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Edizioni ETS Pisa, 2010
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BIOLOGICAL MANAGEMENT OF PLANT DISEASES:
HIGHLIGHTS ON RESEARCH AND APPLICATION
E.C. Tjamos, S.E. Tjamos and P.P. Antoniou
Department of Plant Pathology, Agricultural University of Athens, Faculty of Crop Sciences, 75 Iera odos street,
Votanikos 11855, Athens, Greece
SUMMARY
An updated short account is given of the nature and
practice of biological control. It includes data on key
mechanisms of biocontrol agents (bca’s) being studied,
relationships between bca’s and plants being investigated and commercialization prospects in modern world
agriculture. Based on the voluminous literature on biological control research and application, successful cases
are selected and directions that might lead to the development of more effective bca’s against plant diseases are
discussed. The broad adoptions of organic farming as
an additional agricultural practice to conventional agriculture and IPM have both increased research and application of bca’s already commercially available. However, extensive applications require further developments and greater understanding of the complex interactions among plants, bca’s and the environment. Additionally fast and more efficient procedures compared to
fungicide registration approaches will facilitate bca’s
commercial application.
Key words: biological management, biological control
agents, commercialization of biocontrol agents.
INTRODUCTION
Various approaches are currently available to manage
plant diseases. Although good agricultural practices
help preventing or mitigating disease symptoms, the use
of fungicides, soil fumigants and disease resistant varieties, hybrids or rootstocks, still constitute the major effective tools. Scientific efforts for several decades now
have been focused on developing alternative approaches
for managing plant diseases. A long standing worldwide
effort has been concentrated on biocontrol agents
(bca’s). For several years biological control of plant diseases meant discovery and evaluation of bca’s as alternatives to chemicals. Most scientists expected rather erroCorresponding author: E.C. Tjamos
Fax: +0030 210 5294505
E-mail: ect@aua.gr
neously that bca’s could act independently from and
equally well as chemical applications. This philosophy
has now changed completely, since today chemicals are
mixed with bca’s or with SAR factors. Furthermore, biological control could be an entirely different approach
compared with chemicals being used against a single or
several plant diseases. Indeed biological control of plant
diseases was initially used as a very narrow process to
suppress a single pathogen, by a single antagonist, in a
single cropping system. As the years go by, the cases of
bca’s as microbial inoculants able to suppress a single
type or even a certain group of plant diseases has being
extensively developed. Traditionally, biological control
of plant diseases includes the term “control”. However,
nowadays it seems more appropriate to introduce the
expression “biological management” of plant diseases,
so to cover all aspects referring to suppression rather
than real control of plant diseases accomplished by
bca’s. This change becomes obvious from the application data available worldwide by evaluating effectiveness of bca’s in the everyday practice.
BIOLOGICAL INTERACTIONS AND MECHANISMS
OF BIOLOGICAL MANAGEMENT
Biological control simply means that pathogens are antagonized by the presence, activities or products of other
similar or different organisms that they encounter in the
plant’s rhizosphere or phyllosphere. Direct antagonism
by obligate parasites of a plant pathogen requires a high
degree of selectivity for the pathogen, called hyperparasitism. There are several fungal parasites of plant
pathogens, including those that attack sclerotia (e.g. Coniothyrium minitans), while others attack living hyphae
(e.g. Pythium oligandrum). Single fungal pathogen (e.g.
powdery mildews) could be parasitized by several hyperparasites such as Acremonium alternatum, Ampelomyces
quisqualis, Cladosporium oxysporum, and Gliocladium
virens (Kiss, 2003). On the contrary, indirect antagonisms
could be seen from activities that do not involve sensing a
pathogen by the bca’s. These include antibiotics, enzymes, competition and induced resistance-mechanisms
(Pal and McSpadden Gardener, 2006).
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Antibiotics. Antibiotics produced by microorganisms
have been shown to be effective at suppressing plant
pathogens in planta. Selective examples of bca’s given
below are particularly effective. Pseudomonas fluorescens
F113 produces 2,4-diacetyl-phloroglucinol against Pythium spp. (Shanahan et al., 1992). Agrobacterium radiobacter produces agrocin 84, against Agrobacterium tumefaciens (Kerr, 1980). Bacillus subtilis QST713 produces
iturin A against Botrytis cinerea and R. solani (Paulitz
and Belanger, 2001; Kloepper et al., 2004). B. subtilis
BBG100 produces mycosubtilin against Pythium aphanidermatum (Leclere et al., 2005). B. amyloliquefaciens
FZB42 produces bacillomycin and fengycin against
Fusarium oxysporum (Koumoutsi et al., 2004).
Pseudomonas fluorescens 2-79 and 30-84 produce
phenazines against Gaeumannomyces graminis var. tritici
(Thomashow et al., 1990). Trichoderma virens produces
gliotoxin against Rhizoctonia solani (Wilhite et al., 2001).
Enzymes. Several bca’s produce enzymes able to hydrolyze chitin, proteins, cellulose, and hemicellulose,
thus contributing to direct suppression of plant
pathogens. There are selective examples of bca’s able to
produce enzymes, effective against certain plant
pathogens. Serratia marcescens affects Sclerotium rolfsii
by chitinase expression (Ordentlich et al., 1988). However, such activities are rather indicative of the need to
obtain carbon nutrition. So, bca’s showing a preference
for colonizing and lysing plant pathogens might be classified as biocontrol agents. Lysobacter and Myxobacteria
are known to produce plentiful amounts of lytic enzymes, and some isolates have been shown to be effective at suppressing fungal plant pathogens (Bull et al.,
2002). So, the lines between competition, hyperparasitism, and antibiosis are generally disguised.
Competition. Although difficult to be proven directly,
much indirect evidence suggests that competition between pathogens and non-pathogens for nutrient resources is important for restricting disease incidence
and severity. Soilborne pathogens, such as species of
Fusarium and Pythium, infecting through mycelial contact, are more susceptible to competition by other soiland plant-associated microbes than by those germinating directly on plant surfaces which they invade through
appressoria and infection pegs. Rhizosphere or phyllosphere bca’s generally protect the plant by rapid colonization, thus consuming completely the limited available substrates so that none is left for pathogens to
grow. For example, effective catabolism of nutrients in
the spermosphere has been identified as a mechanism
contributing to the suppression of Pythium ultimum by
Enterobacter cloacae (van Dijk and Nelson 2000;
Kageyama and Nelson, 2003). At the same time, these
microbes produce metabolites that suppress pathogens.
These microbes colonize the sites where water and car-
bon-containing nutrients are most readily available and
utilize root mucilage. To survive in such an environment, microorganisms secrete iron-binding ligands
called siderophores that sequester iron from the microenvironment. Biocontrol based on competition for essential micronutrients, such as iron, has also been examined. Kloepper et al. (1980) were the first to demonstrate the importance of siderophore production as a
mechanism of biological control of Erwinia carotovora
by several plant growth promoting Pseudomonas fluorescens strains.
Induced resistance. Triggering latent host plant defence pathways by non-pathogenic bca’s is the most indirect form of antagonism. Plants respond to a variety of
chemical stimuli produced by bca’s. Such stimuli can either induce or condition host plant defences through
biochemical changes expressing resistance mechanisms
against subsequent infection by pathogens. Induction of
host defences can be localised and/or systemic in nature.
The determinants and pathways of induced resistance
stimulated by bca’s and other non-pathogenic microbes
have being occasionally characterized. The first of these
pathways, called systemic acquired resistance (SAR), is
mediated by salicylic acid (SA), which typically leads to
the expression of pathogenesis-related (PR) proteins including a variety of enzymes. A second case, referred to
as induced systemic resistance (ISR), is mediated by jasmonic acid (JA) and/or ethylene, which are produced
following applications of some non-pathogenic rhizobacteria. Some most striking examples of bacterial determinants and types of disease resistance (ISR) induced by
bca’s include a Bacillus mycoides strain able to produce
peroxidase, chitinase and b-1,3-glucanase in sugar beet
(Bargabus et al., 2002). B. subtilis GB03 and IN937 producing 2,3-butanediolI in Arabidopsis (Ryu et al., 2004).
Pseudomonas putida strains producing a lipopolysaccharide in Arabidopsis (Meziane et al., 2005). Serratia
marcescens 90-166 producing siderophore in cucumber
(Press et al., 2001). A number of strains of root-colonizing microbes have been identified as potential elicitors of
plant host defences. Some biocontrol strains of
Pseudomonas sp. and Trichoderma sp. are known to
strongly induce host plant defences (Harman, 2004). A
number of chemical elicitors of SAR and ISR may be
produced by the PGPR strains upon inoculation, including salicylic acid, siderophore, lipopolysaccharides, and
2,3-butanediol, and other volatile substances (Van Loon
et al., 1998; Ongena et al., 2004; Ryu et al., 2004).
BIOLOGICAL MANAGEMENT OF PLANT DISEASES:
RESEARCH AND DEVELOPMENT
While biological control of plant diseases developed
mainly as an academic discipline over fifty years ago,
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now is being supported by both the public and private
sector. A tremendous number of scientific papers are
currently published in phytopathological journals or
others specifically devoted to the discipline of biological
control. The advancements of sciences in computing,
molecular biology, analytical chemistry, modernize research approaches and elucidate topics regarding structure and functions of bca’s along with plant and
pathogens at certain levels. Thus, topics of biocontrol
research and development related to the ecology of
plant and bca’s, to the discovery of new effective strains
along with the development of more efficient application procedures and practical integration into agricultural systems are essential parameters for moving from
hope to reality in biological management of plant diseases. Below some data are given concerning newly introduced terms on bca’s and cases of successful applications.
Bioinoculants and mechanisms of PGPR plant
growth promotion. Several PGPR bioinoculants are currently used commercially. They are called different names
and operate through a range of mechanisms: (i) bioprotectants, through suppression of plant disease; (ii) biofertilizers, through improved nutrient acquisition; (iii) biostimulants, through phytohormone production. Bioinoculants include bacteria belonging in the genera Bacillus,
Paenibacillus Streptomyces, Pseudomonas, Burkholderia
and Agrobacterium, which are currently used as bca’s
also at commercial level. They suppress plant disease
through induction of systemic resistance, production of
siderophores or antibiotics. Biofertilizers are also available for increasing crop uptake of nitrogen from nitrogen-fixing bacteria (Azospirillium), and iron uptake from
siderophore-producing bacteria (Pseudomonas). Species
of Pseudomonas and Bacillus can produce as yet not well
characterized phytohormones or growth regulators that
cause extensive root growth, thus increasing the absorptive surface of plant roots. These PGPR are referred to as
biostimulants and the phytohormones that they produce
include indole-acetic acid, cytokinins, gibberellins and inhibitors of ethylene production. Current means of delivery of inoculants include peat, granular, liquid and wettable powder formulations. A major determinant of
growth promotion is the magnitude of their ability to colonize rhizosphere. Currently M. Lorito’s group in Portici
(Italy), working on various aspects of biological control in
general and on the use of Trichodermas in particular, has
elucidated the relationships established between bca’s
and plants and the mechanisms involved. Important findings include the discovery of molecular factors that regulate these interactions, and antimicrobial genes useful for
increasing plant disease resistance. These studies have
contributed to the development of new biofertilizer
products that utilize natural antimicrobial compounds
produced by diverse antagonists (Harman et al., 2004;
Tjamos et al.
S4.19
Marra and Lorito, 2006; Navazio et al., 2007; Marra et
al., 2008; Ruocco et al., 2009).
Promising biocontrol cases in Greece with
prospects of commercialization. There are several effective biocontrol cases in Greece with prospects of commercialization. Indeed, Tjamos et al. (2000) selected
and evaluated hundreds of endorhizosphere bacteria
from solarized soils as bca’s against Verticillium dahliae.
They showed the effectiveness of to bacterial isolates,
one from Paenibacillus alvei (designated K-165) and the
other form Bacillus amiloliquefaciens (called 5-127) in
successfully controlling Verticillium wilt in glasshouse
or field experiments. Application of a bacterial potato
seed dusting in infested potato fields reduced Verticillium symptoms by 50% and increased yield by 25%.
Both bacterial isolates produced indolebutyric, indolepyruvic and indole propionic acids and chitinase.
Furthermore, Tjamos et al. (2005) studying induction of
resistance to V. dahliae in Arabidopsis thaliana by bca
strain K-165 demonstrated ISR protection against this
pathogen. Among others the concomitant activation,
and increased transient accumulation of the PR-1, PR-2,
and PR-5 genes were observed in the treatment in which
both the inducing bacterial strain and the challenging
pathogen were present in the rhizosphere of the A.
thaliana plants. As to foliar pathogens Kalogiannis et al.
(2006) studied phyllosphere yeasts as biocontrol agents
against Botrytis cinerea of tomato by evaluating their
ability to suppress grey mould. In greenhouse experiments, an isolate of Rhodotorula glutinis (Y-44) was the
most efficient in controlling grey mould of tomato
plants. In greenhouse-grown tomato plants R. glutinis
Y-44 sprays were as effective as two commercial fungicides in controlling the pathogen. Dimakopoulou et al.
(2008), studying the activity of the phyllosphere
grapevine yeast Aureobasidium pullulans against Aspergillus carbonarius, the cause of sour rot of wine
grapes in Greece, showed that the most effective yeast
isolate of this yeast (Y-1), was as effective as the commercial fungicide fludioxonil + cyprodinil, in reducing
sour rot infection, A. carbonarius presence on berries at
harvest and ochratoxin A contamination in must.
BIOLOGICAL MANAGEMENT OF PLANT
DISEASES: PROBLEMS AND PROSPECTS
IN COMMERCIAL APPLICATIONS
World agriculture is interested in reducing dependence on chemical inputs, so biological management of
plant diseases can be expected to play an important role
in Organic and Integrated Pest Management (IPM) systems when successful. With the growing interest in reducing chemical inputs, companies involved in the manufacturing and marketing of bca’s should experience
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continued growth (McSpadden Gardener and Fravel,
2002). Research has led to the development of a small
but essential commercial sector that produces a number
of biocontrol products. Most of the commercial bca’s
production is handled by relatively small companies
with total sales on the order of 10 to 20 million US dollars annually (Fravel, 2005). Three examples from
Greece of successful field applications, waiting for commercialization, have been given above. Numerous cases
of potential commercial application including mycofungicides and fungal biofertilizers have been promoted, while fungal biofertilizers have also been registered
for application. Selective cases of commercial formulations are common today. Fungal biofertilizers include
plant growth stimulating fungi e.g. Trichoderma, mycorrhizal fungi (ectomycorrhiza e.g. Pisolithus tinctonus
and arbuscular mycorrhizae e.g. Glomus intraradices).
Biocontrol products are either marketed as stand-alone
products or formulated as mixtures with other microbial (e.g., High Stick N/T) or synthetic fungicides (e.g.,
Kodiak). Since the cost of registering a product with the
EPA as a biopesticide can be between $200,000 and
$500,000, a minimum capitalization close to 1 million
dollars is required for establishing a start-up company.
Although this is very expensive and time consuming
procedure (the time to complete registration currently is
around 18 months) several commercial products are
available after undergoing this procedure. Currently,
several biocontrol products although not being registered as such yet, circulate as plant strengtheners or
growth promoters without any specific claim regarding
disease control by the distributors.
CONCLUDING REMARKS
Development and extensive use of biological management of plant diseases requires considerable well-financed research in order to produce reliable consistent
and efficacious products. It has been seen that bca’s
tend to be highly specific and selective in the pathogen
they control and the environment and the sites in which
they are effective. Because of this, the number of different species or strains of bca’s needed as formulated
products to control many plant diseases, across the wide
range of crops and agricultural environments, was initially considerably larger compared with the number of
chemicals that might be required to control the same
diseases. Furthermore, the regulatory policies that required separate approval for each bca with protocols for
registration based on those developed for chemicals significantly hampered or delayed commercialization. Recently, biological management of plant diseases, based
on formulations circulating as biofertilizers or biostimulants etc, overcomes regulation applicable for their registration as disease control products.
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