Pesticides, Microbial

E Montesinos and A Bonaterra, University of Girona, Girona, Spain

ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Discovery of Microbial Pesticides

The Plant as a Microbial Ecosystem Production and Formulation
Plant Disease and Biological Control Methods Monitoring, Environmental Impact, and Biosafety
Microorganisms Active in the Biological Control of Plant Registration and Commercialization
Pathogens and Their Mechanism of Action Further Reading

Glossary competitive exclusion An ecological process by which

antagonism A biological type of interaction between an organism excludes another organism from a habitat
living organisms that has a detrimental effect on one or through more efficient use of nutrients and space
both partners. colonization.
biological control A process derived from antagonistic fermentation The mass production of microorganisms
interactions between a beneficial organism and a by cultivating in solid or liquid culture media for industrial
pathogen or parasite that results in the control of a purposes.
disease or pest. formulation A method of preparing biopesticides for
biopesticide A formulated active ingredient of commercial delivery that consists of the preservation of
biological origin, generally composed of microbial cells, microbial cells to increase shelf-life and enhance field
that is used to control plant diseases or pests. survival and efficacy.
biosafety The property of an active microbial ingredient registration The administrative procedure by which an
related to the lack of adverse effects in plants and active ingredient is authorized for commercial use
animals, including humans. following specific legal regulations.

Defining Statement specialized type of fungi, and have applications in plant

phytostimulation, biofertilization, and bioremediation.
Microbial biopesticides are products made from benefi- Pathogenic or detrimental interactions of microbes with
cial microorganisms that are used to control plant plants involve viroids, viruses, bacteria, and fungi, and lead
diseases. Many products composed of bacteriophage, bac- to infectious diseases affecting only the plant kingdom.
teria, yeast, and fungi are marketed worldwide, and are The microbiota associated with plants has been classi-
used by spraying or drenching plants, by local application cally studied by cultivation-dependent methods, which
of sticks or tablets near the root system, by seed coating or are estimated to recover only 0.01–10% of the total
root bacterization before transplanting, and by using population. However, based on molecular analysis, it has
helper insects for dispersion. been estimated that there are more than 4000 microbial
species per gram of soil in the rhizosphere.
Healthy plants contain a diversity of commensal and
The Plant as a Microbial Ecosystem saprophytic microorganisms that contribute to provide, as
in animals, an environmental equilibrium as the initial
Microorganisms have adapted to the plant environment barriers for entrance of pathogens (Figure 1). Bacteria are
because plants offer a wide diversity of habitats, including abundant in plants because they enable rapid growth and
the phyllosphere (aerial plant part), rhizosphere (zone of are represented by the Gram-negative Pseudomonas
influence of the root system), and endosphere (internal (Pseudomonas fluorescens), Pantoea (Pantoea agglomerans,
transport system). Interactions may be neutral, beneficial, synonymous with Erwinia herbicola), Xanthomonas
or detrimental for either the microorganism or the plant. (Xanthomonas campestris), Flavobacterium, Micrococcus, and
Beneficial interactions for plants are caused by symbiotic in some plants or plant parts by lactic acid bacteria
or nonsymbiotic bacteria and by mycorrhizae, a highly (Lactobacillus and Leuconostoc) or Azospirillum species.

110
 Applied Microbiology: Agro/Food | Pesticides, Microbial 111

(a) (b)

(c) (d)

Figure 1 Microorganisms interact with plant surfaces. (a) A print of a leaf on the surface of nutrient agar into a Petri plate reveals the
presence of microbial colonies after several days of incubation; (b) infection of the stomata by a germinating spore of a pathogenic
fungus; (c) colonization of the surrounding area of the stomata by bacteria; and (d) colonization of the hypanthia surface of a flower.

Plants are also colonized by Gram-positive spore and temperature are environmental parameters that often
formers, which are mainly represented by the Bacillus exhibit strong fluctuations (daily, seasonal, hourly
species that colonize the soil and root system together changes) and greatly affect microbial epiphytic life, as
with actinomycetes. Yeasts such as Aureobasidium, they determine growth rate, germination, taxis, and
Sporobolomyces, Cryptococcus, Torulopsis, Saccharomyces, other essential processes for colonization of the plant
Kloeckera, Rhodotorula, and Candida are part of the micro- surface. The root environment is more stable. The spatial
biota of leaves and develop especially in fruits and flowers distribution of the microbiota on the plant surface is not
with high content of sugars. Different types of molds such homogeneous because it depends strongly on suitable
as Alternaria, Penicillium, Fusarium, and Aspergillus can also sites for colonization and growth. Microcolonies are
be found. Several endophytic microorganisms also inhabit often found in key environments like in the surrounding
the phloem and xylem vessels of plants. area of the stomata on the leaf surface, the nectaria in
The composition and population levels of microorgan- flowers, the calyx end in fruits, or the root tip.
isms in plants are determined by nutrient sources released
by plant organs as root or leaf exudates or specific secre-
tion systems as the nectaria in flowers. The degree of Plant Disease and Biological Control
change in microbial composition is associated with the Methods
type of plant species or cultivar and varies according to
the phenological state and organ (leaves, flowers, fruits, Losses in crop production due to plant disease aver-
seeds, roots). Water availability on the aerial plant surface age 13% worldwide and severely limit production,
112 Applied Microbiology: Agro/Food | Pesticides, Microbial

quality, and safety of food. Several thousands of Microorganisms Active in the Biological
diseases that are caused by 120 genera of fungi, 30 types Control of Plant Pathogens and Their
of viruses, and 10 genera of bacteria have been described Mechanism of Action
in plants.
Disease control is mainly achieved by treating crops Finding beneficial microorganisms to develop microbial
with significant amounts of synthetic chemical pesti- pesticides is a trial-and-error process. The experience
cides. However, social and political concerns have accumulated over several decades of research using
influenced the practice of crop protection, which has different screening strategies indicates that many suitable
been progressively reoriented toward a rational use of sources come from disease-suppressive soils or healthy
pesticides and toward a reduction in the number of plants in epidemic areas affected by a given disease.
registered active ingredients to those that are more However, success has also been obtained with nondir-
selective, less toxic, and with a lower negative environ- ected search, that is finding microorganisms of interest
mental impact. As a consequence, several countries have from habitats far from the plant environment intended to
undertaken regulatory changes in pesticide registration be protected.
requirements, given that consumer health and environ- To be a biological control agent is not an attribute of a
mental preservation prevail over productive or given genera or species of microorganisms, but it is gen-
economic considerations. erally restricted to the strain level. Usually, a few out of
The therapeutic approach used in the past for the thousands of strains that have been isolated result in a
plant protection is changing to a sustainable pest man- good candidate being developed as a commercial biopes-
agement approach. However, this change in crop ticide. Many research projects have been successful and
protection methods is generating additional problems hundreds of strains of microorganisms have been reported
caused by a shortage of tools for control of diseases in as active in the control of plant pathogens, such as bacteria
plants of economic importance. Fortunately, new meth- and fungi causing aerial or root diseases, or are effective
ods of crop protection have emerged from traditional against postharvest rot of products like fresh
practices in agriculture and forestry such as the use of fruits and vegetables. Strains of biological control agents
disease suppressive soils, crop rotation, soil solarization, are distributed among Gram-negative bacteria of
and green manure or compost as organic amendments. the families Rhizobiaceae, Pseudomonadaceae, and
Many of the benefits of these old techniques rely on Enterobacteriaceae, and among Gram-positive bacteria
naturally occurring microorganisms that exert a biolo- such as Bacillaceae, Lactobacillaceae, Leuconostocaceae,
gical control on pests and diseases (Figure 2). and Streptomycetaceae. There are also representatives of
yeasts and fungi within Ascomycota and Basidiomycota,
and also in Oomycota. Finally, there are also hipovirulent
pathogens with biocontrol ability like Fusarium,
Rhizoctonia, Sclerotinia, and Phytophthora, mainly isolated
from suppressive soils (Table 1).
Beneficial microorganisms able to control plant diseases
Disease Biocontrol can colonize or compete with the pathogens for nutrients
and sites of interaction, or exert antagonism through anti-
Environment microbial compounds, develop hyperparasitism or directly
(T,RH,W,R...) attach to the pathogen cells, interfere with pathogen signals,
or induce resistance into the plant host. There are examples
of strains that cover a single mechanism, or it is possible that
a combination of several mechanisms converge within the
Re icrotion, d
(c
...)

m posi

same strain (Table 2; Figures 3 and 4). Antibiosis against

o
den n

sid bio ensi

sity
ce, oge

en ta ty...)

plant-pathogenic bacteria and fungi is very common among

t
th
Pa

bacterial pesticides, for example, by the production of

ulen
(vir

Host opines, a type of toxic derivative of amino acids, in

(supportiveness...)
Agrobacterium radiobacter, phenolic antifungal compounds in
Figure 2 The disease tetrahedron is composed of a Pseudomonas species, antimicrobial peptides and polyenes in
susceptible host plant that can be infected by a virulent Bacillus and actinomycetes, or lytic enzymes in several yeast
pathogen under suitable environmental conditions, and it and fungi like Candida and Trichoderma. Competitive exclu-
is influenced by interactions with the natural microbiota.
This four-level interaction may result either in the disease
sion of the pathogen from sites of infection by better use of
process in the plant host or in the biological control of nutrients and colonization is also a common mechanism
the pathogen. that can accompany other mechanisms. Several
 Applied Microbiology: Agro/Food | Pesticides, Microbial 113

Table 1 Groups of microorganisms that have strains of sequence is available for Debaryomyces hansenii (7 chromo-
biocontrol agents of plant diseases somes and 12.2 Mb) and Pichia guilliermondii (10.54 Mb),
Group Family/Phylum Genus which have strains that control several postharvest and
preharvest fungi. Sequencing is in progress for
Bacteria Bacillaceae Bacillus Trichoderma atroviride (40 Mb), which has strains that can
Paenibacillaceae Brevibacillus
serve as general-purpose microbial biopesticides.
Paenibacillus
Cellulomonadaceae Cellulomonas
Enterobacteriaceae Enterobacter
Pantoea (Erwinia) Discovery of Microbial Pesticides
Rahnella
Serratia
The process of discovery and development of a microbial
Lactobacillaceae Lactobacillus
Leuconostocaceae Leuconostoc biopesticide consists of two stages. The first stage deals with
Pseudomonadaceae Burkholderia the isolation of candidate microorganisms in pure culture,
Pseudomonas the identification and characterization of strains, and the
Rhizobiaceae Agrobacterium measurement of efficacy in laboratory-controlled bioassays
Streptomycetaceae Streptomyces
to finally perform pilot testing under greenhouse or field
Yeast Ascomycota Aureobasidium
Candida conditions. In the second stage, candidate microorganisms
Coniothyrium are cultivated at a large scale, preserved and formulated for
Debaryomyces industrial production, and submitted for toxicological and
Kloeckera environmental impact studies to qualify for registration and
Metschnikowia
authorization for commercial delivery (Figure 5).
Pichia
Basidiomycota Cryptococcus Finding bacteria, fungi, or virus able to interfere with
Pseudozyma the biological cycle of selected plant pathogens requires
Rhodotorula proper sampling to increase the probability of obtaining
Sporobolomyces useful strains. For this reason, samples are taken at places
Fungi (molds) Ascomycota Ampelomyces
or containing materials where there is evidence of bene-
Chaetomium
Epicoccum ficial microorganisms, such as in disease-suppressive soils
Gliocladium or healthy plants or plant parts that survived the disease
Muscodor in epidemic areas.
Myrothecium The screening for antagonistic activity against the target
Penicillium
pathogen is a critical step because the type of microorgan-
Trichoderma
Ulocladium ism selected depends on the method used. Approaches
Basidiomycota Phlebiopsis based on host-mediated selective enrichment and pheno-
Oomycota Pythium typic or molecular marker-assisted selection have
improved the isolation and development of new biocontrol
agents. Usually, screening procedures are specific to a
given mechanism of action, such as antibiosis, competition,
hyperparasites, especially abundant among the yeast and
or induction of plant defenses. Isolation in pure culture
fungi biocontrol agents like Coniothyrium, Gliocladium, Pichia, needs a suitable method of cultivation in synthetic media
Ampelomyces, Streptomyces, and Trichoderma, interact directly or of enrichment in a given biological system (cellular
and degrade the fungal cell wall or spore envelope. Some cultures or trap organisms).
bacteria and fungi are able to induce defense responses in At this stage, several hundreds or thousands of isolates
plants, by producing either elicitors (e.g., cell wall compo- are collected, and after identification the strains are sub-
nents) or messenger molecules (e.g., salicylic acid). Finally, mitted for efficacy bioassays in a small-scale controlled
a new mechanism has emerged based on the finding that laboratory environment using in vitro, ex vivo, or, in planta
there are bacteria that can degrade chemical signal messen- tests in three-partner model pathosystems (putative bio-
gers necessary for quorum sensing (e.g., acyl homoserine logical control agent, target pathogen, host plant)
lactones) used by many plant–pathogenic bacteria patho- (Figure 6). The knowledge of the mechanisms of action
gens to start the infection process in the host. of a biological control agent and the information derived
The basic and applied research on biological control from partially or totally sequenced genomes can provide
agents has stimulated sequencing of their genomes. The data on gene targets that can be developed for high-
genome has been completely sequenced for various bio- throughput screening procedures. Modern techniques of
logical control strains like Bacillus amyloliquefaciens FZB42 assisted selection using fenotypic or genotypic markers
(3.9 Mb), Bacillus subtilis 168 (4.21 Mb), and P. fluorescens contribute to improving the productivity of the screening
Pf-5 (7.1 Mb). In the case of fungal strains, the genome stage. Recent advances in DNA arrays or screening of
114 Applied Microbiology: Agro/Food | Pesticides, Microbial

Table 2 Mechanisms of action in selected biocontrol agents of plant diseases

Microorganism and strain Target pathogen or disease Mechanism involved

Virus
Bacteriophage Xanthomonas and Pseudomonas diseases Lysis
Bacteria
A. radiobacter K84/K1026 A. tumefaciens Ab-opines
B. amyloliquefaciens FZB42 Fusarium oxysporum Ab-AMP
B. subtilis ATCC6633 Pythium Ab-AMP
B. subtilis 6051 P. syringae Ab-AMP
B. subtilis GA1 Botrytis Ab-AMP
B. subtilis UMAF6614 Podosphaera fusca Ab-AMP
P. agglomerans Eh318/C9-1 Fire blight CE, Ab-pseudopeptide
P. agglomerans EPS125/CPA-2 Postharvest fungi CE
P. chlororaphis PCL1391 F. oxysporum Ab-PCA
P. chlororaphis MA342 Pythium, others Ab-DDR, CE
P. fluorescens A506 Fire blight and frost damage CE, Ab
P. fluorescens Pf-5 Pythium, Rhizoctonia Ab-DAPG, Plt, Prn
P. fluorescens Q2-87 Gaeummanomyces graminis Ab-DAPG
P. fluorescens WCS417 F. oxysporum IR
P. fluorescens SS101 Phytophthora Ab-cAMP, IR
P. fluorescens EPS62 E. amylovora CE
P. syringae ESC10/ESC11 Postharvest rot fungi Ab-Syr, CE
S. griseoviridis K61 Soil-borne pathogens CE, HP, Ab-polyenes
Fungi
Ampelomyces quisqualis AQ10 Powdery mildew HP
A. pullulans CF10 Postharvest fungi, fire blight CE, Ab
A. pullulans Ach1-1 Postharvest fungi CE
C. oleophila I-182/Q Postharvest fungi CE, Ab-lytic enzymes
C. sake CPA-1 Postharvest fungi CE
Coniothyrium minitans CON/M/91-08 Fungi HP
Gliocladium catenulatum J1446 Soil-borne fungal pathogens HP, Ab-lytic enzymes
G. virens GL21 Soil-borne fungal pathogens HP, Ab-gliotoxin
P. guilliermondii 87 Fungi CE, HP
T. harzianum T22, P1 Soil-borne pathogens COM

Ab, antibiosis; AMP, cyclic antimicrobial peptides; CE, competitive exclusion; COM, complex; DAPG, diacetyl phloroglucinol; DDR, didehydro-
rhizoxin; HP, hyperparasitism; IR, induced resistance; PCA, phenylcarboxilic acid; Plt, pyoluteorin; Prn, pyrrolnitrin; Syr, syringomicin.

Host-mediated Competitive exclusion

resistance - colonization of entry sites on host
- induced systemic resistance (ISR) - competition for nutrients (C, N, P, iron)
- systemic acquired resistance (SAR) - physical barriers (biofilm)

Mechanisms of action of
microbial pesticides

Direct cell-to-cell Intercellular Antibiosis

interaction signal - lytic enzymes
- hyperparasitism interference - antimicrobial peptides
- interference with cell/hyphal - quorum sensing quenching and proteins
growth - phenolic compounds
- polyketides
- biosurfactants
- others

Figure 3 An overview of the mechanisms of biological control of plant pathogens that have been demonstrated in beneficial
microorganisms interacting with plants.
 Applied Microbiology: Agro/Food | Pesticides, Microbial 115

(a) (b)

(c) (d)

(e) (f)

Figure 4 Antagonism against plant pathogens can be revealed in vitro. (a) Halus of inhibition of confluent growth of a fungus produced
by colonies of selected antagonistic bacteria and yeast. (b) Antibiosis is accompanied by swarming motility of the colonies of the
antagonist bacteria against a plant-pathogenic bacterium. (c) Production of lytic enzymes that hydrolyze chitin, a component of the
fungal cell wall that has been added to the agar plate. (d) Synthesis of siderophores around colonies that are involved in competition for
iron. (e, f) Direct interference and hyperparasitism due to attachment of bacterial cells to the spores of fungi.

metagenomic libraries of cloned DNA can also contribute presentation of a low effective dose, the production of
to developing potent screening methods. During the specific metabolites against the target pathogen, and tol-
selection process, apart from efficacy and consistency of erance to some pesticides commonly used in agriculture,
results between bioassays, additional criteria, such as the are used to retain isolates.
116 Applied Microbiology: Agro/Food | Pesticides, Microbial

Discovery,
Pilot tests are conducted under conditions as close as
screening and possible to the environment under which biological control
isolation in will be practiced. During this stage, the biocontrol agent is
pure culture confronted with several pathosystems to verify its action
spectrum and under environmental conditions sufficiently
Identification and
characterization diverse to guarantee a wide range of applicability. The high
number of different conditions to be tested permit working
with only those isolates that were selected as suitable candi-
Selected Bioassays and
Patenting microbial dates in the previous massive screening stage. Unfortunately,
mechanism of action
strain work on most biological control agents discovered concludes
with this step because of a too narrow action spectrum,
Pilot tests
inconsistency of results between trials, or a low efficacy
under field or practical conditions (Figure 7).
Biosafety and The discovery of biopesticides based on microorgan-
environmental Specific analytical
isms, if successful, is of economic interest because of the
Preservation
and formulation impact methods and possibility of commercial exploitation of the technology.
traceability A few hundreds of patents exist involving strains of
Mass
production microorganisms with potential application as microbial
pesticides, including fungicides, insecticides, herbicides,
and plant growth stimulants.

Registration for commercial use

Production and Formulation
Figure 5 Steps involved in the discovery of microbial
pesticides that ends up with patent filing and registration and For the commercial development of a microbial pesticide,
authorization for commercial delivery of formulated products. the microorganism cells have to be produced at the

(a) (b) (c)

(d)

Figure 6 Pathogen infection in a plant host is controlled by microbial pesticides. (a) Inhibition of brown spot infections on pear leaves
that have been treated with an antagonistic bacterium (upper left panel) in comparison to a nontreated control (lower left panel).
(b) Control of fire blight infections in immature pear disks by several strains of antagonistic bacteria (rows). (c) Inhibition of fungal
infections in strawberry roots by antagonistic bacteria (upper right panel) in comparison to a nontreated control (lower right panel).
(d) A pear fruit wounded, treated with antagonistic bacteria, and inoculated with spores of the blue mold shows strains that inhibit rot.
 Applied Microbiology: Agro/Food | Pesticides, Microbial 117

(a)
industrial scale, preserved, and formulated for storage and
delivery. Depending on the nature of the microorganism
(bacteria, fungi, yeast, or virus), the methods used for indus-
trial scale-up are solid- or liquid-phase fermentation.
Methods consist essentially of the technology used in the
pharmaceutical and food industry. Bacteria and yeast are
produced by liquid fermentation using bioreactors, but
many fungi are fermented by using solid-state procedures.
The objective is to obtain the highest yield possible using a
culture medium with the lowest cost, often achieved using
food-grade molasses, peptones, brewer’s yeast extract, and
protein hydrolysates for industrial use. After liquid fermen-
tation the microbial cells are concentrated by filtration or
centrifugation, and in solid fermentation the material is
(b)
recovered and homogenized to get a concentrated liquid
suspension of cells or spores. In cases in which the micro-
organism is not directly culturable in synthetic media
(bacteriophage), industrial production consists of an initial
step in which the host bacterial cells are produced and a
second step of virus scale-up feeding on the previously
produced bacteria. Concentrated suspensions obtained gen-
erally range from 108 to 1010 cfu ml 1, depending on the
nature of the microorganism (Figure 8).
The storage and preservation of the microbial pesticide
to increase its shelf-life is one of the main limiting factors for
commercialization. The procedure involves stabilizing the
viability of cells in a liquid-phase suspension maintained
Figure 7 Control of blue mold rot in apples (a) and of brown rot under refrigeration or frozen in the presence of crioprotec-
in peach (b) with antagonistic bacteria. Nontreated controls are tant substances, or as a dehydrated product. Dehydration is
shown on the left side and treated fruits on the right side. the best from a practical point of view because it permits

Stock culture Liquid

formulation
Centrifugation

Liquid-phase fermentation
Streak culture

Concentrated
suspension of
cells/spores Drying

Inoculum
preparation
Homogenization Solid
Solid-phase production formulation
Figure 8 Industrial production of microbial pesticides consists of scaled-up fermentation of a suitable strain, downstream processing
of the biomass and supernatant of cultures, and formulation for commercial delivery.
118 Applied Microbiology: Agro/Food | Pesticides, Microbial

optimum storage conditions, handling, and distribution. of around 108 cfu ml 1 is recommended for bacterial bio-
Liophylization is a method that can maintain better viability, control agents and 107 for fungi or yeast. Accordingly,
but the cost is very high. Encapsulation of microbial cells is formulated products are diluted in nonchlorinated water
also used, in which the cells are mixed with a matrix-form- to get the dose recommended.
ing material such as gelatinized polysaccharides or
emulsioned in a lipid material that is finally diluted and
spray-dried to obtain the desired particles. At the industrial
level and in order to obtain a low-cost product, the method Monitoring, Environmental Impact,
preferred is spray or fluidized bed drying. However, spray and Biosafety
drying produces a high loss of viability in some microorgan-
isms (e.g., Gram-negative bacteria) due to the thermal Specific analysis of the microorganism strain of the bio-
treatment that accompanies the process. Although many pesticide is necessary for quality control during
microbial pesticides are prepared as dried products, some production and formulation and also to perform studies
are delivered as liquid suspensions of cells or spores. on traceability, residue analysis, and environmental
Irrespective of whether the final preparation is aqueous or impact. In field studies, the classical microbiological
dried, the active ingredient is formulated to obtain a market- methods may not be suitable because they do not distin-
able product. The formulations are composed of an active guish the strain that contains the microbial pesticide
ingredient (microorganism cells or spores and sometimes from the same species that could be present in the
culture components), carriers or inert materials used to natural microbiota associated with plants. In certain
support and deliver the active ingredient to a target, and cases, selecting an expontaneous mutant resistant to a
adjuvants that promote and maintain the functionality of the suitable antibiotic for which it is rare to find resistance in
active ingredient by including nutrients, chemical agents nature is sufficient. However, genotypic markers are
that filter ultraviolet light radiation, or osmoprotectant com- preferable because they are more stable and their
pounds that prevent damage from cell desiccation. expression does not depend on the type of culture
However, care must be taken because certain components media used for analysis. Specific genotypic markers can
in the formulation can stimulate the pathogen and promote be found by DNA fingerprinting of the biocontrol
disease. In many cases, the technology for formulating bio- agent strain, based on the amplification of gene
pesticides is similar to that used for formulating chemical sequences by PCR methods (RAPD, REP, ERIC,
pesticides and pharmaceutical products, and results in stable AFLP), or by digesting DNA with restriction enzymes
and active products for several months. (RFLP, PFGE). The comparison of fingerprinting pat-
Liquid formulations contain around 107–109 cfu ml 1, terns of the biocontrol strain with those of a wide
whereas powdered products have the active ingredient collection of representative strains of the same species
generally at a higher concentration (around 109–1010 cfu can identify specific bands that can be sequenced and
g 1 for bacteria and yeast), but can be lower in certain characterized (SCAR fragments). The knowledge of the
fungi. The method of application depends very much on nucleotide sequence of SCAR fragments can aid in the
the pathosystem to be controlled. Spraying or drenching design of primers for PCR analysis. Real-time PCR
plants is a common practice when using an inundative (quantitative PCR) is a powerful tool for developing
approach of control. However, inoculative or augmentative methods of highly specific quantitative analyses of
strategies consist of preparing sticks or immobilizing sys- strains of the active ingredient in microbial pesticides
tems (alginate beads) and seed coating or bacterization of under natural conditions.
the root system in seedlings before transplanting. Another important issue that has to be taken into con-
Inundative application methods are essentially the same sideration in microbial pesticides is biosafety. Biosafety is
as used for chemical pesticides. In this strategy, the dose of necessary to avoid nontarget effects on plants or animals, or
biocontrol agent has an important effect on the efficacy of on the environment, because the isolation of a given micro-
control, because the latter depends on the relative amounts organism from plants is not a proof of its inocuity.
of pathogen and biocontrol agent. For many microbial Toxicological studies in mammals, including acute toxicity
pesticides, the median effective dose (ED50) is in the tests, are performed to guarantee safety to consumers and
range of 106–107 cfu ml 1 for bacteria and 105–106 cfu ml 1 handlers of the microbial pesticide, especially if cases of
for fungi and yeast. The ratio between cells of the biocon- opportunistic infections reported at the clinical level or if
trol agent and cells of the pathogen for optimum control certain secondary metabolites of concern are produced.
has been used as a measure of efficiency, which ranges in However, risk evaluation is not a simple task, because the
value from one to several hundreds. The efficiency of risk of non-target effect by a given microorganism is esti-
biocontrol is also dependent on pathogen aggressiveness mated from the intrinsic toxicity–pathogenicity level, the
and host supportiveness. Therefore, to ensure covering a degree of exposure, and the susceptibility of the possible
wide range of conditions of applicability in practice, a dose receiver. Most microbial pesticide strains meet all biosafety
 Applied Microbiology: Agro/Food | Pesticides, Microbial 119

rules. However, in a few cases, there are uncertainties on microorganism identity, biological properties, effi-
regarding the potential risk, because human isolates cacy, specific analytical methods, residues, traceability,
reported in clinical opportunistic infections are not distinct and potential adverse effects on human health and
from environmental isolates. There is scientific contro- nontarget organisms. In the European Union, the regis-
versy regarding the use of strains of Burkholderia cepacia, tration and authorization of microbial pesticides as
Pseudomonas putida, P. agglomerans, and Aureobasidium pull- plant protection products depends on the Health and
ulans as biological control agents, which will need further Consumer Protection Directorate (SANCO), and is
studies of safety and toxicological risk. Interestingly, some regulated by the Directives 2001/36/EC and 2005/
of these microorganisms are part of the normal microbiota 25/EC. In the United States, authorization is regulated
found in plants and are present in several plant-derived by the Environmental Protection Agency. Furthermore,
fresh products. These uncertainties are the reason for spe- specific requirements may change when an authoriza-
cific and differing regulations on risk classification of tion application considers the microorganism as a
microorganisms among countries. biocide or a plant enhancer. Therefore, the availability
of commercial biopesticides depends on the specific
regulations in each country, but several products that
Registration and Commercialization include bacteriophage, bacteria, yeasts, and fungi, which
are active against a wide range of plant pathogens in
Approval of microbial pesticides for commercialization very diverse crops and agroclimatic conditions, are
requires detailed dossiers accounting for scientific data marketed (Table 3).

Table 3 Commercial microbial pesticides

Active ingredient Commercial name Target pathogen/disease Host

A. radiobacter Nogall, Galltrol-A Crown gall Fruit and nut trees, ornamentals
Ampelomyces AQ10 Powdery mildew Apples, cucurbits, grapes,
quisqualis ornamentals, strawberries,
tomatoes
Aspergillus flavus AF36, Aflaguard, NRLL21882 A. flavus Cotton, peanut
B. licheniformis Green releaf, 145F Several fungal diseases Ornamental plants, conifers
of seedlings and tree seedlings,
ornamental turf
B. mycoides Isolate J Cercospora beticola Sugar beet
B. pumilus Ballad, Yield shield Sonata Soil-borne fungi, powdery and Soybean, cucurbits, grapes,
downy mildew, early and late hops, vegetables, peanuts,
blight, brown spot, fire blight pome fruits, stone fruits
B. subtilis Serenade, Rhapsody, Biopro, Bacteria (fire blight) and fungal Grape, hops, vegetables,
BS-F4, Promix, Subtilex, root diseases, Alternaria, peanuts, pome and stone
HiStick N/T, Companion, Kodiak Aspergillus, grey mold fruits, soybean, alfalfa, cotton
B. subtilis var. Taegro, Tae-Technical Soil-borne fungal rots Greenhouses shade and forest
amyloliquefaciens tree seedlings, ornamentals,
and shrubs
Bacteriophage Agriphage Bacterial blight and spot Tomato, pepper
C. oleophila Aspire Postharvest fungal rot Citrus and pome fruits
Coniothyrium minitans Contans WG, Koni Sclerotinia Vegetables, canola, oilseed
rape, sunflower, soybean
Cryptococcus albidus Yield plus Postharvest fungal rot Pome fruits
Gliocladium sp. Gliomix Rhizoctonia solani, Pythium Vegetables and turf
G. catenulatum Primastop, Prestop Soil-borne fungal pathogens Ornamental, vegetable, and
tree crops
G. virens Soilguard Soil-borne root fungal diseases Ornamentals and food crops in
greenhouses and nurseries
Metschnikowia Shemer Fungal rot Preharvest and postharvest
fructicola fruits
Muscodor albus Andante, Arabesque, QST 20799, Bacteria, fungi Food crops, seeds and
Glissade propagules, cut flowers
P. agglomerans Bloomtime, Blossom bless, Fire blight Pome fruit trees, ornamentals
Blightban C9-1
Phlebiopsis gigantea Rotstop Heterobasidium annosus Coniferous trees

(Continued )
120 Applied Microbiology: Agro/Food | Pesticides, Microbial

Table 3 (Continued)

Active ingredient Commercial name Target pathogen/disease Host

P. aureofaciens Spot-less Antracnosis Turfgrass

P. chlororaphis AtEze, Cedomon, Cerall Soil-borne fungi Greenhouse crops and cereals
P. fluorescens Blightban A506 Fire blight, frost damage Fruit trees, almond, potato
and tomato crops
P. syringae Biosave10 LP, Biosave 11 LP, Postharvest fungal rot Pome fruits, citrus, cherries
Esc-10 and potatoes
Pseudozyma flocculosa Sporodex, Pseudozyma Powdery and downy mildew Greenhouses roses and
cucumber
Pythium oligandrum Polyversum, DV74 Soil-borne pathogenic fungi Food crops, ornamentals
and turf
S. griseoviridis Mycostop Root rot, grey mold and foot decay Ornamental and vegetable
crops
S. lydicus Actinovate Root rot, grey mold and Greenhouse and nursery
damping-off crops, turf
T. atroviride Sentinel Grey mold Grape, tomato
T. harzianum Binab T, Trichopel, T-22, Root rot, grey mold and Trees, shrubs, transplants,
Rootshield, PlantShield, Tusal, damping-off ornamentals, vegetables
Trichodex
T. polysporum BinabT Fungal infections through wounds Ornamentals, shade, and
forest trees
Ulocladium oudemansii Botry-zen Grey mold Grape
Verticillium dahliae WCS850 Dutch elm disease Elm

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