Biocontrol of Bacteria and Phytopathogenic Fungi

1. Biocontrol of Bacteria and Phytopathogenic Fungi

2. 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.

3. 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.

4. 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.

5. 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 fromBacillus thuringiensisto plants to control insect pests.

6. 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,andPseudomonas. • Good soil biocontrol systems have been reported for species ofFusarium, Sclerotium, Sclerotinia, Phythium ,andRhizoctonia.

7. The following biocontrol agents have already been registered: Agrobacterium radiobacteragainst crown gall; Bacillus subtilisfor growth enhancement; Pseudomonas fluorescensagainst bacterial blotch; Pseudomonas fluorescensfor seedling diseases; Peniophora giganteaagainstFommes annosus; Pythium oligandrumagainstPhythiumspp.; Trichoderma virideagainst timber pathogens; Trichodermaspp. for root diseases; Fusarium oxysporumagainstFusarium oxysporum;Trichoderma harzianumagainst root diseases; Gliocadium virensfor seedling diseases; Trichoderma harzianum/polysporumagainst 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.

8. 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.

9. 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.

10. II. Mechanisms of Biological Control of Plant Diseases • Induced Resistance and Cross-Protection • Induced resistanceis 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.

11. 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,

12. 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 ofFusariumandVerticilliumspecies, or with other fungi or bacteria, all have shown different levels of cross-protection.

13. B. Hypovirulence • Hypovirulenceis a term used to describe reduced virulence found in some strains of pathogens. • This phenomenon was first observed inCryphonectria (Endothia) parasitica (chestnut blight fungus) on EuropeanCastanea sativain 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

14. Hypovirulent strains ofC. parasiticahave 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, includingRhizoctonia solani, Gaeumannomyces graminivar. triticiandOphiostoma 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.

15. C. Competition • Competitionoccurs 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.

16. 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.

17. 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.

18. 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.

19. 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.

20. Tribe [1957] described the direct attack of sclerotia ofSclerotinia trifoliarumbyConiothyrium mintans. • In a similar way, the mycoparasiteSporidesmium sclerotivorumtraps the sclerotia ofSclerotinia minor; Lifshitz and collaborators [1984] found a new variety ofPythium nunncapable of lysing germinating sporangia ofPythium ultimumin soil. • An example of a different aspect of parasitism is observed inAnquillospora pseudolongissima,which attacks the mycorhizaeGlomus deserticola. • However, most of the published studies on mycoparasitism refer toTrichodermaspp. because they attack a great variety of phytopathogenic fungi responsible for the most important diseases suffered by crops of major economic importance worldwide.

21. 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 includePseudomonas syringae, P. fluorescens, P. cepacia, Erwinia herbicola,andBacillus subtilis. • Fungal genera that have been used for the control of airborne diseases includeTrichoderma, Ampelomyces,and the yeastsTilletiopsisandSporobolomyces. • The mechanisms of action proposed for these biocontrol agents, include competition for sites or nutrients, antibiosis, and hyperparasitism.

22. 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 ofP. syringaepv. tomato,produced byTn5insertional mutagenesis, prevented growth of pathogenic strains in the tomato phyllosphere, presumably by preemptive competitive exclusion.

23. 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.

24. 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 withTrichodermaspp. • 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 theb-tubulins ofTrichoderma viride,the corresponding gene has been cloned and proved to work in otherTrichodermaspecies, thereby producing a biological control agent that could be applied simultaneously or in alternation with the fungicide.

25. 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 pseudomonadsare the most frequently used bacteria for biological control and plant growth promotion, butBacillusandStreptomycesspecies have also been commonly used.

26. Trichoderma, Gliocadium,andConiothyriumspecies are the most frequently used fungal biocontrol agents. • Perhaps the most successful biocontrol agent of a soilborne pathogen isAgrobacterium radiobacterstrain K84, used against crown gall disease caused byA. tumefaciens • Biological control withA. radiobacteris 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 toA. tumefaciens,which would then be resistant to agrocin 84.

27. To prevent this resistance, a transfer-deficient mutant of strain K84 was constructed. • A. radiobacterstrain 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 ofFusarium oxysporumhave been used to control wilt diseases caused by pathogenicFusariumspp.

28. The phytopathogenic bacteriumErwinia carotovorasubsp. carotovorasecretes various extracellular enzymes, including pectinases, cellulases, and proteases. • Pectinases are known to be a major pathogenicity determinant in soft rot disease of potato. • E. carotovorasubsp. carotovoramutants 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 andb-(1,3)-glucanase have been demonstrated.

29. In other studies, chitinase genes fromS. marcescenshave been expressed inPseudomonasspp. and the plant symbiontRhizobium meliloti. • The modifiedPseudomonasstrain controlled the pathogensF. oxysporum f.sp. redolensandGauemannomyces graminisvar. tritici. • The antifungal activity of the transgenicRhizobiumduring symbiosis on alfalfa roots was verified by lysis ofR. solanihyphal tips treated with cell-free nodule extracts.

30. Ab-(1,3)-glucanase-producing strain ofPsuedomonas cepaciasignificantly decreases the incidence of diseases caused byR. Solani, S. rolfsii,andP. ultimum. • The biocontrol ability of thisPseudomonasstrain was correlated with the induction of theb-(1,3)-glucanase by different fungal cell walls in synthetic medium. • Various extracellular antibiotics produced byPseudomonasspp. 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).

31. 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 ofPythium ultimumdamping-off of cucumber was achieved by increasing the number of antibiotic biosynthesis genes inP. fluorescensstrain CHAO. • Constitutive synthesis of oomycin A in HV37a was achieved by insertion of a strong promoter.

32. 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 fromPseudomonasF113 was transferred into variousPseudomonasstrains, one of which was subsequently able to produce PHL and inhibitP. ultimumdamping-off of sugar beet. • This procedure increased activity againstG. graminisvar. tritici, P. ultimim,andR. solani.

33. III. The Trichoderma System • Trichodermaspp. 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 byFusarium, Rhizoctonia,andPythium,and sclerotium-forming pathogens such asSclerotium. • The antagonisticTrichodermawas a mycoparasite.

34. 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 ofTrichodermain 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).

35. Trichoderma coils around the pathogen

36. In this respect, production of appressoria at the tips of short branches has been described forT. hamatumandT. harzianum. • The interaction ofTrichodermawith its host is specific. • The possible role of agglutinins in the recognition process determining the fungal specificity has been recently examined. • Indeed, recognition betweenT. harzianumand two of its major hosts, R. solaniandS. rolfsii,was controlled by two different lectins present on the host hyphae. • R. solanicarries a lectin that binds to galactose and frucose residues on theTrichodermacell walls.

37. This lectin agglutinates conidia of a mycoparasitic strain ofT. 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

38. 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.

39. Biomimics of the Trichoderma-host interaction Trichoderma coils around lectin-coatednylon fibers

40. 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.

41. 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 ofT. harzianuminvolves 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 hydrolyzeBotrytis cinereacell walls in vitro.

42. Trichoderma penetrates the hyphae of its host Rhizoctonia solani.

43. 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 ofT. harzianumappears to be coordinated, suggesting a regulatory mechanism involving substrate induction and catabolite repression.

44. This phenomenon correlates with the ability of eachTrichodermaisolate to control a specific pathogen. • However, the specificity ofTrichodermacannot be simply explained by a difference in enzyme activity, because the nonantagonisticTrichodermaisolates produce lower, but significant, levels of lytic enzymes. • This observation supports the idea that recognition is an important factor in the mycoparasitic activity ofTrichoderma. • The effect of the cell wall-degrading enzymes on the host has been observed using different microscopy techniques.

45. Electron microscopy analysis has shown that during the interaction ofTrichodermaspp. with eitherS. rolfsiiorR. 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. harzianumisolates attack bothS. rolfsiihyphae and sclerotia. • Electron microscopy also showed that the mycoparasite degraded sclerotial cell walls and that the attacked cells lost their cytoplasmic content.

46. It has been proposed thatT. harzianumuses 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 ofTrichodermaas a biocontrol agent. • The process apparently includes: • chemotropic growth ofTrichoderma, • recognition of the host by the mycoparasite, • secretion of extracellular enzymes, • hyphae penetration, and • lysis of the host.

47. The involvement of volatile and nonvolatile antibiotics in the antagonism byTrichodermahas been proposed. • Indeed some isolates ofTrichodermaexcrete growth-inhibitory substances. • A strain ofT. harzianum(T-35) that controlsFusariumspp. on various crops may utilize competition for nutrients and rhizosphere colonization. • Trichodermastimulates growth and flowering of several plant species. • Thus, the biocontrol ability ofTrichodermastrains 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.

48. B. Perspectives • One of the major problems faced when working withTrichodermaspp. is their shaky classification in the species group aggregates established in 1969 by Rifai. • However, recent efforts made to establish a better classification system forTrichodermainclude 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 ofTrichoderma.

49. Using restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD) analyses to estimate the intraspecific divergence among isolates ofT. harzianumand to classify them according to their aggressiveness toAgaricus 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.

50. Perhaps the most exciting subject for persons working in biological control withTrichodermais strain improvement. • From the moment that genetic engineering of biocontrol strains ofTrichodermawas 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 ofTrichoderma.