BIOLOGICAL MANAGEMENT OF PLANT DISEASES: HIGHLIGHTS ON RESEARCH AND APPLICATION

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1 Journal of Plant Pathology (2010), 92 (4, Supplement), S4.17-S4.21 Edizioni ETS Pisa, 2010 S4.17 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 Corresponding author: E.C. Tjamos Fax: ect@aua.gr 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 erroneously 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).

2 S4.18 Biological management of plant diseases Journal of Plant Pathology (2010), 92 (4, Supplement), S4.17-S4.21 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 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 carbon-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 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,

3 Journal of Plant Pathology (2010), 92 (4, Supplement), S4.17-S4.21 Tjamos et al. S4.19 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; 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

4 S4.20 Biological management of plant diseases Journal of Plant Pathology (2010), 92 (4, Supplement), S4.17-S4.21 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. REFERENCES Bargabus R.L., Zidack N.K., Sherwood J.W., Jacobsen B.J., Characterization of systemic resistance in sugar beet elicited by a non-pathogenic, phyllosphere colonizing Bacillus mycoides, biological control agent. Physiological and Molecular Plant Pathology 61: Bull C.T., Shetty K.G., Subbarao K.V., Interactions between Myxobacteria, plant pathogenic fungi, and biocontrol agents. Plant Disease 86: Dimakopoulou M., Tjamos S.E., Antoniou P.P., Pietri A., Battilani Paola, Avramidis N., Markakis E.A., Tjamos E.C., Phyllosphere grapevine yeast Aureobasidium pullulans reduces Aspergillus carbonarius (sour rot) incidence in wine-producing vineyards in Greece. 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5 Journal of Plant Pathology (2010), 92 (4, Supplement), S4.17-S4.21 Tjamos et al. S4.21 Marra R., Lorito M., Study of the three-way interaction between Trichoderma atroviride, plant and fungal pathogens by using a proteomic approach. Current Genetics 50: Marra R., Barbetti M.J., Li H., Woo S.L., Lorito M., A novel role for Trichoderma secondary metabolites in the interactions with plants. Physiological and Molecular Plant Pathology 72: McSpadden Gardener B., Fravel D., Biological control of plant pathogens: Research commercialization, and application in the USA. Online. Plant Health Progress doi: /php rv. Meziane H., Van der Sluis I., Van Loon L.C., Hofte M., Bakker P.A.H.M., Determinants of Pseudomonas putida WCS358 involved in inducing systemic resistance in plants. Molecular Plant Pathology 6: Navazio L., Baldan B., Moscatiello R., Zuppini A., Woo S.L., Mariani P., Lorito M., Calcium-mediated perception and defense responses activated in plant cells by metabolite mixtures secreted by the biocontrol fungus Trichoderma atroviride. BMC Plant Biology 7, pp. 41. Ongena M., Duby F., Rossignol F., Fouconnier M.L., Dommes J., Jhonart P., Stimulation of the lipoxygenase pathway is associated with systemic resistance induced in bean by a nonpathogenic Pseudomonas strain. Molecular Plant-Microbe Interactions 17: Ordentlich A., Elad Y., Chet I., The role of chitinase of Serratia marcescens in the biocontrol of Sclerotium rolfsii. Phytopathology 78: Pal K.K., McSpadden Gardener B., Biological Control of Plant Pathogens. The Plant Health Instructor DOI: /PHI-A Paulitz T.C., Belanger R.R., Biological control in greenhouse systems. Annual Review of Phytopathology 39: Press C.M., Loper J.E., Kloepper J.W., Role of iron in rhizobacteria mediated induced systemic resistance of cucumber. Phytopathology 91: Ruocco M., Lanzuise S., Vinale F., Marra R., Turrà, D., Woo S.L., Lorito M., Identification of a new biocontrol gene in Trichoderma atroviride: the role of an ABC transporter membrane pump in the interaction with different plant pathogenic fungi. Molecular Plant-Microbe Interactions 22: Ryu C.M., Farag M.A., Hu C.H., Reddy M.S., Kloepper J.W., Pare P.W., Bacterial volatiles induce systemic resistance in Arabidopsis. Plant Physiology 134: Shanahan P., O Sullivan D.J., Simpson P., Glennon J.D., O Gara F., Isolation of 2,4-Diacetylphloroglucinol from a fluorescent pseudomonad and investigation of physiological parameters influencing its production. Applied and Environmental Microbiology 58: Thomashow L.S., Weller D.M., Bonsall R.F., Pierson L.S., III Production of the antibiotic phenazine-1-carboxylic acid by fluorescent pseudomonas in the rhizosphere of wheat. Applied and Environmental Microbiology 56: Tjamos E.C., Tsitsigiannis D.I., Tjamos S.E., Antoniou P.P., Katinakis P., Selection and screening of endorhizosphere bacteria from solarized soils as biocontrol agents against Verticillium dahliae of solanaceous hosts. European Journal of Plant Pathology 110: Tjamos S.E., Flemetakis E., Paplomatas E.J., Katinakis P., Induction of resistance to verticillium dahliae in Arabidopsis thaliana by the biocontrol agent K-165 and pathogenesis-related proteins gene expression Molecular Plant Microbe Interactions 18: van Dijk K., Nelson E.B., Fatty acid competition as a mechanism by which Enterobacter cloacae suppresses Pythium ultimum sporangium germination and damping-off. Applied and Environmental Microbiology 66: Van Loon L.C., Bakker P.A.H.M., Pieterse C.M.J., Systemic resistance induced by rhizosphere bacteria. Annual Review of Phytopathology 36: Wilhite S.E., Lumsden R.D., Strancy D.C., Peptide synthetase gene in Trichoderma virens. Applied and Environmental Microbiology 67:

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