (1979) has indicated that during the summer nitrogen-fixation by
azolla decreases. It appears therefore that fertilizers and micro-elements
should be applied. Table 57 gives the results of applying fertilizers and
micro-elements, which shows that all nutrients stimulated (fresh weight)
production of azolla, but nitrogen fertilizers reduced nitrogen-fixation.

Table 57 EFFECT OF VARIOUS FERTILIZERS DURING THE SUMMER PERIOD

UPON THE PROPAGATION AND NITROGEN-FIXING CAPACITY OF AZOLLA
(Chu, 1979)

Increase after Increase Acetylene Increase

Fertilizer 10 days reduction
cultivation % n moles/ %
g fresh wt C H /mg/fresh
2 4
wt/day

Macro-elements

Ammonium sulphate 1% 29.85 162.7 3.58 34.1

Superphosphate 1% 37.45 204.1 10.96 104.3
K SO l% 26.75 134.5 11.87 112.9
2 4
Control 18.35 100 10.5 100

Micro-elements

Sodium molybdate
1 ppm 2.96 197.3 11.13 114.4
Ferrous sulphate
500 ppm 3.01 200.7 10.73 110.3
Control 1.5 100.0 9.73 100
6.10.10 Azolla and Rice Yields
Yields from rice grown with A. mexicana were 550/kg/ha greater than
for A. filiculoides and rice, and 850 kg/ha more than the controls. The
amounts of A. filiculoides and A. mexicana used in the experiments were res-
pectively 30 and 38 kg/ha. Incorporation of 60 kg N/ha into the soil. as
decomposing A. filiculoides increased rice yields by 1 470 kg/ha over yields
of unfertilized controls (i.e. 112%). Combining A. filiculoides as a green
manure in the plots produced an increase of 2 700 kg/ha over controls
(i.e. 216/), compared with increases of 292 and 1 470 kg/ha respectively for
separate treatments with A. filiculoides as a dual crop and green manure
(Talley et al, 1977).

Moore (1969) cites yield increases of 14, 17, 22 and 40% in four
separate experiments where A. pinnata was grown together with rice.

Espinas and Watanabe (1976) evaluated the results of added phosphorus

midseason,puddling to incorporate the azolla, and azolla inoculation on straw
and on grain yields (Table 58). In plots with azolla inoculation, its
incorporation and phosphorus, the grain and straw yield was far higher than
in plots without these treatments. The effect on straw yield of midseason
puddling was significant, and over-shadowed the effect of azolla inoculation.

Table 58 STRAW AND GRAIN YIELD OF RICE VARIETY IR 30 TREATED WITH

PHOSPHORUS, PUDDLED AND INOCULATED WITH AZOLLA
(Espinas and Watanabe, 1976)

Treatments Straw Yield Grain Yield

PUA kg/ha kg/ha

--- 1 018 d 1 478 d

-+- 1 363 bc 2 358 ab
--+ 1 542 ab 1 832 c
-++ 1 316 bc 2 155 abc
+-- 1 233 cd 1 860 c
++- 1 305 bc 2 245 bc
+-+ 1 378 bc 2 020 bc
+++ 1 644 a 2 534 a

P U A: P = phosphorus, U = puddled, A = azolla

In each column, the means followed by a common letter are not significantly different
at 5% level.

Scientists in the Peoples Republic of China (Department of Agriculture,

1975) reported rice yield increases of 0.4 to 158% with an average of 18.6%
from the results of 422 field experiments. Experiments in Sri Lanka
(Kulasooriya and de Silva, 1977), indicated that 32% more grains per panicle
were produced from azolla and rice than in the control.
6.10.11 Azolla as Feed
Azolla can be used as animal feed. E.g. it can be fed to pigs fresh or
fermented. The fodder contains about 1.3% crude protein and is a good green
roughage similar to sweet potato vines. It is estimated that 660 m2 (1 mou)
of good green azolla provides sufficient roughage for 10 to 20 pigs (1 ha is
required for 150 to 300 pigs).

Azolla harvested from the field should also provide good quality
forage. Moore (1969) estimated that it contains 23% protein, 4.4% crude
fats, 9.5% crude fibre and 6.4% starch (dry weight).

The chemical composition of azolla (Singh and Subudhi, 1978) is

presented in Table 59. Protein content is high but ash and calcium contents
are similar to commercial feed.

Based on feeding experiments to leghorn chicks, Singh and Subudhi

(1978) reported that fresh azolla can substitute for 20-25% of commercial
feed (Table 60). The increase in weight of birds was noticed when they were
fed with azolla along with normal diet. There was no mortality of chicks
when these were fed with fresh azolla.

Table 59 CHEMICAL COMPOSITION OF AZOLLA

(Singh and Subudhi, 1978)

Constituents % (dry wt)

Ash 10.5
Crude fat 3.0-3.36
Crude protein 24 - 30
Nitrogen 4-5
Phosphorus 0.5-0.9
Calcium 0.4-1.0
Potassium 2.0-4.5
Magnesium 0.5-0.65
Manganese 0.11-0.16
Iron 0.06-0.26
Soluble sugars 3.5
Crude fibres 9.1
Starch 6.54
Chlorophyll 0.34-0.55
Table 60 EFFECT OF VARIOUS FROPORTIONS OF AZOLLA ALONG WITH
COMMERCIAL FEED ON THE BODY WEIGHT OF CHICKS (FOUR CHICKS FOR
EACH FEEDING GROUP)
(Singh and Subudhi, 1978)

Age of Mean weight / bird/week, g.

birds in
weeks 100% feed 50% feed+ 75% feed+ 100% feed+
16% azolla 12.5% azolla 5% azolla

7 155 120 120 149

8 193 151 149 188
9 234 179 181 245
10 278 201 252 335
11 320 231 312 398
12 373 264 416 480
13 433 309 454 544
14 494 344 518 614

6.10.12 Azolla as Weed

Because of its rapid vegetative reproduction azolla will cover a
water surface very quickly. Cohn and Renlund (1953) reported that
A. caroliniana covered a canal in New Jersey from bank to bank for five miles
and the blanket was so dense that it was impossible to row a boat through it.
Moore (1969) reviewed cases where azolla is considered a weed e.g. in
paddies of the Philippines, in Hawaii, in Japan and in South Africa.

Control of azolla as a weed is acheived by using diesoline, either

undiluted or mixed with water 1:1 (Oosthuizen and Walters, 1961).

6.10.13 Azolla as a Means of Improving Water Quality

High concentrations of nitrates and phosphates are the major
contributors to the deterioration of water quality. Rains and Talley (1978)
suggested that azolla could be a good candidate for the biological treatment
of waters. It grows rapidly, and can fix molecular nitrogen, even in the
presence of nitrate, which can be easily removed.

6.10.14 Other Potential Uses of Azolla

In additon to its potential for rice production, fresh azolla is a
good food and when cooked can be consumed as a leafy vegetable. Fresh azolla
or its compost can also be used for the benefit of field crops and vegetables.
Decomposing azolla has also been used successfully in gobar gas plants.
(Singh, 1978).
 7. NODULATED NON-LEGUMINOUS PLANTS

The nodulated non-leguminous plants are perennial angiosperms which

bear nitrogen-fixing root nodules. The first evidence that such plants
fix nitrogen was obtained by Hiltner in 1896 who studied Alnus glutinosa
(Stewart, 1976). Detailed knowledge of the morphology, physiology and
bio-chemistry of these nitrogen-fixing plants has become available through
the work of several investigators (Becking, 1977; Bond, 1974; Quispel, 1974;
Akkermans, 1978).

7.1 THE MACRO-SYMBIONT. THE PLANTS

The orders, families and genera known to be nodulated are presented
in Table 61 (Becking, 1977). The distribution of these nodulated non-legume
angiosperms is given in Table 62 (Stewart, 1976). The species known to bear
nodules are compiled by Bond (1976).

Table 61 CLASSIFICATION OF NON-LEGUMINOUS NITROGEN-FIXING

ANGIOSPERMS
(Becking, 1977)

Number of symbiotic
Order Family Genus species. In parenthesis
number of species

Casurinales Casurinaceae Casuarina 18 (45)

Myricales Myricaceae Myrica 20 (35)
Comptonia 1 ( 1)
Fagales Betulaceae Alnus 33 (35)
Rhamnales Elaeagnaseae Elaeagnus 14 (45)
Hippophae 1 ( 3)
Shepherdia 2 ( 3)
Rhamnaseae Ceanothus 31 (55)
Discaria 2 (10)
Colletia 2 (12)
Coriariales Coriariaceae Coriaria 13 (15)
Rosales Rosaceae Dryas 3 ( 4)
Purshia 2 ( 2)
Cerecarpus 3 (20)
Table 62 NUMBER OF SPECIES AND DISTRIBUTION OF NODULATED NON-L
LEGUMINOUS ANGIOSPERMS
(Stewart, 1976)

Genus No. of Distribution

species

Casuarina 45 Australia, tropical Asia, pacific islands.

Myrica 35 Tropical, sub-tropical, temperate regions.

Alnus 35 Europe, Siberia, North America, Japan, Andes.

Dryas 4 Arctic, mountains of north temperate zone.

Cercocarpus 20 North America.

Purshia 2 North America.

Coriaria 15 Mediterranean, Japan, New Zealand, Mexico,

Chile.
Discaria 10 South America, New Zealand, Australia.

Ceanothus 55 North America

Elaeagnus 45 Asia, Europe, North America.

Hippophae 3 Asia, Europe.

Sheperdia 3 North America.

Aktostaphyles 70 North-West and Central America, Asia.

7.2 THE ENDOPHYTE. THE MICRO-SYMBIONTS

Becking (1974) described the family Frankiaceae, with one genus
Frankia, including ten species, as the endophytes nodulating the non-legume
angiosperms, as follows:
A true mycelium is produced. The mycelium is septate and branched
but branching is not necessarily correlated with cross-wall formation.

In an active nitrogen-fixing nodule, the centre of the host cell is

filled by a hyphal mass. Near the periphery, spherical or club-shaped
terminal swellings are formed on radially arranged hyphae close to
the plant cell wall. These spherical bodies are often called vesicles
(Fig. 26). The vesicles and club-shaped structures are probably
associated with nitrogen-fixation.
No species has as yet been grown on artificial. medium. In previous
claims of isolation Koch’s postulates were not fulfilled. The
organisms have been grown in root nodule tissue culture, but are
often best in subsequent transplants of the tissue because of the
difficulty of infecting newly formed callus tissue. The endophyte
containing callus tissue does not fix nitrogen.
 LYE- sp ItYLctGq csET LLow %010:-.a but-44 uocinisci upoptILIE
GugobpWca AGefejGa (X 1 000)
(coucLypncGq pX 1.2. bLcuc)

The organisms inhabit cortical paranchyme of root nodule tissues as symbionts,

but are also present in a free state in soil, presumably as a viable rooting
stage, since sterile non-leguminous seedlings nodulate when planted in
suitable soil.

The endophyte is specific for a certain plant genus or a group of related

genera. In contrast to Rhizobium no exceptions are known; the barriers of
incompatibility are strict. It seems appropriate therefore, to give species
status to these specific cross-inoculation groups.
 Becking (1974) named 10 species of Frankia based on the host they
associate with as follows:

Frankia spp Genera of plants as hosts

F. alnii Alnus
F. elaeagni Elaeagnus, Hippophae, Sheperdia
F. discariae Discaria
F. ceanothi Ceanothus
F. coriariae Coriaria
F. dryadis Dryas
F. purshiae Purshia
F. cerocarpi Cerocarpus
F. brunchorstii Myrica, Gale, Comptonia
F. casuarinae Casuarina

7.3 CROSS-INOCULATION GROUPS

Like legumes, non-legumes have cross-inoculation groups or barriers
of incompatibility (Becking, 1968). Thus a suspension of crushed root
nodules of one type will not produce nodulation in another type of non-legume
unless they belong to the same cross-inoculation group.

Although no strict barriers of incompatibility are known within any

genus, grades of incompatibility exist. Becking (1968) used crushed nodules
of Alnus glutinosa (from Wageningen, Netherlands) to inoculate seedlings of
A. cordata.(from Europe), A. incana (from Europe), and A. rubra (from
California, U.S.A.) raised from sterilized seeds grown in water culture.
Table 63 shows that of the Western American species A. rubra, only 28% of
the 35 plants in the experimental series were nodulated. In the control
series, 100% nodulation occurred; in other species 93 to 96% nodulation
occurred.

Table 63 CROSS-INOCULATION IN VARIOUS ALNUS SP (INOCULUM:

SUSPENSION OF GROUND A. GLUTINOSA ROOT NODULES,
(Becking, 1968)

Species Percentage nodulated

plants1

Alnus glutinosa (control) 100.0

A. cordata 95.7
A. incana 92.5
A. rubra 27.7

1
Values for 35 plants of each species

7.4 NODULES
Root nodules of non-leguminous plants can be divided into two main
groups: the Alnus and Myrica-Casuarina types.
 Alnus type root nodules are formed in the genus Alnus and other
genera of the families Elaeagnaceae, Rhamnaceae, Coriariaceae and in Rosaceae.
All these nodules are modified roots with an arrested or very slow growing
apical meristem. The nodular lobes are usually dichotomously branched,
producing nodules of corallaid appearance (Fig. 27).

Nodules of Casuarina sp and Myrica sp show the characteristic type

of nodules in which each root-nodule lobe produces a normal nodule in a
negative geotrophic root. Therefore these root nodules become covered with
upward growing rootlets.

1015- sl VODGe oz. uogniwir otbutAl:sa tuscv,g;uca (cob)

uyq mTumu hintimWia (poppow)
(court.rpnrcq pa ylyr- poccurgyR)
 Becking (1977) reviewed the processes involved in nodule formation
with respect to root hair curling, infection threads, early development of
nodules, and their cytology (Fig. 28). Cross sections of the apex of a
nodule lobe of alder showed that the endophyte is not distributed arbitrarily
in the cortical parenchymat cells of the host. The endophyte is not present
in the cortical parenchymatous layers near the stele.

Anatomically, non-leguminous root nodules consist of a central vascular

cylinder, surrounded in turn by an endodermis, a cortical parenchymatous
layer containing the endophyte and on the outside an epidermal layer. At the
tip of the nodule lobe there is an apical meristem.

LT8* sg bs.plagLA uocinTGe (s nwese UGL TuocriTtirTou) Tu coLcox

trug TuTeTtuTou oL .11yo AlmcnTut. pnuqTGa.
lattiçsmosq luocnTuccq ATcp TeroTorsq cnToLG
(coucl.TpnGq pa v. onTabsT)

7.5 EXTENT OF NITROGEN-FIXATION IN SOME NON-LEGUMES

Bond (1963) furnished citations to test the efficiency of nitrogen-
fixation of non-legume systems of Alnus, Myrica, Elaeagnus, Hippophae,
Shepherdia, Casuarina, Coriaria and Ceanothus. In all cases the results
have been equivocal and often dramatic. The nodulated plants grow well, even
luxuriantly. In species such as Alnus glutinosa as much as 300 mg nitrogen
per plant can be accumulated, by fixation in the first season of growth from
seed. In contrast, nodule-free plants, after exhaustion of the seed nitrogen,
make little further growth. An example of the amount of nitrogen accumulated
per plant is presented in Table 64.

Becking (1977) tabulated the findings, by different authors, on the

nitrogen-fixation measurements of some non-legumes (Table 65). Figures
ranged from 2 to 300 kg N/ha/year depending on the type and age and location
of the plants.
Table 64 ACCUMULATION OF NITROGEN BY NODULATED PLANTS IN ROOTING
MEDIA FREE OF COMBINED NITROGEN
(Bond, 1963)
Nitrogen
accumulated Subsequent
Species per plant accumulation
during first
season’s growth mg
mg
Alnus glutinosa 300 2500 by end of 2nd season
Myrica gale 146 5020 “ “ “ 3rd “
Hippophae rhamnoides 26 200 “ “ “ 2nd “
Casuarina cunninghamiana 70 1400 after 2.5 “
Elaeagnus angustifolia - 180 after 1.5 “
Coriaria myrtifolia 36 -

Table 65 FIELD MEASUREMENTS OF NITROGEN-FIXATION IN SOME NON-

LEGUMES
(Becking, 1977)
Non-legume Nitrogen-fixation
kg N/ha/Yr.
Casurina equisetifolia 229
Myrica cerifera 3.4
M. rubra (3-15 yr old) 15.251
Alnus crispa (50 yr old) 61.5
A. crispa 40.0
A. crispa (5 yr old) 157.0
A. glutinosa (0-7 yr old)
(1 plant /m2) 28.0
2
(5 plants /m ) 100.0
A. glutinosa (12 yr old) 26 - 28.0
A. glutinosa 58
A. incana 40
A. rubra 139
A. rubra up to 300
A. rugosa 193
A. rugosa (natural stand) 85
Hippophae rhamnoides
(0.3 yr old) 27
(13-16 yr old) 179
H. rhamnoides
(1-2 yr old) 2
(10-15 yr old) 15
Geanothus spp (natural shrub community) 60
Drys dummondii 18 - 36
D. drummanondii
Some Shepherdia canadensis 61.5

1
Data from comparison of Myrica pine stands and pure pine stands. Nitrogen-fixation
determined by subtracting total amount N pine stand from total amount N Myrica-pine stand.
 Virtanen (1962) demonstrated that the seven-year total content of
nitrogen in leaves of an alder (Alnus glutinosa) plant was 14.0 g. From
this he calculated that in a grove of alder trees 2.5 m tall with a density
of 5 plants/m2 the total nitrogen gain would be 700 kg/ha.

Lawrence (1958) published results showing that plants associated

with sitka alder (Alnus crispa var. sinuata) such as cotton wood (Popadus
trichocorpa) weighed 22.5 times as much as those of equal age in a non-alder
area. Alder thickets 5 years old and 1.5 m tall added 157 kg/N/ha each year
by leaf fall alone.

Dommergues (1963) estimated the increase in nitrogen level of soil

from the Cape Verde Islands, off Dakar, West Africa afforested with Casuarina
equistifolia. Bare soil contained 80 kg N/ha, but after being under forest
for 73 years, the soil yielded 309 kg N/ha i.e. 137 kg N in the Aoo horizon,
28 kg N in the Ao horizon and 144 kg N in the mixed horizon 0-10 cm. In
addition the standing timber of the Casuarina forest was estimated to contain
531 kg N/ha. This meant an average net nitrogen-fixation of 58.5 kg N/ha/yr.

Akkermans (1971) showed that an annual nitrogen-fixing activity of

approximately 60 kg N/ha/Yr was calculated in alder groves. Since there is
a large variation in both the distribution of the nodules and their activity
in natural vegetation, higher values up to 130 kg N/ha/Yr may be expected in
some places. For Hippophae vegetation a maximum nitrogen-fixing activity of
15 kg N/ha/Yr has been calculated on the basis of the smaller number of
nodules produced per unit area.

7.6 OTHER NON-LEGUME NITROGEN-FIXING PLANTS

The nodulation of the plant Trema cannabina var. scabra of the family
Ulmaccae Order Urticales was found to be caused by Rhizobium which cross-
inoculates with other legumes (Trinick, 1973). Later on this plant was
re-examined and identified as Parasponia rugosa BL. (Akkermans et al, 1978).

Nodulation of Zygophyllaceae was also reported to be caused by

Rhizobium, with the species Zygophillum album, Z. coccineum, Z. decumbens,
Z. simplex, Fagonia arabica and Tribulus alatus (Sabet, 1946; Mustafa and
Mahmoud, 1951).

In the nitrogen-fixing species of the genus Gunnera (Order Myrtoles,

Family Gunnaraceae) no root nodules are formed, but the symbiont (a blue-
green algae Nostoc punctiforme) is an inhabitant of special glands in the
stem (Becking, 1977).

The Cycadaceae (Cycads) bear tuberculate roots developed into coralloid

formations superficially similar to the nodule clusters of Alnus, Ceanthus
etc. The Cycadaceae comprise 9 genera with some 90 species, of which about
one third have been recorded to bear such nodule clusters (Bond, 1967).

The nodules of Cycads have blue-green algae of the genera Anabaena or

Nostoc inside them. The algae form a deep green zone in the middle cortex
of the lobe which is very obvious to the naked eye when a lobe is cut open
(Wittman, Bergersen and Kennedy, 1965)

Evidence has been presented (using 15N) that nitrogen-fixation occurs in.
the nodules and is followed by a rapid export of fixed nitrogen to other parts
of the plants. Bond (1959) showed that Ceratozamia nodules fixed about 1.6%
of the total nitrogen of the sample. Bergersen, Kennedy and Wittman (1965)
showed that nodules of Macrozamia communis produced 5.2 µg N/g fresh weight/hour.
 8. SOME COSTS OF BIOFERTILIZERS

It is generally believed that the immediate gains obtained from using

biofertilizers do not compete favourably with the use of inorganic nitrogen
fertilizers. However, the long-term benefits, such as increased crops yields
together with soil improvement and conservation, increased efficiency of
nitrogen utilization, reduced air and water pollution, qualify biofertilizers
as partial or complete substitutes for inorganic nitrogen fertilizers.

The cost of production of biofertilizers is relatively low compared

to that of mineral fertilizers. Some examples of the cost of legume and
algal inoculants is given below.

8.1 LEGUME INOCULANTS

Burton (1972) calculated the cost of inoculating alfalfa seeds in the
U.S.A. The cost depends on the method used and whether application is made
by the grower or the seedsman. Inoculant for on-farm application costs around
U.S. $0.30/ha. The materials for pelleting (inoculant, acacia gum, and
calcium bicarbonate) cost about U.S. $1.50/ha. Seeds for 4 ha of alfalfa
(50 kg) can be treated in 15 to 30 min. Including labour, the cost of
inoculating alfalfa will range from U.S. $0.50/ha for regular inoculation to
U.S. $2.50 for seed pelleting.

In India (Subba, Rao, 1972) the cost of 1 000 packets of legume

inoculant was given as Rs 600 or 60 paise per packet.

In Egypt, the unit of inoculant is sold to the farmer for U.S. $0.20
(E. £0.15) but the Government subsidises the production costs. From the
farmer’s point of view costs and savings compared to using mineral fertilizers
under Egyptian soil conditions for a crop such as soybean (soils free of
R. japonicum) can be calculated as follows:

Without inoculants
Nitrogenous fertilizers required: 60 kg/N/feddan (144 kg/N/ha)
Total cost 60 x E £0.15 = E £9.00 (E £21.6/ha)

With inoculants
Nitrogenous fertilizers required: 15 kg/N/feddan (36 kg/N/ha)
Cost 15 x E £0.15 = E £2.25/feddan (E £5.4/ha)
Inoculant 2 units x E £0.15 = E £0.30/feddan (E £0.72/ha)
Total cost E £2.55/feddan (E £6.1/ha)

Net saving with inoculation: 9.0-2.55 = E £6.45/feddan

(E £15.48/ha).

8.2 ALGAL INOCULANTS

Algal technology is being introduced in some rice growing states in
India using an open air soil culture method for producing bulk material for
field application, through large scale experiment stations, state seed farms,
and by village level panchyat unions and individual farmers (Ventkataraman,
1972).
 At a state seed farm in Tamil Nadu, algal production costs U.S. $3.65/ha.
When chemical fertilizer is used at the rate of 25 kg/ha, it costs
U.S. $12.20/ha. Thus using algae, a saving of $9.50/ha is achieved.

Results of using algae in Egypt indicate that 1/ to 1/ the required

3 2
mineral fertilizers can be saved. About 60 kg/N/feddan(144 kg/ha)are
required for rice cultivation. The cost of this is E £9.00/feddan (E £21.6/
ha). With algal application a saving of 3.0 to 4.5 E £/feddan (E £7.2 to
10.8/ha) can be achieved.
 Appendix A
METHODS OF ASSESSING BIOLOGICAL NITROGEN-FIXATION
A.l INTRODUCTION
The assessment of the nitrogen-fixing ability of a certain system
requires a suitable method to directly detect gains in nitrogen or indirectly
the nitrogenase activity. The Kjeldahl method for determination of nitrogen
content was introduced in 1883 and was the only available method until the
1940’s when the 15N method was adopted to assess nitrogen-fixation in
Azotobacter. Subsequently, different methods were developed to ascertain the
ability of an organism to fix nitrogen. Table 66 summarizes these methods
(Burns and Hardy, 1975).

A.2 KJELDAHL METHOD

In spite of its limitations the Kjeldahl method is still very useful

for assessing the response to inoculation, especially in laboratories lacking
the specialized equipment required for other methods, although it cannot be
used to present conclusive evidence of the capacity of an organism to fix
nitrogen. Details on equipment needed, procedure and calculations are presented
by Allen (1959) and Vincent (1970). Alternative methods e.g. A value and
isotope dilution are also available. (Rennie, Rennie and Fried, 1978. Use of
isotopes in biological nitrogen-fixation).

15
A.3 N INCORPORATION METHOD
2

The use of stable isotope N was first introduced to confirm nitrogen

2
fixation by azotobacter. The method is 1000-fold more sensitive than the
Kjeldahl method and it contributed to a greater understanding of biochemistry
and bacteriology of nitrogen-fixation during the 1940’s and 1950’s.

The method includes:

15
1) incubation of the test material in the presence of N enriched with N.
2
2) conversion of all N in the sample to ammonia by Kjeldahl digestion.
3) isolation of the ammonia by distillation.
4) oxidation of the ammonia to N .
2
5) mass analysis of N in a mass spectrometer.
2

15
Any N incorporated by the test material is detected as an increase
2
in the abundance of mass 15 above the background value of 0.364% due to
natural abundance of 15N. This increase is expressed as % 15N excess. Samples
giving an atom % 15N excess of more than 0.015 could be accepted as positive
evidence for N fixation.
2

A.4 ACETYLENE REDUCTION METHOD

In 1966 it was discovered by Schollhorn and Burris in Wisconsin, and
Dilworth in Perth, Australia, that the nitrogenase reduced acetylene to
ethylene (Dilworth, 1966). The method based on this principle was soon
developed to measure nitrogenase activity in legumes, non-legumes, algae,
lake samples, soil samples, etc.

Essentially the method involves incubating the test material in a gas

tight container which contains partial pressure of acetylene. At appropriate
sldaT aa 1OITAX11-113DOATIM 2YA22A
enlua) bno ottrum (ayer

ams/ax2
xsaaA bodism bno 1qirntq sacnsgolliN m8ins100 ni b19r1 bns
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times samples of the atmosphere may be withdrawn by syringe for immediate or
eventual analysis by using flame ionization after gas chromatography. The
amount of C H detected is correlated with the intensity of nitrogenase
2 4
activity in the sample. Values for nitrogenase obtained by this method are
conventionally designated as N (C H ) fixing activity and nitrogenase (C H )
2 2 2 2 2
activity.

Burns and Hardy (1975) evaluate the method as follows:

i. Sensitivity: It is about 103 times more sensitive than 15

N .
2

ii Facility: Because both C H are gases the application of substrate C H

2 4 2 2
and recovery of product C H require only simple gas handling techniques.
2 4
The gas chromatograph and incubation equipment are likewise simple to use.

iii. Internal standard: C H is a natural internal standard for the assay and it
2 2
is measured readily in conjunction with the product C H .
2 4

iv. Specificity: Product C H is readily separated and quantified on the gas

2 4
chromatograph.

v. Rapidity: About 80 incubations and about 160 gas chromatographic analyses

per man-day are feasible.

vi. Product stability: The gas samples containing product C H can be stored
2 4
indefinitely in gas-tight containers for chromatographic analyses.

vii. Economy and mobility: All apparatus used is relatively inexpensive and rugged
enough for use in all terrain-type mobile units.

viii. Sample conservation: The test material need not be sacrificed for analysis,
and normally can be repeatedly sampled because each analysis requires only
a small fraction of the incubation atmosphere.

The disadvantages of the method include the explosive nature of C H

2 2
and the indirect nature of the reaction in the sense that C H is not the
2 2
physiological substrate. In most studies the correlation between C H
2 2
reduced and N reduced indicates that the reduction of 3 to 4 mol C H
2 2 2
reflects nitrogenase activity capable of reducing one mole of N . This
2
relationship is consistent with the amount of reductant needed, since C H
2 2
reduction requires 2 e-, and N reduction requires 6 e-. However, variability
2
in the conversion factor has been observed, and the most reliable data for
aiven conditions are obtained by establishing a factor for each particular
variation in experimental procedure.

Sample Preparation
Assays should be made immediately after sampling (within 2 hours).
Nodules are best assayed while still attached to the roots. Removing the
tops has no measurable effect on short term assays.

Some N -fixing systems are apparently cold labile and therefore

2
samples should not be stored cold before assay. For comparisons, sampling
and assays should take place at the same time each day because the nitrogen-
ase activity of nodulated roots may vary with the light intensity (photosyn-
thetic activity) and temperature throughout the day. When possible,
nodulated roots should be shaken free from soil and assayed without washing.
Activity is diminished when the surface film of water remaining after washing
nodulated roots is not carefully removed and drying of nodules also diminish-
es activity. Samples are assayed in conveniently sized containers, e.g. 50
ml syringes or glass bottles, with a rubber septum in the stopper to allow
it to be pierced by a hypodermic needle.
 The activity of nodules is decreased as the partial oxygen pressure
(pO ) falls below 0.20 atm. For rhizosphere, non-symbiotic organisms, a pO
2 2
of about 0.04 atm is often optimal. Assay vessels should be large enough to
minimize change in pO . Liquid samples should be well shaken during assay.
2
It is preferable to assay soil as intact cores. Samples containing blue-green
algae should be illuminated at light intensities corresponding to those in
situ in the field.

For assays of soil-plant systems, soil cores can be placed in an air

tight plastic container (such as a plastic plant pot with a clear plastic
bag of rigid, clear plastic cover) connected to the base by a water seal.
The containers should be incubated in the light as the activity of rhizosph-
ere-associated nitrogen-fixing bacteria is influenced by photosynthesis. A
known amount of propane to give a concentration of 100 ppm is injected and
used as an internal calibration standard to measure the volume of gas in the
incubation chamber and to monitor leaks and other losses of C H .
2 4

Assay Procedure
For active systems e.g. nodules or blue-green algae, assays can be
made in air, provided a concentration of 10% C H is used. The atmosphere
2 2
in the vials can be altered by alternately evacuating to 30 mm Hg and flush-
ing with an appropriate gas mixture (usually Ar/O ) two or three times.
2

Some gas withdrawn from the sample container by syringe and

replaced with acetylene to give a final concentration of 10% of acetylene
may be premixed with the gas phase introduced into the sample container.
Samples without C H added should be assayed for C H produced endogenously.
2 2 2 2
Samples are best incubated at temperatures which are either constant or
related to the in vivo temperature.

After a suitable time (30 min for legume root nodules, up to 24 hours
for soil) gas samples 0.5-2 ml are taken with a syringe. For periods up to
about 3 hours, the gas sample can be stored by sticking the syringe needle
into a rubber bung. For storing gas samples over longer periods, samples can
be transferred into pre-evacuated containers.

The gas colume of the assay vessel is measured by displacement with

water (using a burette) after assay. This is not necessary with propane
used as an internal standard.

Gas Chromatography
Gas mixtures can be separated by gas chromatography and then quanti-
fied by flame ionization or thermal conductivity detectors. For acetylene,
ethylene and propane, various column packing materials can be used. A
convenient system to use is 80-100 mesh porapak N or T in a 2m x 0.003m
diameter, stainless steel column at 100 °C, with a nitrogen gas flow rate of
25 ml/min using a hydrogen/air flame ionization detector. Another convenient
system is 100:0 NO PO on spherosil x 0.3 cm glass column at 35°C with a
3 4
nitrogen carrier gas flow rate of 4 ml/min. Most detectors can routinely
detect 0.1 ppm C H in a 0.5 ml gas sample. For porapak N or T, CH (propane)
2 4 4
has the shortest retention time followed by C H (ethylene), C H (acetylene)
2 4 2 2
and C H (acetone). For a Na PO column, however, propane is eluted before
3 8 3 4
the acetylene. At most concentrations encountered in the assay, peak heights
can be taken linearly and be related to the concentration with fair accuracy.
Calculation

A suitable calculation for ethylene (C H ) produced in moles C H /h is

2 4 2 4
as follows:

(C H sample CU x Vol gas in sample container x assay time (h) x K)

2 4
Vol injected into GLC

- (C H blank CU x Vol gas in blank container x K)

2 4
Vol injected into GLC

where: CU = Chart units used to measure peak height.

blank = Sample container with added C H only.
2 2
K = Conversion factor obtained using a standard C H gas mixture
2 4
to calibrate the chromatograph.

For example, for 100 ppm C H standard, K is derived as follows:

2 2

1 ml of 100 ppm C H contains

2 2

-6
100 x 10 ml C H and = X CU
2 4

22.4 C H at STP = 1 mole C H

2 4 2 4

1 ml of 100 100 x 10-6 moles C H

ppm C H =
2 2 44
22.4 x 103
= 0.00446 m moles C H = X CU
2 4
Then K (or CU) = 0. 00446 m moles C H
2 4
X

Precautions and Comments

i. C H is soluble in silicon rubber and autoclaving produces C H for rubber such as
2 4 2 4
subaseals. C H contamination in rubber can be decreased by steaming with water for
2 4
10-15 minutes and leaving to stand 24 hours before further use.

ii. Disposable plastic syringes and 25 g needles (0.5 mm diameter) are suitable
for samples and gas transfers. Contaminating C H can be decreased by
2 4
evacuating and flushing the syringes with air about 6 times.

iii. C H can be bought in cylinders (Welding grade is suitable) or it can be

2 2
produced by reacting calcium carbide with water. C H from either source
2 2
contains variable amounts of contaminating C H (in addition to CH and
2 4 4
phosphine) and suitable control blank samples should be taken to monitor
this.

iv. C H can be used as an internal standard in the assay to monitor sampling and
2 2
injection errors. Propane can also be used in the assay atmosphere as a
standard, specially for assays of soil-plant systems.

v. The chromatography of each set of assays should be calibrated. A standard,

premixed cylinder of C H (C. 100 ppm) in Ar or N is useful for this.
2 4 2

vi. C H is a plant growth hormone, and most responses are saturated by 2 ppm
2 4
C H . Plant membrane integrity is affected by C H and/or C H .
2 4 2 4 2 2

vii. Because C H is more soluble than N in water, the theoretical conversion

2 2 2
factor of 3 may not apply and the actual ratio should be found by using
15
N or Kjeldahl methods.
 Appendix B

RHIZOBIA
B.l. RECOGNITION AND CHARACTERIZATION OF RHIZOBIUM
The decision as to whether a culture is or is not a rhizobium usually
depends on a plant test. Certain conformable and contradictory characteristics
are worth noting (Vincent, 1970).

Some distinction between groups of Rhizobium and the recognition of

Agrobacterium is likely to be possible on the basis of cultural and biochemical
characteristics (Graham and Parker, 1964). The general characteristics are
outlined in Table 67. Typical R. japonicum - R. lupini-Cowpea rhizobia produce
very small colonies and utilize citrate poorly. They commonly produce
penicillinase, and do not cause a presipitate in Ca glycerol phosphate medium.

Plant tests that permit reasonable distinction between present species

(Graham and Parker, 1964) are presented in Table 68. On some occasions
strains of R. phaseoli could be expected to show rather more cross reactivity
with other hosts of the clover-pea-vetch-bean group. However, the three
species, or symbionts, in this group can be generally distinguished by their
preferred host where the reaction often results in a much more effective
association than in other seemingly less compatible cases. Similarly, among
the slow growers, separation into the present subgroups is indicated by the
nature of the preferred host. R. meliloti can almost always be readily
distinguished from the other rhizobia, as well as from the agrobacteria by
its nodulation of Medicago sativa.

Table 67 CULTURAL AND BIOCHEMICAL CHARACTERISTICS OF MAJOR GROUPS OF

RHIZOBIA AND DISTINCTION FROM AGROBACTERIUM
(Collected from Graham and Parker, 1963).

R. trifolii R. japonicum
R. legumino- R. meliloti R. lupini Agrobacterium
serum Cowpea
R. phaseoli rhizobia

i. Colony size, mm 2+ 2+ 1 2+
ii. Raffinose used 0.84 0.73 0.19 0.89
iii. Citrate used 0.03 0.27 0 0.72
iv. Growth pH 4.5 0.65 0.09 0.83 0.88
v. Growth pH 9.5 0 0.91 0 0.56
vi. Growth 2% NaCl 0 0.45 0 0.61
vii Growth 39°C 0 0.72 0 0.06
viii.Response to thiamine 0.65 0 0 0.11
ix. Response to pantothenate 0.89 0 0.03 0
x. Response to biotin 0.59 0.36 0.23 0
xi. H S produced 0 0.81 0 1.0
2
xii. Penicillinase produced 0.08 0.08 0.77 0.11
xiii.Precipitate from
Ca glycerophosphate 0 0.55 0 1.0

Entries, except in the case of i. represent the proportion of rhizobia strains in each
group, or species, showing the characteristics.
Table 68 Nodulation specificity of Rhizobial species
(Collected from Graham and Parker, 1964).

R.legum- R.trifo- R.phase- R.lup- R.japo- Cowpea R.meli-

Test host inosarum lii oli ini nicum rhizo- loti
bia

Vicia sativa 0.85 0.1 0 0 0 0 0

Trifolium repense 0.4 0.72 0 0 0 0 0
Phaseolus vulgaris 0.15 0.05 1.0 0.2 0.7 0.5 0
Macroptillium pathyroides3 0 0 0.8 0 0.3 0.2 0
Ornithopus sativus 0 0 0 1.0 0.3 0.05 0
Glycine max 0 0 0 0.1 1.0 0.3 0
Vigna sinensis 0 0 0 0.2 0.3 0.5 0
Medicago sativa 4 0 0 0 0 0 0 1.0

1/ Entries represent the proportion of rhizobial strains in each species nodulating the
host.
2/ Partly reflects poor compatibility of isolates from Agrican clovers; those from T.ambiguum
are poorly compatible with Mediterranean clovers.
3/ Formerly Phaseolus: M.atropurpureum is widely susceptible to slow growing rhizobia
including isolates from Lotoraonis and Leucaena which are host specific.
4/ Whereas M.sativa appears to be always invasible by R.meliloti, other species of Medicago
are likely to be more strain specific. Some strains from Leucaena nodulate M.sativa.

B.2. LITMUS MILK REACTION

Reactions in litmus milk (after 1-6 weeks) have been used to record
changes in the acid or alkaline direction, and the development of a digested
“serum” zone which is a feature of some strains of Rhizobium. Non-rhizobial
forms or contamination will often be revealed by their rapid growth in this
medium, frequently accompanied by rapid change of reaction and decolouriza-
tion of litmus.

In general, litmus milk reactions provide more reliable evidence for

purity of rhizobia than for identification of species. With the exception
of R.meliloti, fast growing strains tend to produce an alkaline reaction in
litmus milk accompanied by the formation of a wide serum zone. An acid
reaction and acid serum zone in litmus milk are produced by strains of
R.meliloti. No serum zone accompanies the alkaline reaction brought about by
members of slow growing rhizobia as exemplified by R.lupini, R.japonicum,
cowpea rhizobia and related forms. None of the rhizobia peptonize or
coagulate milk. All reactions in litmus milk are produced slowly and are
best judged after several weeks incubation at 25-30°C.

B.3. TECHNIQUES APPLIED IN THE STUDY OF RHIZOBIAL ECOLOGY

i. Selective media for rhizobia
A medium which selects rhizobia and inhibits other microorganisms in soil
has not yet been formulated. Attempts have been made to develop media to
select rhizobia from soil but none have been satisfactory.
ii. Plant dilution (plant infection) techniques
The selective media most widely used to detect rhizobia in soil has been
the legume host itself. The method depends on the eventual appearance of
a nodule on the roots of a legume test plant as the indicator of an
appropriate Rhizobium in the soil. Numbers of rhizobia are usually
estimated by preparing 10 fold dilutions as inocula for replicate rhizobia-
free seedlings. This “plant dilution” technique is thus a variation of
the statistical most probable number (MPN) assay and based on the assump-
tion that a single Rhizobium can initiate nodulation.

iii. Serological methods

Serology has been used in ecological studies of rhizobia primarily to
identify different strains of rhizobia as to:
a. Survival of inoculant strain.
b. Competition among inoculant strains, and
c. Nature of indigenous soil rhizobia.

The test is usually formed by exposing the systems to a legume host plant,
isolating rhizobia from the nodules that are formed, and studying the
occurrence of the serotype(s) among the nodule isolates. Agglutination
technique is widely used. Immuno diffusion technique has also been
applied (Vincent, 1970).

iv. Immunological approaches

The fluorescent antibody (FA) technique is a general one for ecological study
of microorganisms in natural environments. Antibodies against the micro-
organisms to be studied are prepared and after the addition of a fluorochrome,
the antibody is used as a highly selective stain for the direct microscopic
examination of the natural environment. The antigen-antibody reaction is
visualized by fluorescence microscopy to allow for the simultaneous detection
and recognition of the microorganisms of interest. The technique is now
developed to detect and recognize specific strains of rhizobia in soil and
to observe some qualitative interrelationships of that strain with its
environment. To use the FA to quantify rhizobia in soil, population should
not be less than 103 to 104 depending on the properties of the soil.

v. Mutant markers and phage typing

Mutants resistant to the antibiotic streptomycin were first proposed by
Obaton (1971). Streptomycin resistant mutations are readily obtained
spontaneously at adequately high level in a single step and are stable.
Rhizobia so marked must first be tested to ensure that the mutant has not
lost symbiotic effectiveness, as some strains may loose efficiency.

After inoculation of soil or seed the mutant may be identified by

recovery from nodules plated on appropriate media containing antibiotic.
Other antibiotics e.g. spectinomycin, can be used alone or with strepto-
mycin as double markers.

Phage typing is used as a means of distinguishing among closely related

strains of rhizobia. Rhizobiophage commonly occurs in nodules and field
soils cropped to legumes.
B.4. LEGUME SEED PELLETING
Lime pelleting of legume seeds is becoming a commonplace operation on
the farm. Its main purpose is to protect the root nodule bacteria from harm-
ful conditions and thereby produce an improved environment for their survival
on the seed. Several advantages are obtained by lime pelleting (Roughley,
Date and Walker, 1965) as follows:

it counteracts soil or fertilizer acidity, or both, in the

micro-environment of the seed, allowing improved survival of
the added rhizobia:

it makes aerial sowing of inoculated seed a practical

proposition:

it ensures better survival of the bacteria when days between

inoculation (pelleting) and sowing are unavoidable:

it improves the chance of obtaining promptly nodulated plants

by ensuring better survival of rhizobia after sowing especially
when germination is delayed.

Adhesive. A high grade gum arabic or gum acacia is the best adhesive agent.
It is essential that the gum is free from preservatives. Very finely ground
gum is hard to dissolve in water (8-mesh ground dissolves satisfactorily).
Other adhesives are methofas R, cellofas A, and methocel R. These do not
provide the same degree of protection for the rhizobia as does gum arabic.

Pelleting material. A finely ground form of calcium carbonate that will pass
at least 300 mesh seive is required.

The inoculum. Peat inoculants are preferred or other satisfactory inoculants

can be used.

Equipment. Small batches of seed (up to 5 lb) can be pelleted satisfactorily

by hand, in a dish. Larger quantities can be pelleted very satisfactorily in
a clean cement mixer or revolving drum.

In the absence of these facilities, reasonable pellets have been made

by simply rolling the ingredients in a canvas sheet by alternately lifting
one end toward the other until smooth pellets are formed.

Preparing the pellets

A summary of the steps to be taken and rates of materials recommended
for pelleting seed are given below.

Ingredient Quantity Operation

Gum arabic 3.5 fl oz (99 ml) Dissolve gum arabic in water.
Drinking water 8 fl oz (227 ml) This makes ½ pint (0.28 1) of gum
solution.
Inoculum 1 packet Add inoculum to gum solution and
mix thoroughly.
Seed:
- small, e.g. white clover 15 lb (6.8 kg) Add the gum inoculum mixture to the
- medium, e.g. sub clover 30 lb (13.6 kg) seed, and mix until seed is evenly
- large, e.g. vetch 60 lb (27.3 kg) coated.

Fine lime 7½ lb (3.4 kg) Add the lime all at once and mix
rapidly for 1 to 1½ min only.
Characteristics of a good pellet
- The seed should be evenly coated by the lime.
- After the mixing, the pellet should appear dry but without
loose lime left on the surface giving it a powdery look.
- Free lime should not be left in the mixer.
- Pellets should be firm enough to drop on the floor without
damage.
- When dry, the pellets should withstand light rolling between
the fingers.

B.5. TECHNIQUES FOR QUALITY CONTROL OF LEGUME INOCULANTS

(Roughley, personnel contact)
Quality control must begin with the selection and maintenance of
mother culture, and include control of the broth before addition to the
carrier and the finished product. All broths must be numbered and the
batch of inoculant prepared from them given the same number or numbers.
Each container of inoculum should also carry this number so that if there
is a nodulation failure, the source of the inoculum can be traced.

CONTROL OF MOTHER CULTURES

i. Each year strains used for commercial production should be streaked
onto yeast extract mannitol agar.

ii. If pure, subculture onto YMA in a screw-capped bottle or test tube.

iii Check the resulting growth by:

a. Gram stain
b. Growth on glucose-peptone agar
c. Check by agglutination that the culture is true to type

iv. Subculture onto enough screw-capped tubes to supply mother cultures

for 1 year to reduce subculturing strains to a minimum.

v. Inoculate 12 plants growing in N-free agar in tubes with a loop

of culture taken from each of the tubes prepared in iv. Compare
growth with uninoculated control plants.

CONTROL OF BROTH CULTURES

Each broth must be tested before impregnating the carrier. Because
some tests take more than 2 days, the peat will be impregnated before all
the results are known. Should the results of these later tests fail to meet
the minimum standard the inoculant must be discarded.

Preliminary Tests Which Must be Completed Before the Carrier is Impregnated:

i. Streak broth into glucose peptone brom-cresol purple agar in duplicate
tubes. Incubate at 30°C. Examine f or growth and whether or not acid
or alkali was produced after 24 hours. Most rhizobia fail to grow on
this media and usually do not produce acid (yellow). Some strains make
a little growth after 48 hours and may turn the media alkaline (purple).

ii. Gram-stain: rhizobia are gram negative. Reject any broth with contaminants
present.
iii. Total cell count: broth must contain at least 1000 x 106 cells/ml. If fewer,
reject the broth.
iv. Serological check: to verify that the correct strain is present, mix 3 ml
of broth with 3 ml of 0.85% saline in a test tube and boil for 1/ hour
2
(to remove “H” antigen).
Test by using 3 serology tubes:
a. 2 for test
b. 1 for control
Use a pasteur pipette to add to an agglutination tube:
a. 18 drops of boiled broth-saline plus 2 drops of
antisera, and
b. 18 drops of boiled broth-saline plus 2 drops of
0.85% saline.
Incubate in water bath at 45° C for 4-5 hours.
Result: Agglutination of cells compared with control idicates a positive
result.

Tests Which Require More Time for the Results

Total viable count. This is an extremely important test:
To make satisfactory inoculants a broth must contain at least 500 x
106 viable rhizobia/ml. If there are fewer, the impregnated carrier
must be discarded.

Dilute the broth in sterile tap water to a final concentration of 10-8.

Plate out aliquots at 10-6, 10-7 and 10-8 onto duplicate plates of YMA.
Incubate at 26°C. Count the most suitable dilution after the appropriate
time.

CONTROL OF THE FINAL INOCULANT

At least five containers from each batch should be subjected to the
tests detailed below. If more than one fails, reject the batch. If one
only fails test a further 5 containers. Should any of the second set of 5
fail reject the batch.

Viable Counts of Rhizobia by Plate and Most Probable Number Count

(MPN) Methods
i. Add 30 g carrier to 270 ml sterile water and shake for 10-15 minutes
(10 g carrier to 90 ml sterile water may be used as an alternative
i.e. 10-1dilution).
ii. Prepare further dilutions of 10-6,10-7,10-8,10-9,10-10
iii. Plate out 1 ml aliquots from the 10-6 and 10-7 dilutions onto YMA +
Congo red. Incubate at 26°C for appropriate time.
iv. Transfer 1 ml from dilutions 10-7,10-8,10-9,10-10 into each of 2 tubes
with a sterilized seedling of the appropriate host growing in N-free agar.
Examine for nodulation after 4-6 weeks and calculate the number of rhizobia
in the inoculant from the most probable number tables.

Cultures in unsterilized carriers should contain a minimum of 500 x

106 rhizobia/g carrier. In sterilized carriers the minimum number should
be 1000 x 106/g.
CULTURE MEDIA UTILIZED FOR RHIZOBIA
Medium for Use in Fermentation
K HPO 0.75 g
2 4
MgSO .7H O 0.4 g
4 2
CaCO 0.4 g
3
Yeast extract1 3.0 g
Sucrose 10.0 g
Water 1 litre
pH 6.8
1
/ Commercial yeast extract preparation. Alternatively yeast extract may he prepared
using
compressed yeast (described under yeast extract mannitol agar recipe). In this
case add 100 ml of extract and reduce the water to 900 ml.

Yeast Extract Mannitol Agar with Congo Red Indicator for

Plate Counts of Broth and Inoculants
Constituents
Yeast extract 3.0 gm (dehydrated) or 100.0 ml of yeast extract
MgSO .7H O 0.2 solution.
4 2
K HPO 0.5
2 4
Mannitol 10.0
Agar 15.0
Congo red (1:400 aqueous
solution) 10.0 ml
Water (pH 6.8) 1 000.0 ml

Preparation of Yeast Extract Solution

Constituents
Compressed yeast 400.0 gm
Deionised water 4.0 litres

Method
i. Add yeast to water and let stand for 2 hours
ii. Autoclave for 3 mins
iii. Stand for 2 days
iv. Decant clear liquid and dispense into flasks
v. Autoclave for 30 mins
To use: 100 ml/litre media
Glucose Peptone Agar
Constituents
Glucose 5.0 gm
Bacteriological peptone 10.0 gm
Agar 15.0 gm
Water 1 litre
1.6% alcoholic brom-cresol purple

Method of preparation:
i. Add peptone and agar to water. Autoclave at 10 lb/in2 for 5 minutes.
ii. Add glucose, brom-cresol purple, dispense in tubes and autoclave
at 10 lb /in2 for 10 minutes.

N.B. Do not autoclave glucose twice.

Plant Medium (Nitrogen Free) Used for Tests of Effectiveness and

Counting Rhizobia (MPN)
Constituents
CaHPO 1.0 gm
4
K HPO 0.2
2 4
MgSO . 7H O 0.2
4 2
NaCl 0.2
FeCl 0.1
3
Agar 15.0
Water 1 litre
Other salts added as required e.g. trace elements

B.6. METHOD FOR SEED STERILIZATION FOR PLANTS USED IN COUNTING

RHIZOBIA. (MPN)
Mercuric Chloride Sterilization
i. Wash seed with alcohol for ½ minute.
ii. Pour off alcohol and wash in mercuric chloride for 2½ minutes.
iii. Pour off mercuric chloride and wash seeds with sterile water six
quick washes then leave seeds soaking in water for several hours.
iv. Spread seeds thinly in petri dishes containing plain agar. Incubate.

Mercuric chloride (HgCl2) - for seed sterilization

HgCl2 1.0 gm
Conc. HCl 5.0 ml
Distilled water 500.0 ml
Seeds which may be sterilized with mercuric chloride
Clovers-sub, white, rose, red, strawberry
Medics - lucerne, paragosa
 Vetch - for good germination, leave seeds soaking in water,
after sterilization overnight.
Lotus bean
Seed may have to be washed free of fungicide before sterilizing.
After sterilizing, place seed in closed container on damp cotton
wool until germinated. This may take 4-5 days.

Acid Sterilization - Using Concentrated Sulphuric Acid

i. Wash seeds with acid for 10 minutes
ii . Pour off acid carefully, then wash seeds quickly in six washes
of sterile water. Leave seeds soaking for several hours.
iii. Spread seeds thinly in petri dishes containing plan agar. Incubate.
Seeds which may be sterilized with concentrated sulphuric acid
Lotononis sp
Siratro
Desmodium
Fine stem stylo
Trifolium semipilosum
Soak in acid for 20 minutes then proceed as described above.
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 Appendix D
BLUE-GREEN ALGAE
15
D.l. N TRACER
Williams and Burris (1952) developed a method for testing Calothrix
parietina as follows.

After the algal cultures have used most of the fixed nitrogen, they are concentrated
aseptically by decantation or centrifugation and are resuspended in 100 ml of fresh Chu 10
medium containing one mg fixed nitrogen per ml. The medium is modified by substituting CaCl
2
for Ca(NO ) and the nitrogen is supplied as NH NO . The 250 ml bottles are closed with
3 2 4 3
sterile rubber stoppers carrying cotton plugged inlet tubes.

The bottles are evacuated and assed with 0.25 atom 15N-enriched N , 0.50 atom argon,
2
0.20 atom O and 0.05 atom CO . These cultures are illuminated and remain stagnant. They are
2 2
not opened until time for harvest (4-30 days). At harvest, cells are sedimented, resuspended
in distilled water and washed by centrifugation.

The cells are subjected to Kjeldahl digestion, the ammonia is distilled and then
converted to N with alkaline hypobromide. The N is analysed for 15N with a mass
2 2
spectrometer.

By this method, 0.003 atom per cent of 15N excess can be detected and 0.015 atom per
15
cent N excess will serve as a conservative level for establishing fixation of 15N.

D.2. DETERMINATION OF AMMONIA IN CULTURES OF BLUE-GREEN ALGAE

(Solorzano, 1969)
Reagents
i. Phenol alcohol solution, 10 g phenol in 100 ml of 95% C H OH.
2 5
ii. Sodium nitoprusside solution, 0.5% W/V in water.
iii. Alkaline hypochlorite solution. This is prepared by mixing two
solutions in a ratio of 4 vols of a: 1 vol of b.
a. 100 g of trisodium citrate + 5 g NaOH in 500 ml H O.
2
b. Sodium hypochlorite solution.
Reagent i will keep well for some months.
Reagent ii will keep for one month if kept in brown bottles.
Reagent iii should be prepared daily.
Solution a will keep for some months as well as b if stored in a
refridgerator (40°C).

Procedure
Standard curve: using a range of concentrations of NH Cl in distilled
4
water, pipette 8 ml of solution into triplicate test tubes (range from 0 to 1 mmol).
Add 0.2 ml of phenol alcohol solution, followed by 0.2 ml of sodium nitropruside
solution followed by 0.5 ml of alkaline hypochlorite solution. Shake well between
each addition. Allow to stand at room temperature (22-27°C) for 1 hour and read
absorbence at 640 nm using a reagent blank as zero. The colour will remain
unchanged over 24 hours. Glassware should be acid washed before use.

Samples
Use 5 ml (or smaller volume made up to 5 ml with distilled water) of
supernatant prepared by centrifuging or filtering the cultures. Add reagents
and treat as for standard curve. Read values of ammonia concentration from
standard curve.
D.3. IN SITU ASSAY OF NITROGEN FIXING ACTIVITY
An in situ acetylene reduction assay technique for use in paddy fields, using
plastic bags and bottomless metal frames has been developed. The plastic bag chamber
is convenient for addition and removal of gas. This technique has been used by Watanabe
et al (1977) to estimate nitrogen fixation in rice fields.

D.4. NITROGENASE (C H ) ACTIVITY OF BLUE-GREEN ALGAE

2 2
As an example (El-Sayed, 1978). 25 ml samples of algal suspension were
transferred into 420 ml capacity serum bottles. The bottles were then sealed with
tight silicon rubber subseals. The suitable volume of acetylene was injected into
the bottles to satisfy an acetylene concentration of 10% as recommended, without
removing air, and the pressure was then restored to atmospheric by pricking the seals
with a syringe needle. The bottles were incubated under illumination at 30°C for
2 hr after which samples of gas phase were withdrawn into a syringe and stored until
subsequent analysis for ethylene production. Two ml of gas samples were injected
into a Pye Unicum GLC Model 104, fitted with 183 cm long and 0.4 cm diam stainless
steel column, filled with 10% NO PO on 100-120 mesh Spherosil X OB and with dual
3 4
flame ionization detection. The carrier gas was nitrogen at a flow rate of 28 ml/min,
hydrogen and air at rates of 30 and 300 ml/min, respectively. Temperatures were 35°C
for column, 60°C for injection and 150°C for detector.

During analysis 0.5-2 ml of standard gas mixture (air and acetylene 9:1
containing 100 ppm ethylene gas) were injected.

To calculate the amount of ethylene produced by the samples, the heights of

the samples and standard peaks were compared. Results were presented as namomol
C H /mg dry wt/h.
2 4

D.5. SILICA GEL PLATES

Silica gel plates are used in one or more steps for isolating and purifying blue-
green algae. To each petri dish 0.2 g of powdered CaCO is added followed by the addition
3
of 20 ml of sodium silicate solution 8.5%. Acid solution (composed of 20 ml of liquid
modified Chu 10 medium, 300 ml of HCL 30% v/v and 5 ml of bromothymol blue 0.04%) is
added to the dish from a burette with continuous mixing until the gel is obtained
(Shalan, 1974).

To remove any excess of acidity, the silica gel plates are dipped for 2 days in
a current of tap water, then the plates are sterilized by flame.

D.6. CHLOROPHYLL (A) CONTENT

Chlorophyll (a) content is generally used as an index of growth of blue-green
algae. Golterman and Cylmo (1968) developed the following procedure to estimate algal
growth.

The algal growth is vigorously macerated together with washed sand. The chlorophyll
(a) is then extracted with 90% v/v aqueous acetone. After centrifugation (500 rpm/min) the
coloured supernatant is measured at a wave length of 665 nm using a one cm thick cuvette of
the spectrophotometer (special zeiss, Jena) and 90% acetone as a blank. The amounts of
chlorophyll (a) are then calculated as mg chlorophyll (a)/mg algal dry weight using the
following equation:
µg chlorophyll (a)/mg dry wt = Ex x 11.9 x Volume of acetone
Dry wt of the alga/mg

Where Ex = extension of 665 nm.

D.7. ALGAL BIOMASS IN SOIL
Lynn (1974) derived an equation to convert chlorophyll (a) to algal
biomass:
Algal biomass = chlorophyll (a) mg/m2 x 3.15 kg/ha

The method of determining algal biomass in soil involves:

i. The air dried algal crust of a limited area is homogenized in 10 ml
of 90% acetone and extracted in the dark at 50°C for 24 hrs.
ii. The extract is cleared of debris by centrifuge spinning at full speed
for 2-3 min.
iii. Solvent and contained chlorophyll are decanted to a spectrophotometer
tube and read at 665 nm and 750 nm before and after acidifying with one
drop of concentrated HCl.
iv. The corrected 665 nm readings are then used to calculate the
concentration of chlorophyll (a) and algal biomass in the sample
according to the following equation:

26.73 (665 -750 ) x (665 -750 )V

b b a a
Chlorophyll (a) (mg/m2) =
A
Algal biomass (kg/ha) = Chlorophyll (a) mg/m2 x 3.15
V = volume in litres of extracting solution
A = area of the sample
665 and 665 are optional densities of 90% acetone extract
b a
before and after acidification, respectively.

D.8. CULTURE SOLUTIONS FOR BLUE-GREEN ALGAE

Various nutrient solutions have been used for isolation and maintenance
of blue-green algae. Essentially all these solutions have the following
characteristics:
i. Weakly alkaline reaction is usually obtained by the use of K HPO or carbonate.
2 4
ii Nitrogen, if used, is in the form of Ca or KNO .
3
iii. Mg and Fe are present.
iv. Other elements are used.
v. The concentration of the different elements is variable with the different
culture media.

Common media used with blue-green nitrogen-fixing algae are summarized

as follows:
Medium for tolypothrix (Watanabe, 1960) g/l
KNO 3.0
3
Na HPO .12H O 0.5
2 4 2
MgSO .7H O 0.5
4 2
CaCl 0.02
2
FeSO 0.02
4
As solution 1 ml
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3 2
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D.9. MANAGEMENT OF ALGAL PRODUCTION FOR PADDY INOCULATION

(Indian Technology)
Singh (1978) outlined the following recommendation for the production
and application of blue-green algae:

Algal Propagation
i. Prepare shallow trays of galvanized iron sheet (4' x 3' x 9") or of bricks
and mortar so as to have permanent units. The size and number of trays
(tanks) depends upon the amount of inoculum produced. It is advisable to
have permanent structures with proper irrigating and draining facilities at
the block level.
ii. Keep 17 kg of soil in the trays and mix it well with 10 g of superphosphate.
About 2 g of Na molybdate may alsobe added if there is a response to its
addition in a particular type of soil.
iii. Keep the soil flooded after the addition of water. Lime is recommended
for acidic soils to raise the pH.
iv. When the water becomes clear sprinkle the starter culture on the surface
of the standing water. The starter culture must be in a healthy condition
(about 20 g fresh material equivalent to 2 g dry wt.), for rapid growth.
The trays should be kept in the open field since algae grown under field
conditions multiply rapidly after inoculation.
v. Algae multiply rapidly during the summer and cover the area in 15 days.
Harvest the algal mat and use it for inoculation. In off seasons the
algal mat is collected, dried in the shade and is kept for further possible
use. As far as possible fresh algae should be used as inoculum.
vi. Continue cultivation and harvesting with the above amendments and change
the soil after 3-4 harvests. A single harvest from a tray 85 cm x 65 cm
x 15 cm, after 20 days, weighs around 250-400 g of fresh algae.
vii. To control the insects which eat blue-green algae add 3-5 g of diazinon
or cyeolane or furadan.
viii. Nitrogen-fixing blue-green algae growing in fallow flooded rice fields
before ploughing can also be collected and used as a starter culture or
inoculum. For this purpose some experience of identifying promising
blue-green algae by visual observation is required.

Field Application
i. Inoculate fresh algal material as fast as possible, rather than dried
algae, at the rate of 25-50 kg/ha (90% moisture) on standing water
which corresponds to 3-5 kg/ha on a dry weight basis. The addition of
excess algal material helps to produce more algae in a shorter time.
ii. Apply algal material with each crop for 3-4 consecutive seasons.
iii. Inoculate the algae after a week of planting in clear water. Inoculation
should be avoided during rains.
iv. Apply superphosphate at the rate of 20-40 kg P O /ha in two doses
2 5
during inoculation. The second one should be applied after 20 days.
v. Algae can be used with 20-40 kg N-fertilizer safely to obtain higher
yields.
vi. Recommended rates of pesticides or herbicides and other agronomic
practices do not generally interfere with algal establishment.
 Appendix E

AZOLLA

E.l. GUIDELINES FOR AZOLLA MULTIPLICATION AND UTILIZATION

Singh (1978, personal communication) suggested a few guidelines for
the management of Azolla. (Fig. 29).

Multiplication
i. Ploughed, levelled and bunded fields must be used for multiplication.
ii. Maintain 5-10 cm standing water continuously while multiplication is
in progress. Raising the water level to 30 cm will not have any adverse
effect.
iii. Inoculate Azolla at the rate of 0.2 to 0.4 kg per m2 depending upon the
availability of inoculum.
iv. Mix superphosphate at the rate of 4 to 20 kg P O /ha together with inoculum.
2 5
Ratna phosphate and rock phosphate are not recommended for Azolla cultivation.
A few grams (0.1 to 1.0 g/kg of pesticide furadan) should also be mixed with
the inoculum or both superphosphate and furadan may be applied after
inoculation.

LIE- bd unitibirciwou
(coutIpnicq PW WIC VOW)
v. After the formation of a layer (in a week) harvest the Azolla with the
help of a bamboo stick and again inoculate at the same rate for further
multiplication.
vi. Harvested Azolla should be re-inoculated in another field otherwise
it will decompose and turn into compost (5% N) which can also be used.
vii. Generally fresh Azolla (0.2 - 0.3% N) is used for manuring.
viii. Under conditions at Cuttack (India) Azolla multiplies throughout the
year while the water temperature is between 17 - 29 and 28 - 33°C
(night/day) at the rate of 2 - 4 times per week. Its multiplication
is affected adversely when the water temperature exceeds 32-40°C
(night/day).
ix. Azolla grows well in soils of slightly acid to alkaline pH (6 - 8).
Very acid soils of Kerala (pH 2.9 - 3.6) do not support its growth.
x. It can also be multiplied in concrete tanks containing 15 cm of soil.
xi. The multiplication programme should be started about a month in advance
so as to have sufficient inoculum for one acre.
The multiplication programme may be summarized as follows.
4 kg azolla + 36 g superphosphate (SP) inoculate 4 x 3 m incubate one week
12 kg azolla + 90 g SP inoculate 5 x 6 m incubate one week 36 kg azolla +
300 g SP inoculate 10 x 10 m incubate one week 110 kg azolla + 900 SP
inoculate 20 x 15 m incubate one week 330 kg azolla + 3 kg SP inoculate
45 x 20 m incubate one week 1000 kg azolla inoculate into one acre
with SP.
(Note: A few grams of the pesticide furadan may be mixed with each
inoculum).

E.2. MULTIPLICATION OF AZOLLA ON LARGE AREAS

i. Divide fields into (not larger than) 300 or 400 m2 subplots. Azolla
grows better in smaller plots since it gets blown about by wind if
the plots are too large.
ii. When pests occur apply 2 - 3 kg/ha of furadan on the spots where damage
has started. Azolla pests do not attack rice plants and in fact help to
foster Azolla decomposition.

E.3. UTILIZATION OF AZOLLA

As Green Manure:
Grow Azolla before planting crops in ploughed and levelled fields, for
which inoculant required is about 2000 kg/ha. After 7-14 days, drain
the field and mix the Azolla layer in the soil, with a plough if possible.
Then transplant rice seedlings into the plot within a week. Application
of a second layer of Azolla after incorporation of the first will provide
more nitrogen. One layer of Azolla provides 10 tonnes green matter/ha,
which is equivalent to 25-30 kg N. The increase in yield ranges from
0.5 to 1.5 tonnes/ha over control.
 As Dual Cropping with Rice
If water is not available before planting, inoculate the Azolla on
standing water at the rate of 500 to 1 000 kg/ha a week after planting
when seedlings are established. Recommended doses of superphosphate
for rice may be applied in split doses, i.e. the first half as a basic
dose and the second half before addition of Azolla with furadan.
After the mat has formed, drain the field, if possible, and incorporate
the Azolla with the paddy water. If this is not possible, leave it and
it will decompose in due course and increase the crop yield. It is
advisable to use a basal dose of nitrogen fertilizer when the Azolla is
grown with rice to encourage better tillering. Floating Azolla also
checks weed growth.
Use of Azolla with Inorganic Nitrogen Fertilizer
Azolla is also used along with nitrogen fertilizer. Both of them are
used as a basic dressing or as topdressing to obtain maximum yield.

E.4 NUTRIENT SOLUTIONS USED FOR AZOLLA GROWTH

Espinas and Watanabe (1976)
so bbw
VO bbw
C9 vo bbw
We vo bbw
Hu 02 bbw
WO 012 bbw
13 0-5 bbw
S9 O'OT bbw
C9 0-01 bbw
Es (s2 LGLLIC CI4LSf6) 5 bbw
bH 2.2
r6 A9u ow' Tae3 (Insu suci IpnXW T88)
J\TO HGTTLTeGT 20J(1OU
T\JO Co T\2 Kuob 2ojnpou
Ineu sug Ipm1Gc (Ta.N8)
T\TO Kuob 2ornfrou
bsq0 m946L + Is0 we\T 101-1b0"
Ao WTuP 10-19' TaT (Insu sug juriXW Ja.Ne)
KI-13b0" e.e wv\I
KCT ..\*2 we\T
We20" e0 we\1
CgCJ3 33'2 we\T
K320" V*3 we\T
E6CPI-130 52 we\T
WTCLOGT6W6Uf2 1'0 we\T
bu 2'2-2'0
 cPn (J8.8) pqsu VL 3o5
T' 101-1,1.66 2orri4rou
cs2o'*sH3o O'TTA n
WE20AH30 wove n
E6CT1 00051 E
H23o2 00003 E
AgHlbo' wovle
KCT wosse
Wpm° 00003 E
DT24TJjsq m9g6L I 000 LIJI

IT' m-cnignLs eoTruTou

Vqg I 000 bbw 01. 1411140' ço CP6KT9LIV 01-* nos'
 Appendix F

CULTURE COLLECTIONS
OF
NITROGEN-FIXING ORGANISMS

The following list of addresses covers most

centres carrying nitrogen-fixing organisms
F.l. CULTURE COLLECTIONS OF RHIZOBIUM
Australia
Mr. J. Brockwell
CSIRO, Division of Plant Industry
Box 1600 Canberra City, A.C.T.

Dr. R.J. Roughley

AIRC Service
Dept. of Agriculture, PMB 10
Rydalmere, N.S.W. 2116

Dr. C.A. Parker

Dept. of Soil and Plant Nutrition
Institute of Agriculture
Nedlands, W. Australia, 6009

Brazil
Dr. J. Dobereiner
Institute de Pesqurae Experimentacae
do Sentro sul (IPEACS)
Km 47 via Campo Grande Zc-26, GB.

Canada
Dr. M. Hauser
Dept. of Microbiology
University of Guelf

Dr. F.B. Roslycky

Res. Institute, Canada Agriculture
University Sub. P.O.
London, Ontario

Czechoslovakia
Dr. Helena Jakubcova
Dept. of Microbiology
Central Res. Inst. of Plant Production
16106 Praha 6, Ruzynev, 507

France
Dr. M. Obaton
Station de Recherches de Microbiologie des Sols,
7 rue Sully - 21 - Dijon

India
Dr. R.B. Patil
Dept. of Microbiology
University of Agricultural Sciences
Bangalore 24

Dr. R.B. Rewari

IARI
Division of Microbiology
New Delhi, 12
 Dr. M. Sulaiman
BNF Scheme
Poona - 5

Japan
Dr. S. Ishizawa
Dept. of Soils and Fertilizers
National Inst. of Agricultural Sciences
Nishigahara, Kita-kir, Tokyo

Dr. S. Yoshida
Dept. of Agricultural chemistry
Nagoya University
Chekusa-Ku, Nagoya

The Netherlands
Miss E.O. Biewenga
Institute of Soil Fertility
Oosterweg 41, Haren, Groningen

Dr. E.G. Mulder

Laboratory of Microbiology
University of Wageningen, Wageningen

Poland
Dr. Z. Lorkiewicz
Institute of Microbiology and Biochemistry
University of Marie Curie
Skiodowska, Lublin

Rhodesia
Dr. W.P.L. Sandman
Glasslands Res. Station
P. Bag 701, Marandelles
Rhodesia, Africa

South Africa
Dr. B. Strijdom
Plant Protection Res. Institute
Private Bag 134
Pretoria, S-Africa

United Kingdom
Dr. D. Gareth Jones
Dept. of Agricultural Botany
University College of Wales
Panglais, Aberystwyth, Wales

Dr. M. Dye
Rothamsted Experimental Station
Harpenden, Hertfordshire
U.S.A.
Dr. B.E. Caldwell
USDA
Crops Res. Division
Beltsville, Maryland 20705

Dr. F.E. Lessel

ATCC
12301 Parklawn Dr.
Rockville, Md, 20852

U.S.S.R.
Dr. F.S. Matevosian
Institute of Microbiology
Armenian S.S.R. Academy of Sciences
Tsharentso 19,
Erevan 25, Armenian S.S.R.

Dr. L.M. Dorossinsky

Al1 Union Scientific Res. Institute of Agric. Microbiology
Leningrad, Gerzen St. 42

Yogoslavia
Dr. Vera Petrovic
Institute of Soil Science
Beograd, Teodova Drajzera 7

In addition to the above collection centres, there are two MIRCENS which
are dealing with rhizobia cultures:

i. Porto Alegre (Brazil)

Dr. J.R. Jardin Freire
UFRGS Faculdade de
Agronomia,Caixa Postal 776
90000 Porto Alegre, Brazil.
ii. Dr. S.O. Keya
Soil Science
. University of Nairobi
P.O. Box 30197
Nairobi, Kenya.

F.2. CULTURE COLLECTIONS OF BLUE-GREEN ALGAE

Canada
National Res. Council
Ottawa, Canada

Czeckoslovakia
Prof. B. Fott
Charles University
Chair of Botany, Cryptogamological Dept.
Praha
 Dr. J. Komarek, Dr. O. Lhotsky
Czechoslovak Academy of Sciences
Botanical Institute
Laboratory of Hydrobotanics
Treban

Prof. S. Prat
Czechoslovak Academy of Sciences
Institute of Experimental Botany
Collection of Cultures of Autotiophic Organisms
Praha

Federal Republic Germany

Dr. W. Koch
Universitat Gottingen
Planzenphysiologisches Institut
Gottingen

France

Dr. R. Pourriot
Centre de Recherches Hydrobiologiques
Gif - sur - Yvette

Prof. R.Y. Stanier

Institut Pasteur
25 Rue du Doteur Roux
XVe Arrond, Paris

German Democratic Republic

Prof. H. Borriss
Ernst-Moritz-Arndt Universitat
Botanisches Institut
Greifswald

United Kingdom
Dr. E.A. George
The Culture Centre for Algae and Protozoa
36 Storeys Way, Cambridge

India
Prof. G.S. Venkataraman
Indian Agricultural Res. Institute
Culture Collection of Microalgae
New Delhi

Prof. T.V. Desikachary

University of Madras
Centre for Advanced Study in Botany,
Madras
Japan
Dr. I. Ichimura
University of Tokyo
Institute of Applied Microbiology
The Algal Collection
Tokyo

Philippines
Dr. J. Watanabe
IRRI
Los Banos, Philippines

U.S.A.
Prof. R.C. Starr
Indiana University
Culture Collection of Algae
Bloomington, Indiana

Prof. R.W. Castenholz

University of Oregon
Department of Biology
Eugene, Oregon

U.S.S.R.
Dr. B.V. Gromov
State University of Leningrad
Institute of Biology
Leningrad - Petergol
U.S.S.R.

F. 3. CULTURE COLLECTIONS OF AZOTOBACTER AND AZOSPIRILLUM

Holland
Dr. J.H. Becking
Institute for Atomic Sciences in Agriculture
Potsbus 48. Wageningen

Brazil
Dr. F. Dobereiner
Institute de Pesqurae Experimentacae
do Sento Sul (IPEACS)
Km 47 Via Campo Grande Zc-26 GB

United Kingdom
Dr. J. Postgate
Unit of Nitrogen Fixation
ARC University of Sussex
Brighton
F. 4. CULTURE COLLECTIONS OF AZOLLA
U.S.A.
Dr. G.A. Peters
C.F. Kettering Lab
Yellow Springs, Ohio 45387

Holland
Dr. J.H. Becking
Institute for Atomic Sciences in Agriculture
Posbus 48, Wageningen

India
Dr. P.K. Singh
Central Rice Res. Institute
Cuttack, Orissa

Philippines
Dr. I. Watanabe
The International Rice Res. Institute
Los Banos, Laguna

China
Dr. L.C. Chu
Soil & Fertilizer Res. Institute
Fukien Acadamy of Agriculture Sciences
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