Biocontrol of Bacteria and Phytopathogenic Fungi

Biocontrol of Bacteria and Phytopathogenic Fungi Alfredo Herrera Estrella Uniahd Impuato. Impuato, Mixico

llan Chet The Hebrew Universiry of Jemakm, Rehovot, Israel

I. INTRODUCTION Despite the many achievements of modem 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 Waard et al., 19931. wide variety of fungal diseases that threaten agricultural crops Recently, Ritter [I9901 reported that over 70 pesticides, including soil fumigants, have been detected in groundwater in 38 states in the United States. Thirty-two of these pesticide detections were attributed to point sources or misuse. A landmark study published by the U.S. Environment Protection Agency (EPA) indicates that,in the United State alone, 3ooO4000 cancer cases are induced annually by pesticide residues on foods, and another 50-100 by exposure to pesticides during application. This type of findings have made the governments of many countries increasingly aware of the drawbacks of many chemical pesticides, in terms of their effect on the environment, as well as on the growers and the consumers of agricultural products. Studies aimed at replacing pesticides with environmentally safer methods are currently being conducted at many research centers. The heightened scientific interest in biological control of plant pathogens is partly a response to growing public concerns over chemical pesticides. In this context, it is important to mention that few areas of research within plant pathology have attracted more interest during the past 28 years than has the use of introduced microorganisms for biological control of plant pathogens [Cook, 19931. This follows an over #year period, starting in the mid 19205, when biological control of plant diseases moved from the discovery of suppression in response to organic materials added to the soil, to attempts at biological control with single cultures of microorganisms added to soil, to disappointment and the emergence of an attitude that plant pathology has made little progress in biological control [Baker, 1987; Cook, 19911.

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Herrera Estrella and Chet

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. Although intensive activity is currently being geared toward the introduction of an increasing number of biocontrol agents into the market [Elad and Chet, 19951, 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 [Mukerji and Garg, 19881. Usually, results so far are intermediate and inconsistent. 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. No practical or economical chemical control was replaced by Agrobacterium mdiobacter strain K-84 [Kerr, 19801 for biological control of crown gall, nor would a chemical pesticide necessarily be replaced by ice-minus bacteria for control of the ice-nucleation active bacteria [Wilson and Lindow, 19931 responsible for the frost damage on potatoes. Biological control should and can be justified on its own merits, without giving it importance at the expense of chemical controls. Potential agents for biocontrol activity are rhizosphere-competentfungi 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 marmanet al., 19891. 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 marmanet al., 19891. Existing biological control attributes can be enhanced by improving existing, known biocontrol agents, with genetic manipulation. Genetic manipulations of biocontrol agents not only can enhance their activity, but also can expand their spectrum. 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 endotoxinencoding gene transferred from Bacillus thuringiensis to plants to control insect pests [Vaeck et al., 19871. 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: Ahemaria, Epicoccum, Sckrotinia, Septoria, Drechskm, Venturia, Plasmopam, Erwinia, and Pseudomom. Good soil biocontrol systems have been reported for species of Fusarium, Sckrotium, Sckrotinia, Phythium, and Rhizoctonia. The following biocontrol agents have already been registered: Agrobacterium radiobacter against crown gall (USA. Australia, NZ); Bacillus subtilis for growth enhancement (USA); Pseudomom @omscens against bacterial blotch (Australia); Pseudomom fluorescens for seedling diseases (USA); Peniophom gigantea against Fommes annosus (UK);Pythium oligandnun against Phythium spp. (USSR); Tnchodem v i d e against timber pathogens (Europe); Trichodem spp. for root diseases (USSR); Fusarium oxysponun against Fusarium oxysponun (Japan); Trichodem hatzionum against root diseases (USA); Gliocadium virens for seedling diseases (USA); Trichodem hatzianudpolysponunagainst wood decay (USA) [Elad and Chet. 19951. 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 sepa-

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ration of plant response or antagonistic activity is not always possible or in soil where direct observation and chemical analysis are difficult. These limitations must be kept in mind when extrapolating results obtained in the laboratory to the natural environment. Furthermore, apart from the antagonistic activity of the applied agent, effective biocontrol involves the ability to survive in the habitat in which it is applied. 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 Elad and Chet, 19951.

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 [de Wit, 19851. 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 [de Wit, 19851. 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 [Hamme~schmidtet al., 19841, and correlations between resistance and lignin formation, peroxidase activity, and protease inhibitors have been found [Dean and Kuc, 1987; Roby et al., 19871. In systemically protected tobacco or cucumber, increases in newly formed pathogenesisdated (PR) proteins have also been recorded, and these may be chitinase-, glucanase-, or osmotin-like Fritig et al., 1987; Gianinazzi et al., 1980; Metraux et al., 19881. 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 fonnae speciales of Fusanum and Venicillium species, or with other fungi or bacteria, all have shown different levels of cross-protection mllocks, 1986; Matta and Garibaldi, 1977; Ogawa 'and Kornada, 1985; Sneh et al., 19851.

B.

Hypovirulence

Hypmitulence is a term used to describe reduced virulence found in some shahs of pathogens. This phenomenon was fmt observed in Cryphonectria (Endothia) pamsitica (chestnut blight fungus) on European Castanea sativa in Itaiy, where naturally occurring hypovirulent strains were able to reduce the effect of virulent ones [Grente and Sauret, 1%9b]. These slowergrowing hypovirulent strains contain a single cytoplasmic element of double-stranded RNA

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(dsRNA). similar to that found in mycoviruses, that was transmitted by anastomosis (fusion of the hyphae of two strains) in compatible strains through natural virulent populations of C. parasitica [Grente and Sauret. 1%9a; Van Alfen et al.. 19751. In more recent studies, 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 [Choi and Nuss, 19921. Hypovirulent strains of C. parasitica have been used as biocontrol agents of chestnut blight [hagnostalcis, 19821. This may be considered a specialized form of cross-protection that is limited to the control of only established compatible svains plan Alfen and Hansen, 19841. Hypovirulence has also been reported in many other pathogens, including Rhizoctonia sohni. 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 [Koltin et al.. 1987; Rogers et al., 19861.

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 [Weller, 19881. Implicit in this definition is the understanding that combative interactions, such as antibiotic production or mycoparasitism, or the occurrence of induced resistance in the host are not included, even though these mechanisms may form an important part of the overall processes occurring in the interaction. In the rhizosphere competition for space as well as nutrients is of major importance. 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. Mywrrhizal fungi can also be considered to act as a sophisticated fonn 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 [Chaluavarty and Unestam, 1987; Marx, 19721. Similarly, plants with endomyco~hizalassociations can be more resistant to pathogens than nonmycorrhizal plants of similar size and developmental stage [Dehne, 19821. Occasionally, the effect may be partially systemic [Rosendahl, 19851.

D.

Antibiosis

The production of antibiotics by actinomycetes, bacteria, and fungi is very simply demonstrated in vivo. Numerous agar plate tests have been developed to detect volatile and nonvolatile antibiotic production by putative biocontrol agents and to quantify their effects on pathogens [Whipps, 19871. In general, however, the role of antibiotic production in biological control in v i m remains unproved. Secondary metabolite production is influenced by culnrral 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. Even so, 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 [Howell and Stipanovic, 1980; Papavizas and Lumsden, 1980; Williams and Vickers, 19861.


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Species of Gliocadium and Trichodenna are well-known biological control agents that produce a range of antibiotics that are active against pathogens in vitro [Claydon et al., 1987; Dennis and Webster, 1971a. b; Ghisalberti and.Sivasithampararn. 19911 and, consequently, antibiotic production has commonly been suggestedas a mode of action for these fungi, Within bacterial biocontrol agents several species of the genus Pseudomoms produce antibiotics involved in their ability to control plant pathogens [Fravel, 19881.

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 b w i s et al., 19891. 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 [Baker, 1987; Chet, 1993; Handelsrnan and Parke. 19891. There are several examples of this phenomenon. Tribe [I9571 described the direct attack of sclerotia of Sclerotiniu t r i f o l i a ~ nby Coniothyriwn mintam. In a similar way, the mycoparasite Sporidesrniwn sclerorivomrraps the sclerotia of Sclerotiniu minor [Ayers and Adam, 19791; Lifshitz and collaborators [I9841 found a new variety of Pythiwn nwvt capable of lysing germinating sporangia of Pythium ultimum in soil. An example of a different aspect of parasitism is observed in Anquillospom pseudolongissima, which attacks the mycorhizae Glomus &se~icola [Paulitz and Baker, 19871. 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 [Andrews, 1992; Blakeman and Fokkema, 19821. The more common bacterial species that have been used for the control of diseases in the phyllosphere include Pseudomonus syringae, P.fluarescens, P. cepacia, Erwinia herbicola, and Bucilh subrilis. 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. Phytopathogenic bacteria possess several genes that encode phenotypes that allow them to parasitize plants and overcome defense responses elicited by the plant [Panopoulos and Peet, 19851. In addition, phytopathogenic bacteria possess pathogenicity genes such as hrp [Willis et al., 19911. Isogenic, avirulent mutants can be produced by insertional inactivation of genes involved in pathogenicity. 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 [Cooksey, 19881. Nonpathogenic mutants of Enviniu amylovom, produced by transposon mutagenesis, have also been used in the biological control of fire blight [Norelli et al., 19901.


Antibiosis has been proposed as the mechanism of control of several bacterial Wanneste et al.. 19921 and fungal [Levy et al.. 19921 diseases in the phyllosphere. 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. Elad et al., 1993; Gullino and Garibaldi, 1988; Sivan and Chet, 19931 and P. syringae pv. tomato [Cooksey, 19881. 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 P-tubulins of Trichodem vin'de, the corresponding gene has been cloned and proved to work in other Trichoderma species [Goldman et al., 19931, thereby producing a biological control agent that could be applied simultaneously or in alter- . nation with the fungicide. Molecular techniques may eventually be used to transfer several beneficial traits, such as the production of one or more antibioticsand pesticide tolerance, to an aggressive phyllosphere colonizer.

,

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. Microorganisms have been used extensively for the biological control of soilborne plant diseases as well as for promoting plant growth [Bakker et al., 1991; Thomashow and Weller, 1990; Weller, 19881. 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. Trichakrma, Gliocadium, and Cmiothyrium species are the most frequently used fungal biocontrol agents. Perhaps the most successful biocontrol agent of a soilborne pathogen is Agrobacterium mdiobacter strain K84, used against crown gall disease caused by A. tmfaciens. Biological control with A. mdiobacter is mediated primarily by the bacteriocin agrocin 84 synthesis, which is directed by genes carried by the plasmid pAgK84. This plasmid also cames the genes needed for resistance to agrocin 84 and has conjugal transfer capacity. Consequently, pAgK84 may be transferred to A. turnefaciens, which would then be resistant to agrocin 84. To prevent this resistance, a transferdeficient mutant of strain K84 was constructed. A. mdiobacter strain K1026 is identical with the parental strain, except that the agrocin-producing,plasmid, pAgK1026, has had the vansfer region deleted [Jones et al., 19881. 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 oxysponun have been used to control wilt diseases caused by pathogenic Fusarium spp. [Alabouvette and Couteaudier, 19921. Molecular techniques have been used to remove various deleterious traits of soilborne phytopathogenic bacteria to construct a competitive antagonist of the pathogen. Random Tn5 insertions into the genome of Pseudomom

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solanacearum [Trigalet and Demery, 1986; Trigalet and Trigalet-Demery, 19901or insertion of an interposon into the hrp cluster [Frey et al., 19931 produced avirulent mutants. The avirulent mutants exhibited various levels of invasiveness of tomato plants and provided protection against bacterial wilt disease caused by the pathogen [Frey et al., 19931. The phytopathogenic bacterium Erwinia carotovom 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 woberts et al.. 19861 have been used in the biocontrol of this disease [Stromber et al., 19901. Molecular techniques have also facilitated the introduction of beneficial traits into rhizosphere-competent organisms to produce potential biocontrol agents. Chitin and P-(1,3)-glucan are the two major structural components of many plant pathogenic fungi. except by Oomycetes, which contain cellulose in their cell wall and no appreciable levels of chitin. Biological control of some soilborne fungal diseases has been correlated with chitinase production [Buxton et al., 1%5], bacteria producing chitinases or glucanases exhibit antagonism in vitro against fungi Wdlender et al., 1993; Gay et al., 19921, inhibition of fungal growth by plant chitinases and dissolution of fungal cell walls by a streptomycete chitinase and P-(1.3>glucanase have been demonstrated [Schlumbaum et al., 1986; Skujins et al., 1%5]. The importance of chitinase activity was further demonstrated by the loss of biocontrol efficacy in Serratia marcescens mutants in which the chiA gene had been inactivated [Jones et al., 19861. Other organisms from which chitinase-encoding genes have been isolated include: rice [Zhu and Lamb, 19911, tobacco r a p e et al., 19901, cucumber [Metraux et al., 19881, potato [Gaynor and Unkenholz, 19891, Bucillus circulans [Joshi et al., 19881, Streptomyces [Robbins et al., 19881, and Cellvibrio Wynne and Pemberton, 19861. A recombinant Escherichia coli expressing the chiA gene from S. marcescens was effective in reducing disease incidence caused by Sclerotium rolfsii and Rhizoctonia s o h [Oppenheim and Chet, 1992; Shapira et al., 19891. In other studies, chitinase genes from S. marcescens have been expressed in Pseudomonas spp. and the plant symbiont Rhizobiwn metiloti. The modified Pseudomonas strain controlled the pathogens F. oxysporum f. sp. redolens and Gbuernannomyces gmminis var. tritici [Sundheim, 1990, 19921. The antifungal activity of the transgenic Rhizobiwn during symbiosis on alfalfa roots was verified by lysis of R. solani hyphal tips treated with cell-free nodule extracts [Sitrit et al., 19931. A p(1.3)-glucanase-producing strain of Pseudomonas cepacia significantly decreases the incidence of diseases caused by R. solani, S. rolfsii, and P. ultimwn. The biocontrol ability of this Pseudomonas strain was correlated with the induction of the P-(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 [Fravel, 19881, including phenazine-l-carboxi l k acid (PCA) [Thomashow and Weller, 1988; Thomashow et al., 19901, oomycin A [Gutterson et al., 1986; Howie and Suslow, 19911, pyoluteorin (PLT) maas et al., 1991; Keel et al., 19921, and 2.4-diacetyl-phloroglucinol (PHL) [Haas et al., 1991; Keel et al., 19921. 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. This is particularly interesting for bacteria in which the biosynthetic genes are arranged either in operons or clusters, or when only one enzyme is missing, to achieve the synthesis of the antibiotic. For example, increased production of PLT and PHL and superior control of Pythiwn ultimum damping-off of cucumber was achieved by increasing the number of antibiotic biosynthesis genes in P. fluorescens strain CHAO [Haas et al., 1991; Maurhofer et al., 19921. Constitutive synthesis of oomycin A in P.fluorescens strain HV37a was achieved by

270


insertion of a strong promoter from E. coli upstream of the ajkE locus. The recombinant P. fluorescens strain containing the tac-afuE construct produced significantly higher levels of wmycin than the parental strain and provided greater control of P. ultimum infection [Gutterson, 1990; Gutterson et al., 19901. ~ l t e m a t i v e lbiosynthetic ~, 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. Cloned PCA biosynthetic genes were transferred from P. fluorescens 2-79, which exhibits poor rhizosphere competence, into P. putida and P. fluorescens strains that exhibit superior rhizosphere competence. The recombinant strains, which synthesized PCA in vitro, are potentially superior biocontrol agents because of their ability to colonize the rhizosphere [Bull et al., 19911. In a similar study, a cloned genomic fragment from Pseudomoms 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 [Fenton et al., 19921. The spectnun of activity through expression of an additional antibiotic increased when genes conferring PHL synthesis were mobilized from P. aureofaciens into P. fluorescens 2-79, ... which normally produces only PCA. This procedure increased activity against G.gmminis var. tritici, P. ultimim, and R. s o b i [Vincent et al., 19911.

Ill. THE TRICHODERMA SYSTEM Trichodem 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 (Chondrosterewn purpurewn), on plum, peach, and nectarine; Dutch elm disease (Ophiostoma ulmi) on elm, honey fungus (Annillaria mellea) on a range of tree species; and against rots on a wide range of crops, caused by Fusariwn, Rhizoctonia, and Pythium, and sclerotium-forming pathogens such as Sclerotinia and Sclerotiwn [Chet, 1987, Dubos, 1987; Papavizas, 19851. In many experiments, showing successful biological control, the antagonistic Trichodem was a mycoparasite [Bwsalis, 1%4; Chet and Elad, 1982; EIad et al., 19831.

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 [Chet et al., 19811. This phenomenon appears as a chemotropic growth of Trichodem in response to some stimuli in the host's hyphae or toward a gradient of chemicals produced by the host [Chet and Elad, 19831. Chemotactic responses in host-parasite relationship have been found in other systems, such as in lytic bacteria [Chet et al., 19711, nematode-trapping fungi [Jansson and Norbring-Hertz, 19791, and plant pathogenic bacteria [Ashby et al., 19871. When the mycoparasite reaches the host, its hyphae often coil around it or are attached to it by forming hwk-like structures (Fig. 1). In this respect, production of appressoria at the tips of short branches has &en described for T. hamatum and T. hariirmum. The interaction of T n c h o d e m with its host is specific [Chet and Baker, 1981; Elad et al., 1980; Sivan et al., 19841. The possible role of agglutinins in the recognition process determining the fungal specificity has been recently examined. Indeed, recognition between T. hatzianum and two of its major hosts, R. solani and S. rolfsii, was controlled by two different lectins present on the host hyphae. R. s o b i canies a lectin that b i d s to galactose and fucose residues on the Tricho& m a cell walls Elad et al., 1983al. This lectin agglutinates conidia of a mycoparasitic strain of T. harzianum, but did not agglutinate two nonparasitic strains Darak et al., 19851. This


27 1

Fig. 1 Ttichoderma coils around the pathogen.

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 Trichodenna species to attack different R. s o h i isolates Elad et al., 1983al. 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. This lectin has been isolated and purified from the culture filtrate of this plant pathogen [Barak and Chef 19901. Inbar and Chet [1992; 19941 were able to mimic the fungus-fungus'interaction in vitro using nylon fibers coated with either concanavalin A or the purified S. rolfsii lectin. As previously shown in vivo for the fungal hyphae Elad et al., 1983 b,c; Hannan et al., 1980; Henis and Chef 1975; Wells et al., 19721, during the interaction Trichodenna recognized and attached to the coated fibers, coiling around them and forming other mycopamsitism-related structures, such as appresorium-like bodies and hyphal loops (Fig. 2) [Inbar and Chet, 1992; Inbar and Chet, 19941. Following these interactions, the mycopamsite sometimes penetrates the host mycelium (Fig. 3). apparently by partially degrading its cell wall Elad et al., 1983b,c]. Microscopic observations [Benhamou and Chet, 1993; Hadar et al., 1979a.b; Liu and Baker, 1980; Weinding. 19321 led to the suggestion that Trichodenna spp. produced and secreted mycolytic enzymes responsible for the partial degradation of the host's cell wall. Results supporting this hypothesis have shown that indeed Trichodenna produces extracellularly p-(1.3)-glucanases. chitinases, lipases, and proteases when grown on cell walls of R. s o h i [Chet and Baker. 1981; Chet et al.. 1979; Elad et al.. 1982; Geremia et al.. 1991; Hadar et al.. 1979al. The production of all cell wall-degrading enzymes secreted by Trichodenna has been studied during its growth

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Fig. 2 Biomirnics of the Tricho&mur-host interaction. Tnchodcma coils around lectincoated nylon

fibers.

in different carbon sources. Maximal p(1.3)-and p-(1.6)-glucanasespecific activities were detected in media supplemented with either pustulan [p-(1.6)-glucan], nigeran [a-(1.3)-glucan alternating with a-(1.4)-glucan] or Botrytis cinema purified cell walls [De la Cruz et al., 1993; Geremia et al.. 19911. In contrast with the results of Geremia and coworkers I19911 where p(13)-glucanase activity was not observed in media supplemented with chitin, De la Cruz and collaborators [I9931 obtained high levels of specific activity of this enzyme in medium containing chitin as sole carbon source. These experiments, however, are not equivalent because two different T.hurziunum strains were used and chitin was pretreated in a different way. In 1993, Geremia and coworkers, reported the isolation of a 31-kDa basic protease that is secreted by T.hurziunum during simulated mycopamsitism, an interesting observation was that chitin also appeared to suongly induce proteinase activity. The corresponding gene @rbl) was cloned and characterizLd [Geremia et al., 19931. That was the first report of cloning of a myparasitism-related gene. Recently, Flores et al. [I9961 showed that the gene is induced during fungus-fungus interaction and used it to generate transgenic Trichodem 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. s o h i on cotton plants to only 6%. whereas the disease incidence for the nontransfonned strain was 30% mores et al., 19961. Although the results obtained with the protease-overproducingstrains were clear, the use of such strains may be even more useful in the control of F. oxyspom for which it has been suggested that proteins in its cell wall may make them more resistant to degradation by the extracellular enzymes of T. huniunum[Sivan and Chet, 1989al.


Fig. 3 Tnchodema penetrates the hyphae of its host Rhizoctonia solani.

The purification and characterization of three chitinases fmm T. harzianum has been reported [De la Cruz et al., 19921. These authors reported the isozymes to be 37, 33, and 42 kDa, respectively. Only the purified 42-kDa chitinase hydrolyzed Botrytis cinerea-purified cell walls in vitro, but this effect was heightened in the presence of either of the other two isoenzymes [De la Cnu.et al., 19921. However, the chitinolytic system of T. hardMum was recently found to be more complex [Haran et al., 1995], consisting of six distinct enzymes. The system is apparently composed of two ~-(1,4)-N-acetylglucoSarninidaSesof 102 and 73 kDa,respectively, and four, endochitinases of 52, 42, 33, and 31 kDa,respectively. All the chitinolytic enzymes were induced and secreted during growth of Trich&rma on chitin as the sole carbon source. Only the 102-kDa p-(1.4)-N-acetylglucosaminidase was expressed intracellularly, at a low constitutive level, when Trichoderma was grown on glucose. 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 bmad spectrum of chitin-containing plant pathogenic fungi. Pmbably 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. Since the report of the purification of this enzyme, the corresponding gene has been cloned [Carsolioet al., 1994; Hayes et al., 19941. Expression of the gene (ech42) 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 [Carsolio et al., 19941. Recently, a second chitinase and a p-(1,3>glucanase genes have been cloned [De la Cruz et al., 1995; Lim6n et al., 19951. In summary, expression of all enzymes fmm the cell wall-degrading system of T. harzicurum appears to be coordinated, suggesting a regulatory mechanism involving substrate induction

274


and catabolite repression. Regulation of the expression of the system is most likely at the level of transcription, as indicated by the repression of enzyme synthesis by &hydroxyquinoline. an inhibitor of transcription P e la Cnu, et al., 19931 and Northern blot analysis of the available genes [Carsolio et al., 1994; De la CNZ et al., 1995; Flores et al., 19%; Geremia et al., 1993; Lim6n et al., 19951. However, whether expression of all genes coding for the cell walldegrading enzymes is switched on by a key molecule produced by the host during in vivo interaction remains to be investigated. The level of hydrolytic enzymes produced differs for each host-parasite interaction analyzed. This phenomenon correlates with the ability of each Trichodenna isolate to control a specific pathogen. However, the specificity of Trichoderma cannot be simply explained by a difference in enzyme activity, because the nonantagonistic Trichodenna isplates produce lower, but significant, levels of lytic enzymes Elad et al., 19821. This observation supports the idea that recognition is an important factor in the mycoparasitic activity of Trichodenna. The effect of the cell wall-degrading enzymes on the host has been observed using different microscopy techniques. Interaction sites have been stained by fluorescein isothiocyanateconjugated lectins or calcofluor. The appearance of fluorescence indicated the presence of localized cell wall lysis at points of interaction between the antagonist and its host Elad et al., 19831. Electron microscopy analysis has shown that during the interaction of Trichoderma spp. with either S. rolfsii or R. solrmi, the parasite hyphae contacted their host and enzymatically digested their cell walls. In addition, an extracellular fibrillar material was deposited between the interacting cells and a mobilization of the organelles inside the invading cells toward the cellular region in contact with the host has been visualized [Chet et al., 19811. In response to the invasion, the host produced a sheath matrix which encapsulated the penetrating hyphae and the host cells became empty of cytoplasm Elad et al., 19831. The susceptible host hyphae showed rapid vacuolation, collapse, and disintegration [Chet et al., 19811. T. hunianum isolates attack both S. rolfsii hyphae and sclerotia [Artigues et al., 1984; Henis et al., 19831. 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. hunianum uses sclerotial cell content to sporulate on sclerotial surfaces and inside the digested regions [Chet and Henis, 1985; Elad et al., 19841. Therefore, it is considered that mycoparasitism is one of the main mechanisms involved in the antagonism of Trichodem as a biocontrol agent. The process apparently includes (1) chemotropic growth of Trichodem, (2) recognition of the host by the mycoparasite, (3) secretion of extracellular enzymes, (4) hyphae penetration, and (5) lysis of the host. The involvement of volatile and nonvolatile antibiotics in the antagonism by Trichoderma has been proposed [Dennis and Webster, 1971a,b]. Indeed some isolates of Trichuderma excrete growth-inhibitory substances [Claydon et al., 1987; Ghisalberti and Sivasitharnparam, 1991; Sivan et al., 19841. Claydon et al. [I9871 identified volatile alkyl pyrons produced by T. hrziMwn that were inhibitory to several fungi in vim. When these metabolites were added to a peat-soil mixture, they reduced the incidence of R. sohi-induced darnping-off on lettuce. Recently, Ordentlich and 'Chet (unpublished data) isolated a novel inhibitory substance, 3-(2hydroxypropyl), 4-(24hexadienyl,2(5H)-furanone, produced by T. harzimrum that suppresses growth of F. oxysponun and may be involved in the biocontrol of fusarium wilts. However, there is not sufficient evidence for their contribution to pathogen suppression and disease reduction in situ. A strain of T. harziunum (T-35) that controls Fusariwn spp. on various crops may utilize competition for nutrients and rhizosphere colonization [Sivan and Chef 1989bl. Trichodenna stimulates growth and flowering of several plant species [Chang et al., 1986; Windham et al., 19861. Thus, the biocontrol ability of Trichoderma strains is most likely


27 5

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 Trichodenna 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 Trichodem include electrophoretic k q otypes of different species and strains of this genus and their possible variability [Hayes et al.. 1993; Herrera Estrella et al., 19931. In addition, Meyer et al. [I9921 used a DNA fingerprinting technique to analyze the nine species aggregates of Trichodenna. Muthumee~akshiet al. [I9941 used restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD)analyses and sequencing of the internal transcribed spacer 1 region in the ribosomal DNA gene block to estimate the intraspecific divergence among isolates of T. hurzianum and to classify them according to their aggressiveness to Agaricus bispoms. This also represents the first step toward distinguishing aggressive strains from less aggressive ones without a bioassay. Another possibility is the use of the mycoparasitisrn-related genes as molecular probes to identify aggressive strains. However, major efforts should still be made to' allow a clear classification of the genus. Recently, we have been able to make a direct correlation between the genome structure of different Trichodem isolates, their in vitro biocontrol efficiency, and their capacity to undergo interstrain hyphal fusion [G6mez I., Chet I., and Hen-era Estrella A., manuscript in preparation]. This data together with other cell-to-cell communication studies could have a major influence on the selection of Trichodem isolates for their combined use in the formulation of products for their successful use in the field. Perhaps the most exciting subject for persons working in biological control with Tricho&mis strain improvement. From the moment that genetic engineering of biocontrol strains of Trichodenna was made feasible at the beginning of the 1990s [Goldman et al., 1990; Hen-era Estrella et al., 19901, enonnous 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 [Pe'er et al., 19911 or the phylloplane Wgheli et al., 19941. This was followed by the introduction of foreign genes that could potentially enhance the biocontrol capacity of Trichodem. An example of this is the work of Haran et al. [19931, in which the strong chitinase of the bacteria Serratia marcescens was introduced into T. hurzianum 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 nontransfonned strain. Many of this type of genes are available and could be tested in Tricho& m . On the other hand, cloning of the genes coding for the different cell wall-degrading enzymes produced by Trichodem will allow us to test their relevance in mycoparasitism through their overexpression and disruption. However, a very exciting issue for the basic biologists working with Trichodem is the cascade of intracellular events that follow the initial recognition of the host. Studies on this subject would lead to the understanding of a series of signal transduction events that must take place after the initial detection of the host, resulting in gene expression and the regulation of gene expression. Thus, it is clear that the Trichodem 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.

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