Biocontrol of Bacteria and Phytopathogenic Fungi

• Despite the many achievements of modern agriculture, certain cultural practices have actually enhanced the destructive potential of diseases.

• These practices include use of genetically similar crop plants in continuous monoculture, use of plant cultivars susceptible to pathogens, and use of nitrogenous fertilizers at concentrations that enhance disease susceptibility.

• Plant disease control, therefore, has now become heavily dependent on fungicides to combat the wide variety of fungal diseases that threaten agricultural crops.

• It is reported that over 70 pesticides have been detected in groundwater in 38 states in the United States.

• U.S. Environment Protection Agency (EPA) indicates that, in the United State alone, 30006000 cancer cases are induced annually by pesticide residues on foods, and another 50100 by exposure to pesticides during application.

• Studies aimed at replacing pesticides with environmentally safer methods are currently being conducted at many research centers.

• This follows an over 40-year period, starting in the mid 1920s, when biological control of plant diseases moved from the discovery of suppression in response to organic materials added to the soil.

• Biological control is a potent means of reducing the damage caused by plant pathogens.

• Commercialized systems for the biological control of plant diseases are few.

• The performance of a biocontrol agent cannot be expected to equal that of an excellent fungicide; although some biocontrol agents have been reported to be as effective as fungicide control.

• Nevertheless, a moderately effective, but consistent agent, seems to be sufficient to establish nonchemical control of plant disease or to reduce the level of chemical residues in agricultural products.

• However, there is an equally great or greater need for biological control of pathogens that presently go uncontrolled or only partially controlled.

• Potential agents for biocontrol activity are rhizosphere-competent fungi and bacteria which, in addition to their antagonistic activity, are capable of inducing growth responses by either controlling minor pathogens or by producing growth-stimulating factors.

• Before biocontrol can become an important component of plant disease management, it must be effective, reliable, consistent, and economical.

• To meet these criteria, superior strains, together with delivery systems that enhance biocontrol activity, must be developed.

• The growing interest in biocontrol with microorganisms is also a response to the new tools of biotechnology.

• Plants and microorganisms can now be manipulated to deliver the same mechanism of biological control, as has been done for the production of the delta endotoxin-encoding gene transferred from Bacillus thuringiensis to plants to control insect pests.

• We can now think of microorganisms with inhibitory activity against plant pathogens as potential sources of genes for diseases resistance.

• The successful control by biological means in the phyllophane that have been reported in the literature involve mainly rusts, powdery mildews, and diseases caused by the following genera of pathogens: Alternaria, Epicoccum, Sclerotinia, Septoria, Drechslera, Venturia, Plasmopara, Erwinia, and Pseudomonas.

• Good soil biocontrol systems have been reported for species of Fusarium, Sclerotium, Sclerotinia, Phythium , and Rhizoctonia.

• The following biocontrol agents have already been registered: Agrobacterium radiobacter against crown gall; Bacillus subtilis for growth enhancement; Pseudomonas fluorescens against bacterial blotch; Pseudomonas fluorescens for seedling diseases; Peniophora gigantea against Fommes annosus ; Pythium oligandrum against Phythium spp.; Trichoderma viride against timber pathogens; Trichoderma spp. for root diseases; Fusarium oxysporum against Fusarium oxysporum; Trichoderma harzianum against root diseases; Gliocadium virens for seedling diseases; Trichoderma harzianum/polysporum against wood decay.

• Biocontrol agents may employ several modes of action; therefore, it is important to know the proportion and timing of each mode of action that may occur.

• Information of this type can be obtained from in vitro studies or by using plants grown under gnobiotic conditions during which the potential activity of biocontrol agents can be assessed.

• However, such studies do not provide information on their mode of action in vivo, particularly within plants for which separation of plant response or antagonistic activity is not always possible or in soil where direct observation and chemical analysis are difficult.

• Unfortunately, insufficient research efforts have been directed toward the selection of characteristics that enhance survival of the biological control agents.

• However, several techniques developed by microbial ecologist and the fermentation industry are now available to select for survival and to manipulate beneficial microorganisms under given environmental conditions, including temperature, osmotic pressure, radiant flux, and pH.

• Moreover, proper formulation of the biocontrol product can provide a preparation with long shelf life, the ability to withstand adverse conditions, and even with the necessary ingredients to induce its specific activity.

II. Mechanisms of Biological Control of Plant Diseases

A.Induced Resistance and Cross-Protection• Induced resistance is a plant response to challenge

by microorganisms or abiotic agents such that following the inducing challenge de novo resistance to pathogens is shown in normally susceptible plants.

• Induced resistance can be localized, when it can be detected only in the area immediately adjacent to the inducing factor, or systemic, when resistance occurs subsequently at sites throughout the plant.

• Both localized and systemically induced resistance are nonspecific and can act against a whole range of pathogens, but whereas localized resistance occurs in many plant species, systemic resistance is limited to some plants.

• Cross-protection differs from induced resistance in that, following inoculation with avirulent strains of pathogens or other microorganisms, both inducing microorganisms and challenge pathogens occur on or within the protected tissue.

• During localized resistance, the plant reacts to the environmental stimulus by the activation of a variety of defense mechanisms that culminate in various biochemical and physical changes, including phytoalexin production and alterations to plant cell walls,

• such as increased production of suberin, hydroxyproline-rich glycoproteins, and lignification, and correlations between resistance and lignin formation, peroxidase activity, and protease inhibitors have been found.

• In systemically protected tobacco or cucumber, increases in newly formed pathogenesis-related (PR) proteins have also been recorded, and these may be chitinase-, glucanase-, or osmotin-like.

• The most commonly reported examples of cross-protection involving fungi are probably those used against vascular wilts.

• Inoculation with nonpathogenic strains or weakly virulent strains of pathogenic formae speciales of Fusarium and Verticillium species, or with other fungi or bacteria, all have shown different levels of cross-protection.

B. Hypovirulence• Hypovirulence is a term used to describe reduced

virulence found in some strains of pathogens. • This phenomenon was first observed in

Cryphonectria (Endothia) parasitica (chestnut blight fungus) on European Castanea sativa in Italy, where naturally occurring hypovirulent strains were able to reduce the effect of virulent ones.

• These slower-growing hypovirulent strains contain a single cytoplasmic element of double-stranded RNA )dsRNA).

• It was demonstrated that a full-length cDNA copy of the hypovirulence-associated virus (HAV) conferred the hypovirulence phenotype when introduced into virulent strains by DNA-mediated transformation

• Hypovirulent strains of C. parasitica have been used as biocontrol agents of chestnut blight.

• This may be considered a specialized form of cross-protection that is limited to the control of only established compatible strains.

• Hypovirulence has also been reported in many other pathogens, including Rhizoctonia solani, Gaeumannomyces gramini var. tritici and Ophiostoma ulmi, but the transmissible elements responsible for hypovirulence or reduced vigor of the fungi are subject to debate and may be due to dsRNAs, plasmids, or viruses.

C. Competition• Competition occurs between microorganisms

when space or nutrients (i.e., carbon, nitrogen, and iron) are limiting, and its role in the biocontrol of plant pathogens has been studied for many years, with special emphasis on bacterial biocontrol agents.

• Thus, an important attribute of a successful rhizosphere biocontrol agent would be the ability to remain at high population density on the root surface, providing protection of the whole root for the duration of its life.

• Mycorrhizal fungi can also be considered to act as a sophisticated form of competition or cross-protection, decreasing the incidence of root disease.

• With ectomycorrhizas, antibiosis against the pathogen, physical protection by the mantle, competition with the pathogen for nutrients coming from the roots, stimulation of antagonistic microflora associated with the mantle, and induction of host plant resistance, all have been suggested as possible mechanisms involved in the protection of roots.

• Similarly, plants with endomycorrhizal associations can be more resistant to pathogens than nonmycorrhizal plants of similar size and developmental stage.

D. Antibiosis• The production of antibiotics by actinomycetes,

bacteria, and fungi is very simply demonstrated in vivo.

• In general, however, the role of antibiotic production in biological control in vitro remains unproved.

• Secondary metabolite production is influenced by cultural conditions and, although many microorganisms produce antibiotics in culture, there is little evidence that antibiotics are produced in natural environments, except after input of organic materials.

• It is possible that detection techniques are insensitive, that antibiotics are rapidly degraded, or that they are bound to the substrate, such as clay particles in soil, preventing detection.

• Species of Gliocadium and Trichoderma are well-known biological control agents that produce a range of antibiotics that are active against pathogens in vitro and, consequently, antibiotic production has commonly been suggested as a mode of action for these fungi.

• Within bacterial biocontrol agents several species of the genus Pseudomonas produce antibiotics involved in their ability to control plant pathogens.

E. Mycoparasitism• Mycoparasitism occurs when one fungus exists

in intimate association with another from which it derives some or all its nutrients while conferring no benefit in return.

• Biotrophic mycoparasites have a persistent contact with or occupation of living cells, whereas necrotrophic mycoparasites kill the host cells, often in advance of contact and penetration.

• Mycoparasitism is a commonly observed phenomenon in vitro and in vivo, and its mode of action and its involvement in biological disease control has been reviewed.

• There are several examples of this phenomenon.

• Tribe [1957] described the direct attack of sclerotia of Sclerotinia trifoliarum by Coniothyrium mintans.

• In a similar way, the mycoparasite Sporidesmium sclerotivorum traps the sclerotia of Sclerotinia minor; Lifshitz and collaborators [1984] found a new variety of Pythium nunn capable of lysing germinating sporangia of Pythium ultimum in soil.

• An example of a different aspect of parasitism is observed in Anquillospora pseudolongissima, which attacks the mycorhizae Glomus deserticola.

• However, most of the published studies on mycoparasitism refer to Trichoderma spp. because they attack a great variety of phytopathogenic fungi responsible for the most important diseases suffered by crops of major economic importance worldwide.

F. Biocontrol of Airborne Diseases• Many naturally occurring microorganisms have

been used to control diseases on the aerial surfaces of plants.

• The more common bacterial species that have been used for the control of diseases in the phyllosphere include Pseudomonas syringae, P. fluorescens, P. cepacia, Erwinia herbicola, and Bacillus subtilis.

• Fungal genera that have been used for the control of airborne diseases include Trichoderma, Ampelomyces, and the yeasts Tilletiopsis and Sporobolomyces.

• The mechanisms of action proposed for these biocontrol agents, include competition for sites or nutrients, antibiosis, and hyperparasitism.

• Several phytopathogenic bacteria exhibit an epiphytic phase before invasion, during which time they are susceptible to competition from other microorganisms.

• Although preemptive competitive exclusion of phytopathogenic bacteria in the phyllosphere can be achieved using naturally occurring strains, avirulent mutants of the pathogen, in which deleterious phenotypic traits have been removed, may be more effective because they occupy the same niche as the parental strain.

• A nonpathogenic strain of P. syringae pv. tomato, produced by Tn5 insertional mutagenesis, prevented growth of pathogenic strains in the tomato phyllosphere, presumably by preemptive competitive exclusion.

• Molecular biology techniques could be used to enhance the efficacy of biocontrol agents that use antibiosis as a mode of action.

• The transcriptional regulation of genes conferring antibiotic production could be altered by replacing its promoter region by one known to direct high levels of transcription.

• It may also be possible to transfer the genes required for antibiotic production from a poor-colonizing organism to one that colonizes more aggressively.

• Biocontrol agents must normally achieve a high population in the phyllosphere to control other strains, but colonization by the agent may be reduced by competition with the indigenous microflora.

• Application of a bactericide to which most members of the microflora are sensitive, but to which the control agent is tolerant, can maximize colonization by the biocontrol agent.

• Integration of chemical pesticides and biocontrol agents has been reported with Trichoderma spp.

• Biocontrol agents tolerant to specific pesticides could be constructed using molecular techniques.

• Resistance to the fungicide benomyl is conferred by a single amino acid substitution in one of the b-tubulins of Trichoderma viride, the corresponding gene has been cloned and proved to work in other Trichoderma species, thereby producing a biological control agent that could be applied simultaneously or in alternation with the fungicide.

G. Biocontrol of Soilborne Diseases• Chemical control of soilborne plant diseases is

frequently ineffective because of the physical and chemical heterogeneity of the soil, which may prevent effective concentrations of the chemical from reaching the pathogen.

• Biological control agents colonize the rhizosphere, the site requiring protection, and leave no toxic residues, as opposed to chemicals.

• Fluorescent pseudomonads are the most frequently used bacteria for biological control and plant growth promotion, but Bacillus and Streptomyces species have also been commonly used.

• Trichoderma, Gliocadium, and Coniothyrium species are the most frequently used fungal biocontrol agents.

• Perhaps the most successful biocontrol agent of a soilborne pathogen is Agrobacterium radiobacter strain K84, used against crown gall disease caused by A. tumefaciens

• Biological control with A. radiobacter is mediated primarily by the bacteriocin agrocin 84 synthesis, which is directed by genes carried by the plasmid pAGK84.

• This plasmid also carries the genes needed for resistance to agrocin 84 and has conjungal transfer capacity.

• Consequently, pAgK84 may be transferred to A. tumefaciens, which would then be resistant to agrocin 84.

• To prevent this resistance, a transfer-deficient mutant of strain K84 was constructed.

• A. radiobacter strain K1026 is identical with the parental strain, except that the agrocin-producing plasmid, pAgK1026, has had the transfer region deleted.

• Competition as a mechanism of biological control has been exploited with soilborne plant pathogens as with pathogens on the phylloplane.

• Naturally occurring, nonpathogenic strains of Fusarium oxysporum have been used to control wilt diseases caused by pathogenic Fusarium spp.

• The phytopathogenic bacterium Erwinia carotovora subsp. carotovora secretes various extracellular enzymes, including pectinases, cellulases, and proteases.

• Pectinases are known to be a major pathogenicity determinant in soft rot disease of potato.

• E. carotovora subsp. carotovora mutants defective in the production of pectate lyase have been used in the biocontrol of this disease.

• Biological control of some soilborne fungal diseases has been correlated with chitinase production, bacteria producting chitnases or glucanases exhibit antagonism in vitro against fungi, inhibitition of fungal growth by plant chitinases and dissolution of fungal cell walls by a streptomycete chitinase and b-(1,3)-glucanase have been demonstrated.

• In other studies, chitinase genes from S. marcescens have been expressed in Pseudomonas spp. and the plant symbiont Rhizobium meliloti.

• The modified Pseudomonas strain controlled the pathogens F. oxysporum f. sp. redolens and Gauemannomyces graminis var. tritici.

• The antifungal activity of the transgenic Rhizobium during symbiosis on alfalfa roots was verified by lysis of R. solani hyphal tips treated with cell-free nodule extracts.

• A b-(1,3)-glucanase-producing strain of Psuedomonas cepacia significantly decreases the incidence of diseases caused by R. Solani, S. rolfsii, and P. ultimum.

• The biocontrol ability of this Pseudomonas strain was correlated with the induction of the b-(1,3)-glucanase by different fungal cell walls in synthetic medium.

• Various extracellular antibiotics produced by Pseudomonas spp. are involved in the biocontrol ability of soilborne plant pathogens, including phenazine-1-carboxilic acid (PCA), oomycin A, pyoluteorin (PLT), and 2,4-diacetyl-phloroglucinol (PHL).

• In systems in which antibiosis plays a primary role, molecular techniques can be used to enhance biocontrol efficacy by increasing levels of antibiotic synthesis, either by increasing the copy number of the biosynthetic genes or by modifying the regulatory signals that control their expression.

• For example, increased production of PLT and PHL and superior control of Pythium ultimum damping-off of cucumber was achieved by increasing the number of antibiotic biosynthesis genes in P. fluorescens strain CHAO.

• Constitutive synthesis of oomycin A in HV37a was achieved by insertion of a strong promoter.

• Alternatively, biosynthetic genes can be introduced into a strain deficient in antibiotic production, or into one that produces a different antibiotic, to increase the spectrum of activity.

• A cloned genomic fragment from Pseudomonas F113 was transferred into various Pseudomonas strains, one of which was subsequently able to produce PHL and inhibit P. ultimum damping-off of sugar beet.

• This procedure increased activity against G. graminis var. tritici, P. ultimim, and R. solani .

III. The Trichoderma System• Trichoderma spp. act against a range of

economically important aerial and soilborne plant pathogens.

• They have been used in the field and greenhouse against silver leaf) Chondrostereum purpureum), on plum, peach, and nectarine; Dutch elm disease )Ophiostoma ulmi) on elm; honey fungus (Armillaria mellea) on a range of tree species; and against rots on a wide range of crops, caused by Fusarium, Rhizoctonia, and Pythium, and sclerotium-forming pathogens such as Sclerotium.

• The antagonistic Trichoderma was a mycoparasite.

A. Mechanism of Action• From recent work, it appears that

mycoparasitism is a complex process, including several successive steps.

• The first detectable interaction shows that the hyphae of the mycoparasite grows directly toward its host.

• This phenomenon appears as a chemotropic growth of Trichoderma in response to some stimuli in the host's hyphae or toward a gradient of chemicals produced by the host.

• When the mycoparasite reaches the host, its hyphae often coil around it or are attached to it by forming hook-like structures (Fig. 1).

Trichoderma coils around the pathogen

• In this respect, production of appressoria at the tips of short branches has been described for T. hamatum and T. harzianum.

• The interaction of Trichoderma with its host is specific.

• The possible role of agglutinins in the recognition process determining the fungal specificity has been recently examined.

• Indeed, recognition between T. harzianum and two of its major hosts, R. solani and S. rolfsii, was controlled by two different lectins present on the host hyphae.

• R. solani carries a lectin that binds to galactose and frucose residues on the Trichoderma cell walls.

• This lectin agglutinates conidia of a mycoparasitic strain of T. harzianum, but did not agglutinate two nonparasitic strains.

• This agglutinin may play a role in prey recognition by the predator.

• Moreover, because it does not distinguish among biological variants of the pathogen, it enables the Trichoderma species to attack different R. solani isolates.

• The activity of a second lectin isolated from S. rolfsii was inhibited by d-glucose or d-mannose residues, apparently present on the cell walls of T. harzianum.

• As previously shown in vivo for the fungal hyphae, during the interaction Trichoderma recognized and attached to

• the coated fibers, coiling around them and forming other mycoparasitism-related structures, such as appresorium-like bodies and hyphal loops (Fig. 2).

• Following these interactions, the mycoparasite sometimes penetrates the host mycelium (Fig. 3), apparently by partially degrading its cell wall.

• Microscopic observations led to the suggestion that Trichoderma spp. produced and secreted mycolytic enzymes responsible for the partial degradation of the host's cell wall.

Biomimics of the Trichoderma-host interaction Trichoderma coils around lectin-coated nylon fibers

• In 1993, Geremia and coworkers, reported the isolation of a 31-kDa basic protease that is secreted by T. harzianum during simulated mycoparasitism, an interesting observation was that chitin also appeared to strongly induce proteinase activity.

• The corresponding gene (prbl) was cloned and characterized.

• That was the first report of cloning of a mycoparasitism-related gene.

• Recently, Flores et al. [1996] showed that the gene is induced during fungus-fungus interaction and used it to generate transgenic Trichoderma strains carrying multiple copies of prbl.

• The resulting strains produced up to 20 times more protease, and one of them reduced the disease incidence caused by R. solani on cotton plants to only 6%, whereas the disease incidence for the non-transformed strain was 30%.

• The complexity and diversity of the chitinolitic system of T. harzianum involves the complementary modes of action of six enzymes, all of which might be required for maximum efficiency against a broad spectrum of chitin-containing plant pathogenic fungi.

• Probably the most interesting individual enzyme of the system is the 42-kDa endochitinase because of its ability to hydrolyze Botrytis cinerea cell walls in vitro.

Trichoderma penetrates the hyphae of its host Rhizoctonia solani.

• Expression of its gene (ech-42) encoding Ech42 is strongly induced during fungus-fungus interaction.

• Its expression is apparently repressed by glucose and may be affected by other environmental factors, such as light, nutritional stress, and may even be developmentally regulated.

• In summary, expression of all enzymes from the cell wall-degrading system of T. harzianum appears to be coordinated, suggesting a regulatory mechanism involving substrate induction and catabolite repression.

• This phenomenon correlates with the ability of each Trichoderma isolate to control a specific pathogen.

• However, the specificity of Trichoderma cannot be simply explained by a difference in enzyme activity, because the nonantagonistic Trichoderma isolates produce lower, but significant, levels of lytic enzymes.

• This observation supports the idea that recognition is an important factor in the mycoparasitic activity of Trichoderma.

• The effect of the cell wall-degrading enzymes on the host has been observed using different microscopy techniques.

• Electron microscopy analysis has shown that during the interaction of Trichoderma spp. with either S. rolfsii or R. solani, the parasite hyphae contacted their host and enzymatically digested their cell walls.

• In response to the invasion, the host produced a sheath matrix which encapsulated the penetrating hyphae and the host cells became empty of cytoplasm.

• The susceptible host hyphae showed rapid vacuolation, collapse, and disintegration.

• T. harzianum isolates attack both S. rolfsii hyphae and sclerotia.

• Electron microscopy also showed that the mycoparasite degraded sclerotial cell walls and that the attacked cells lost their cytoplasmic content.

• It has been proposed that T. harzianum uses sclerotial cell content to sporulate on sclerotial surfaces and inside the digested regions.

• Therefore, it is considered that mycoparasitism is one of the main mechanisms involved in the antagonism of Trichoderma as a biocontrol agent.

• The process apparently includes: • chemotropic growth of Trichoderma, • recognition of the host by the mycoparasite, • secretion of extracellular enzymes, • hyphae penetration, and • lysis of the host.

• The involvement of volatile and nonvolatile antibiotics in the antagonism by Trichoderma has been proposed.

• Indeed some isolates of Trichoderma excrete growth-inhibitory substances.

• A strain of T. harzianum (T-35) that controls Fusarium spp. on various crops may utilize competition for nutrients and rhizosphere colonization.

• Trichoderma stimulates growth and flowering of several plant species.

• Thus, the biocontrol ability of Trichoderma strains is most likely conferred by more than one exclusive mechanism.

• In fact, it seems advantageous for a biocontrol agent to suppress a plant pathogen using multiple mechanisms.

B. Perspectives• One of the major problems faced when working

with Trichoderma spp. is their shaky classification in the species group aggregates established in 1969 by Rifai.

• However, recent efforts made to establish a better classification system for Trichoderma include electrophoretic karyotypes of different species and strains of this genus and their possible variability.

• In addition, using a DNA fingerprinting technique to analyze the nine species aggregates of Trichoderma.

• Using restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD) analyses to estimate the intraspecific divergence among isolates of T. harzianum and to classify them according to their aggressiveness to Agaricus bisporus.

• Another possibility is the use of the mycoparasitism-related genes as molecular probes to identify aggressive strains.

• However, major efforts should still be made to allow a clear classification of the genus.

• Perhaps the most exciting subject for persons working in biological control with Trichoderma is strain improvement.

• From the moment that genetic engineering of biocontrol strains of Trichoderma was made feasible at the beginning of the 1990s, enormous possibilities for modifying strains were opened.

• The first practical use of these techniques was to introduce dominant selectable markers into them to monitor their behavior after release either in soil or the phylloplane.

• This was followed by the introduction of foreign genes that could potentially enhance the biocontrol capacity of Trichoderma.

• An example of this is the work, in which the strong chitinase of the bacteria Serratia marcescens was introduced into T. harzianum and expressed constitutively.

• In vitro tests of the ability of these strains to overgrow the plant pathogen Sclerotium rolfsii in dual cultures showed wider lytic zones along the contact front between the transformants and the pathogenic fungus than those of the nontransformed strain.

• Many of this type of genes are available and could be tested in Trichoderma.

• On the other hand, cloning of the genes coding for the different cell wall-degrading enzymes produced by Trichoderma will allow us to test their relevance in mycoparasitism through their overexpression and disruption.

• Thus, it is clear that the Trichoderma field is still in its childhood and more and more researchers should join the efforts made to obtain natural alternatives for the control of plant diseases.