7 Microbial Enzymes in the Biocontrol of Plant Pathogens and Pests Leonid Chernin and Ilan Chet The Hebrew University of Jerusalem, Rehovot, Israel I. INTRODUCTION Despite many achievements in modern agriculture, food crop production continues to be plagued by disease-causing pathogens and pests. In many cases, chemical pesticides effec- tively protect plants from these pathogens. However, public concerns about harmful effects of chemical pesticides on the environment and human health have prompted a search for safer, environmentally friendly control alternatives (1–3). One promising approach is biological control that uses microorganisms capable of attacking or suppressing pathogens and pests in order to reduce disease injury. Biological control of plant pathogens offers a potential means of overcoming ecological problems induced by pesticides. It is an eco- logical approach based on the natural interactions of organisms with the use of one or more biological organisms to control the pathogen. Generally, biological control uses specific microorganisms that attack or interfere with specific pathogens and pests. Because of their specificity, different microbial biocontrol agents typically are needed to control different pathogens and pests, or the same ones in different environments. Agriculture benefits, and is dependent on, the resident communities of microorgan- isms for naturally occurring biological control, but additional benefits can be achieved by introducing specific ones when and where they are needed (4–9). Many agrochemical and biotechnological companies throughout the world are increasing their interest and investment in the biological control of plant diseases and pests. For plant pathogens alone, the current list of microbial antagonists available for use in commercial disease biocontrol includes around 40 preparations (9–11). These are all based on the practical application of seven species of bacteria (Agrobacterium radiobacter, Bacillus subtilis, Burkholderia cepacia, Pseudomonas fluorescens, Pseudomonas syringae, Streptomyces griseoviridis, Streptomyces lydicus) and more than 10 species of fungi (Ampelomyces quisqualis, Can- dida oleophila, Coniothyrium minitans, Fusarium oxysporum, Gliocladium virens, Phlebia gigantea, Pythium oligandrum, Trichoderma harzianum, and other Trichoderma species). The current market for biological agents is estimated at only $500 million, which is about 1% of the world’s total output for crop protection. The largest share of this market involves biopesticides marketed for insect control (mainly products based on Bacillus thuringiensis Copyright © 2002 Marcel Dekker, Inc. strains that produce a protein toxin with strong insecticidal activity), and these bioinsecti- cides represent around 4.5% of the world’s insecticide sales. Other agents used for bio- control exist on a much smaller scale commercially. However, the biopesticides market is expected to grow over the next 10 years at a rate of 10% to 15% per annum, vs. 1% to 2% for chemical pesticides (12). Several modes of action have been identified in microbial biocontrol agents, no two of which are mutually exclusive. Biological control may be achieved by both direct and indirect strategies. Indirect strategies include the use of organic soil amendments and com- posts, which enhance the activity of indigenous microbial antagonists against a specific pathogen (13), and the use of indirect modes of the microbial-biocontrol-agent action. These include two main mechanisms. One is cross-protection, which involves the activa- tion of physical and chemical self-defense responses (induced resistance) within the host plant against a particular pathogen by prior inoculation of the plant with a nonvirulent strain of that pathogen, resulting in partial or complete resistance to a variety of diseases in several types of plants (14,15). The other is plant growth promotion by root-colonizing bacteria and fungi that are able to stimulate plant growth and development; some of these also are capable of inducing resistance (16–18). The direct approach involves the introduction of specific microbial antagonists into the soil or plant material. These antagonists need to proliferate and establish themselves in the appropriate ecological niche in order to be active against a pathogen or a pest. A beneficial organism used to protect plants is referred to as a biological control agent (BCA) or, often, as an antagonist, because it interferes with the target organisms that damage the plant. Antagonists generally are naturally occurring, mostly soil microorganisms with some trait or characteristic that enables them to interfere with pathogen or pest growth, survival, infection, or plant attack. Usually they have little effect on other soil organisms, leaving the natural biological characteristics of the ecosystem more balanced and intact than would a broad-spectrum chemical pesticide. Some BCAs have been modified geneti- cally to enhance their biocontrol capabilities or other desirable characteristics. There are four general direct mechanisms of biological control of plant diseases. The first is competition with the pathogen for limited resources such as nutrients or space. Antagonists capable of more efficiently utilizing essential resources (e.g., carbon, nitrogen, volatile organic materials, plant residues, iron, microelements) effectively compete with the pathogen for an ecological niche and colonization of the rhizosphere and/or phyllo- sphere, leaving the pathogen less able to grow in the soil or to colonize the plant. Many plant pathogens require exogenous nutrients to germinate, then penetrate and infect host tissue successfully. Therefore, competition for limiting nutritional factors, mainly carbon, nitrogen, and iron, may result in the biological control of plant pathogens (19,20). The second mechanism is antibiosis, which is the inhibition or destruction of the pathogen by a metabolic product of the antagonist. That is, the antagonist produces some compound that is toxic or inhibitory to the pathogen, resulting in destruction of the latter’s propagules or suppression of its activity. Antibiosis is restricted for the most part to those interactions that involve low-molecular-weight diffusible compounds (e.g., antibiotics or siderophores) produced by a microorganism that inhibit the growth of another microorgan- ism (21–26). However, this definition excludes proteins or enzymes that kill the target organism. Hence, Baker and Griffin (19) extended its scope to ‘‘inhibition or destruction of an organism by the metabolic production of another,’’ thereby including small toxic molecules, and volatile and lytic enzymes. The impact of antibiosis on biological control under greenhouse and field conditions is still uncertain. Even in cases in which anti fungal Copyright © 2002 Marcel Dekker, Inc. metabolite production by an agent reduces disease, other mechanisms also may be op- erating. Hypovirulence is another mechanism that reduces virulence in some pathogenic strains. Some natural- or laboratory-source hypovirulent strains were able to reduce the effect of the virulent ones. Hypovirulent strains of Cryphonectria parasitica, Fusarium spp., Rhizoctonia solani, Sclerotinia homoeocarpa, and others have been used as biocon- trol agents of chestnut blight, wilt, rots, and other fungal diseases caused by the wild type of these pathogens. Some of these hypovirulent strains contain a single cytoplasmic ele- ment of double-stranded ribonucleic acid (dsRNA), which can be introduced into virulent strains by deoxyribonucleic acid– (DNA)-mediated transformation. This may be consid- ered a specialized form of cross-protection that is limited to the control of only established compatible strains (27–29). The fourth mechanism is predation/parasitism, which occurs when the BCA feeds directly on or inside the pathogen. In this case, the antagonist is a predator or parasite of the pathogen. When one fungus feeds on another fungus, generally it is called mycoparasi- tism. This process results in the direct destruction of pathogen propagules or structures (30–35). All known BCAs utilize one or more of these general indirect or direct mechanisms. At the product level, this includes the production of antibiotics, siderophores, and cell wall lytic enzymes, and the production of substances that promote plant growth. Additionally, successful colonization of the root surface is considered a key property of prospective antagonists (9). The most effective BCAs use two or three different mechanisms. Antago- nists also can be combined to provide multiple mechanisms of action against one or more pathogens. An understanding of this mechanism of action is important because it provides a wealth of information that can be useful in determining how to maintain, enhance, and implement this form of biological control. Numerous comprehensive reviews on specialized topics, as well as proceedings and books describing the biocontrol activities of different microorganisms against plant patho- gens and pests in laboratories, greenhouses, and the field, appeared in the late 1990s (9,10,34,36–41). However, the biological control of plant diseases is not as well estab- lished as biocontrol of insects in commercial agriculture. The latter has been a successful approach for decades and continues to be a rapidly developing area of research. In this chapter, we limit our discussion to enzymatic mechanisms of microbial control of plant pathogens and pests. II. THE ROLE OF FUNGAL ENZYMES IN THE BIOLOGICAL CONTROL OF PLANT DISEASES A. Gliocladium and Trichoderma Species Systems The fungus Gliocladium virens Miller, Giddens and Foster (ϭTrichoderma virens, Miller, Giddens, Foster, and von Ark) is a common soil saprophyte and one of the most promising and studied fungal biocontrol agents. It originally was isolated from a sclerotium of the plant pathogenic fungus Sclerotinia minor and then was found to be active against several fungal plant pathogens. Trichoderma, a genus of hyphomycetes that is an anamorphic Hypocreaceae (class Ascomycetes), also is common in the environment, especially in soils. Many Gliocladium and Trichoderma spp. isolates obtained from natural habitats have been used in biocontrol trials against several soil-borne plant pathogenic fungi under Copyright © 2002 Marcel Dekker, Inc. both greenhouse and field conditions. In particular, isolates of G. virens, G. roseum, T. viride, T. harzianum Rafai, and T. hamatum have been reported to be antagonists of phyto- pathogenic fungi, including Botrytis cinerea, Fusarium spp., Phytophthora cactorum, Pythium ultimum, Pythium aphanidermatum, Rhizoctonia solani, Sclerotinia sclerotiorum, and Sclerotium rolfsii. These cause soil-borne and foliage diseases in a wide variety of economically important crops in a range of environmental conditions. The antagonists kill the host by direct hyphal contact, causing the affected cells to collapse or disintegrate; vegetative hyphae of all species have been found susceptible. The biological and ecological characteristics and potential of these closely related genera for the biological control of plant pathogens have been reviewed extensively (4,9,31,34,35,42–48). Among the biocontrol mechanisms proposed for Gliocladium and Trichoderma spp. are competition, antibiosis, and mycoparasitism. The last mechanism is based mainly on the activity of lytic exoenzymes (chitinases, glucanases, cellulases, and proteases) re- sponsible for partial degradation of the host cell wall. Barnett and Binder (30) divide mycoparasitism into necrotrophic (destructive) parasitism, which results in death and de- struction of the host fungus, and biotrophic (balanced) parasitism, in which the develop- ment of the parasite is favored by a living host structure. The sequential events involved in mycoparasitism have been described in several comprehensive reviews (31–35). Briefly, mycoparasitism is a complex process that involves ‘‘recognition’’ of the host, positive chemotropic growth, attachment, and de novo synthesis of a set of cell-wall-degrading enzymes that aid the parasite in penetrating the host and completing its destruction. Lec- tins, the sugar-binding proteins or glycoproteins of nonimmune origin, which agglutinate cells and are involved in interactions between the cell surface components and its extracel- lular environment, have been shown to play a role in the recognition and contact between necrotrophic mycoparasites of Gliocladium and Trichoderma spp. and soil-borne patho- genic fungi. This contact, in turn, initiates a signal transduction cascade toward the second, most important step of mycoparasitism, the induction of lytic enzymes able to degrade fungal cell walls. Most fungi attacked by Gliocladium and Trichoderma spp. have cell walls that con- tain chitin as a structural backbone and laminarin (β-1,3-glucan) as a filling material. The other minor cell wall components are proteins and lipids. The ability to produce lytic enzymes has been shown to be a crucial property of these and other mycoparasitic fungi. Several contemporary reviews discuss the role of, in particular, chitinolytic enzymes of Trichoderma spp. in fungal mycoparasitism and biocontrol activity (33,49–51). In the last few years, the enzymatic patterns of various strains of Trichoderma and Gliocladium spp. have been determined, the corresponding genes cloned, and their products characterized. Some of these enzymes have been studied in more detail, with the goal of understanding their role in fungal biocontrol activity and principles of their expression regulation. In general, fungal cell-wall-degrading enzymes produced by G. virens and Trichoderma spp. are strong inhibitors of spore germination and hyphal elongation in a number of phyto- pathogenic fungi. The excretion of lytic enzymes enables Trichoderma spp. to degrade the target fungal cell wall and utilize its nutrients (52–55). A considerable amount of recent research has been devoted to studying the indi- vidual lytic systems produced by Trichoderma spp. Most of the studies on the expres- sion and regulation of these lytic enzymes have been performed in liquid cultures supple- mented with different C sources (e.g., chitin, glucose, β-1,4-linked N-acetylglucosamine [GlcNAc], fungal cell walls) and their antifungal effects determined in vitro. These growth Copyright © 2002 Marcel Dekker, Inc. conditions facilitated the identification of the lytic enzymes induced in Trichoderma spp. to hydrolyze the polymers constituting the fungal cell walls. However, they did not reflect the exact conditions existing during the antagonistic interactions between Trichoderma spp. and its hosts. Thus, using T. harzianum–R. solani and T. harzianum–S. rolfsii interac- tions as model systems, Elad et al. (52) revealed lysed sites and penetration holes in the hyphae of the host fungus caused by the antagonist’s attachment and coiling around it (Fig. 1). In the presence of the protein synthesis inhibitor cycloheximide, antagonism was prevented and enzymatic activity reduced. These observations suggested that the lytic enzymes whose synthesis de novo was induced as a result of early stages of interaction with the target phytopathogen excreted by Trichoderma spp. degrade R. solani and S. rolfsii cell walls at the interaction sites. According to more recent data obtained by electron microscopy of the interaction between T. harzianum and the arbuscular mycorrhizal fungus Glomus intraradices, chitinolytic degradation was seen only in areas adjacent to the sites of Trichoderma spp. penetration. The interaction between T. harzianum and G. intrara- dices involves the following events: (i) recognition and local penetration of the antagonist into mycorrhizal spores, (ii) active proliferation of antagonist cells in mycorrhizal hyphae, and (iii) release of the antagonist through moribund hyphal cells (56). 1. Chitinolytic Enzymes Chitin, an unbranched insoluble homopolymer consisting of GlcNAc units, is the second (after cellulose) most common biodegradable polysaccharide in nature, being the main structural component of cell walls of most fungi and arthropods (insects, nematodes, and other invertebrates) including many agricultural pests (57–59). Many species of bacteria, streptomycetes and other actinomycetes, fungi, and plants produce chitinolytic enzymes that catalyze the hydrolysis of chitin. Chitinases produced by various microbes differ con- siderably in their molecular masses, high-temperature optima, and degrees of stability, probably because of glycosylation; they generally are active in a rather wide pH range. In recent years, soil-borne microorganisms that produce chitinases have become consid- ered as potential biocontrol agents against fungal pathogens, insects, and nematodes that Figure 1 Scanning electron micrograph of Trichiderma spp. hyphae interacting with those of S. rolfsii. Hypha of S. rolfsii, from which a coiling hypha of T. harzianum was removed, showing digested zone with penetration sites caused by the antagonists (ϫ5, 500). (From Ref. 52.) Copyright © 2002 Marcel Dekker, Inc. causediseasesanddamageinagriculturalcrops.Chitinasesalsoplayanimportantphysio- logicalandecologicalroleinecosystemsasrecyclersofchitin,bygeneratingCandN sources.Someproducersofchitinases,includingTrichodermaspp.,arealsosourcesof mycolyticenzymepreparations(51,59,60). ChitinolyticenzymesaredefinedasenzymesthatcleaveabondbetweentheC1 andC4oftwoconsecutiveGlcNAcunits.Onthebasisofaminoacidsequencesimilarities, allchitinaseshavebeengroupedintofamilies18,19,and20,underthemainclassof glycosylhydrolases.Mostofthemicrobialchitinasesbelongtofamily18(61,62).Even withinthesamefamily,chitinasesshowwidelydifferingpropertieswithrespecttosub- stratespecificity,reactionspecificity,andpHoptimum.Thechitinolyticenzymesaredi- videdintothreeprincipaltypesdependingontheiractiononchitinsubstrates.According tothenomenclaturesuggestedbySahaiandManocha(59),endochitinases(EC3.2.1.14) aredefinedasenzymescatalyzingtherandomhydrolysisof1,4-βlinkagesofGlcNAcat internalsitesovertheentirelengthofthechitinmicrofibril.Theproductsofthereaction aresoluble,low-molecular-massmultimersofGlcNAcsuchaschitotetraose,chitotriose, anddiacetylchitobiose.Exochitinases,alsotermedchitobiosidasesorchitin-1,4-β-chito- biosidases(63),catalyzetheprogressivereleaseofdiacetylchitobioseunitsinastepwise fashionasthesoleproductfromthechitinchains,suchthatnomonosaccharidesoroligo- saccharidesareformed. Thethirdtypeofchitinolyticenzymeischitobiasealsotermedashexosaminidase (EC3.2.1.52)orN-acetyl-β-1,4-d-glucosaminidase(EC3.2.1.30)belongstofamily20 andalsoactsinexosplittingmodeondiacetylchitobioseandhigheranalogsofchitin, includingchitotrioseandchitotetraose,toproduceGlcNAcmonomers.Rapidandspecific methodshavebeendevelopedfordetectionandquantitativeassaysofN-acetyl-β-gluco- saminidase,chitobiosidase,andendochitinaseinsolutionsusingp-nitrophenyl-N-acetyl- β-d-glucosaminide,p-nitrophenyl-β-d-N,N′-diacetylchitotriose,andp-nitrophenyl-β-d- N,N′,N″-triacetylchitotrioseorcolloidalchitinassubstrates,respectively(64).Procedures alsoaredescribedforthedirectassayofthesethreeenzymesaftertheirseparationby sodiumdodecylsulfate(SDS)-polyacrylamidegelelectrophoresis(PAGE)inwhichthe enzymesarevisualizedasfluorescentbandsbyusinganagaroseoverlaycontaining4- methyl-umbelliferylderivativesofN-acetyl-β-d-glucosaminide,β-d-N,N′-diacetyl- chitobioside,orβ-d-N,N,N″-triacetylchitotriose,respectively(65). AsetofchitinolyticenzymessecretedbyvariousstrainsofT.harzianum(e.g.,TM, T-Y,39.1,CECT2413,P1ϭT.atroviride),whengrownonchitinasthesoleCsource, consistsofN-acetylglucosaminidases,endochitinases,andexochitinases(chitobiosidases). Intotal,10separatedchitinolyticenzymeswerelistedbyLorito(50);onlyonestepin themicroparasiticprocessofT.harzianum,whichisthedissolutionofthecellwallof thetargetfungusbyenzymeactivity,mayinvolvemorethan20separategenesandgene productssynergisticonetoanother(Table1).TwoN-acetylglucosaminidaseswithappar- ent molecular masses of 102 to 118 kD (depending on the isolate and the procedure used) and 72 to 73 kD (ϭNAG1) have been described by Ulhoa and Peberdy (66), Lorito et al. (67), and Haran et al. (68). The 102-kD enzyme (CHIT102) is the only chitinase of T. harzianum to be expressed constitutively when the fungus is grown with glucose instead of chitin as the sole C source (69). Four endochitinases—CHIT31, CHIT33, CHIT52, and CHIT42 (ϭECH42)—have been reported by De La Cruz et al. (70), Ulhoa and Peberdy (66), Harman et al. (63), and Haran et al. (68). Additionally, a glycosylated chitobiosidase of 40 kD is secreted by strain P1 when grown on crab-shell chitin as the sole C source (63), and a 28-kD exochitinase releasing GlcNAc only was purified from the culture filtrate Copyright © 2002 Marcel Dekker, Inc. Table 1 Examples of Lytic Enzymes Produced by Mycoparasitic Fungi which May Be Involved in Disease Biocontrol Molecular mass Encoding Enzyme (kDa) gene Fungus/strain Reference N-Acetylglucosaminidase 102–118 ND Trichoderma harzianum (66, 68) (EC 3.2.1.30) (TM, 39.1) N-Acetylglucosaminidase 72–73 nag1 T. harzianum (TM, P1) (67, 68, 88) (EC 3.2.1.30) Endochitinase (EC 52 ND T. harzianum (TM) (68) 3.2.1.14) Endochitinase (EC 41–42 ech42 T. harzianum (39.1, P1, (63, 70, 78, 3.2.1.14) CEST2413); G. vir- 79, 84, ens (41) 106) Exochitinase (chitibiosi- 40 ND T. harzianum (P1) (63) dase) Endochitinase (EC 37 ND T. harzianum (CEST 68, 70) 3.2.1.14) 2413, TM) Endochitinase (EC 33 chit33 T. harzianum (CEST (68, 70) 3.2.1.14) 2413, TM) Proteinase 31 prb1 T. harzianum (55) β-1,3-endoglucanase (EC 78 bgn13.1 T. harzianum (109) 3.2.1.6; EC 3.2.1.39) (CECT2413) β-1,3-endoglucanase 17 ND T. harzianum (113) (CECT2413) β-1,3-endoglucanase 36 ND T. harzianum (39.1) (110) β-1,3-exoglucanase (EC 77–110 lam1.3 T. harzianum (P1, T-Y, (67, 111, 3.2.1.58) IMI1206040) 112) β-1,6-endoglucanase 43 ND T. harzianum (117, 118) (CECT2413) β-1,4-endoglucanase 51 egl1 T. longibrachiarum (290) β-1,3-exoglucanase 84 exgA Ampelomyces quis- (141) qualis Endochitinase 40 ND Fusarium chlamy- (130) dosporum β-1,3-glucanase ND ND Trametes versicolor, (131) Pleurotus eryngii β-1,3-glucanase, β-1,6- ND ND Penicillium purpuro- (132) glucanase, chitinase genum β-1,3-glucanase ND ND Tilletiopsis spp. (136) of strain T. harzianum T198. This particular enzyme displayed activity on a wide array of chitin substrates of more than two GlcNAc units in length (71). Lorito et al. (72,73) studied the antifungal activities of a 42-kD endochitinase and a 40-kD chitobiosidase from T. harzianum strain P1 in bioassays against nine different fungal species. Both spore germination and germ-tube elongation were inhibited in all chitin-containing fungi. The degree of inhibition was proportional to the level of chitin in the cell wall of the target fungus. Combining the two enzymes resulted in a synergistic increase in antifungal activity. A variety of synergistic interactions have been found when different enzymes were combined or associated with biotic or abiotic antifungal agents. Copyright © 2002 Marcel Dekker, Inc. The levels of inhibition obtained by using enzyme combinations were, in some cases, comparable with those of commercial fungicides. Moreover, the antifungal interaction between enzymes and common fungicides allowed up to 200-fold reductions in the re- quired chemical doses. These two enzymes, separately or in combination, substantially improved the antifungal ability of a biocontrol strain of Enterobacter cloacae (74). In an in vitro bioassay, different classes of cell-wall-degrading enzymes (glucan 1,3-β-glucosi- dase [EC 3.2.1.58], N-acetyl-β-glucosaminidase, endochitinase, and chitin 1,4-β-chitobio- sidase) produced by T. harzianum and G. virens inhibited spore germination of B. cinerea. The addition of any chitinolytic or glucanolytic enzyme to the reaction mixture synergisti- cally enhanced the antifungal properties of five different fungitoxic compounds against B. cinerea (73). Some of the combinations showed a high level of synergism, suggesting that the interaction between membrane-affecting compounds and cell-wall-degrading en- zymes could be involved in biocontrol processes and plant self-defense mechanisms (75). A correlation between high capacity to produce chitinolytic enzymes and the superior biocontrol potential of the mycoparasitic fungi was also reported by Lima et al. (76). In general, chitinolytic enzymes from Trichoderma spp. appeared to be more effective in vitro against a number of fungal plant pathogens than were similar enzymes from plants or bacteria (72). The ech42 chitinase gene was shown to be highly conserved within the genus Trichoderma (77) and its product, the 42-kD chitinase, is believed to be one of the most crucial for mycoparasitic interactions between Trichoderma spp. and target pathogens. A similar endochitinase was purified from G. virens (78). Carsolio et al. (79) cloned and characterized ech42 (previously named ThEn42) encoding a 42-kD endochitinase in the biocontrol strain T. harzianum IMI206040. Expression of the complementary deoxyribo- nucleic acid (cDNA) clone in Escherichia coli produced bacteria with chitinase activity. This chitinase displayed lytic activity on B. cinerea cell walls in vitro. The ech42 gene was assigned to a double-chromosomal band (chromosome V or VI) upon electrophoretic separation and Southern analysis of the chromosomes. Expression of ech42 was strongly enhanced during direct interaction of the mycoparasite with a phytopathogenic fungus when confronted in vitro and when it was grown in minimal medium containing chitin as sole C source. Similarly, light-induced sporulation resulted in high levels of transcript, suggesting developmental regulation of the gene. T. virens strains in which the 42-kD chitinase gene was disrupted or constitutively overexpressed were constructed through genetic transformation. The resulting transformants were stable and showed patterns simi- lar to those of the wild-type strain with respect to growth rate, sporulation, antibiotic production, colonization efficiency on cotton roots, and growth/survival in soil. However, biocontrol activities of the ‘‘disrupted’’ and constitutively overexpressed strains were sig- nificantly decreased and enhanced, respectively, against cotton seedling disease incited by R. solani when compared with those of the parental strain (80). However, several recently reported experiments have put into question the role of CHIT42 endochitinase as the only key enzyme in mycoparasitism. The biocontrol strain T. harzianum P1, recently attributed to T. atroviride (81), was genetically modified by targeted disruption of the single-copy ech42 gene. A mutant, lacking the 42-kD endochi- tinase but retaining the ability to produce other chitinolytic and glucanolytic enzymes of this strain expressed during mycoparasitic activity, was unable to clear a medium contain- ing colloidal chitin but grew and sporulated similarly to the wild type. In vitro antifungal activity of the ech42-disruptant culture filtrate against B. cinerea and R. solani was reduced by about 40% relative to that of the wild type, but its activity in protecting against P. Copyright © 2002 Marcel Dekker, Inc. ultimum and R. solani in biocontrol experiments was the same or even better than that of strain P1. In contrast, the mutant’s antagonism against B. cinerea on bean leaves was significantly reduced compared with that of strain P1. These results indicate that the antag- onistic interaction between strain P1 and various fungal hosts is based on different mecha- nisms (82). Corresponding results were obtained with several transgenic T. harzianum strains carrying multiple copies of ech42, and the corresponding gene disruptants were con- structed. The level of extracellular endochitinase activity when T. harzianum was grown under inductive conditions increased up to 42-fold in multicopy strains relative to that of the wild type, whereas gene disruptants exhibited practically no activity. However, no major differences in the efficacies of the strains generated as biocontrol agents against R. solani or S. rolfsii were observed in greenhouse experiments (83). One possible explana- tion for these results is that other enzymes of Trichoderma’s chitinolytic system are suffi- cient to control these fungal phytopathogens and that the lack of a certain protein can be compensated for by altering the levels of other proteins with similar activity. In view of the results showing efficient synergism between different chitinolytic enzymes produced by the same Trichoderma sp. isolate, it is not surprising that overexpression of one of these enzymes does not necessarily lead to an increase in biocontrol activity. Moreover, to achieve the highest level of antagonism toward target pathogens, a combination of several enzymes gives a better effect than the overproduction of only one of them. Several groups have reported cloning genes ech42 (79,84–86), chit33 (87), and nag1 (88). Very little is known, however, about the regulation of these genes and the roles of the corresponding enzymes in fungi during mycoparasitism. Generally, products of chitin degradation are thought to induce chitinolytic enzyme expression, and easily metaboliz- able C sources serve as repressors (59,89,90). Fungal cell walls, colloidal chitin, and C starvation have been shown to be inducers of the cloned chitinase genes (79,84,87,88,91). To study the regulation of chitinolytic enzyme synthesis during the Trichoderma sp.–host mycoparasitic interaction, more specific confrontation assays (dual culture) on plates were developed (53,69,92). The differential expression of chitinolytic enzymes dur- ing the interaction of T. harzianum with S. rolfsii and the role of fungal–fungal recognition in this process were studied by Inbar and Chet (92). A change in the chitinolytic enzyme profile was detected during the interaction between the fungi grown in dual culture on synthetic medium. Before contact with one another, both fungi contained a protein with constitutive 1,4-β-N-acetylglucosaminidase activity. As early as 12 h after contact, the chitinolytic activity in S. rolfsii disappeared, while that in T. harzianum (a protein with a molecular mass of 102 kD, CHIT102) greatly increased. After 24 h of interaction, the activity of CHIT102 diminished concomitantly with the appearance of a 73-kD 1,4-β-N- acetylglucosaminidase, which became clear and strong at 48 h. This phenomenon did not occur if the S. rolfsii mycelium was autoclaved prior to incubation with T. harzianum, suggesting its dependence on vital elements from the host. Cycloheximide inhibited this phenomenon, indicating that de novo synthesis of enzymes takes place in Trichoderma spp. during these stages of the parasitism. A biomimetic system based on the binding of a purified surface lectin from the host S. rolfsii to nylon fibers was used to dissect the effect of recognition. An increase in CHIT102 activity was detected, suggesting that the induction of chitinolytic enzymes in Trichoderma sp. is an early event that is elicited by the recognition signal (i.e., lectin–carbohydrate interactions). Experiments with T. harzia- num and the host lectin–covered nylon threads indicated that mere physical contact with the host triggers both the mycoparasitism-specific coiling of Trichoderma sp. hyphae Copyright © 2002 Marcel Dekker, Inc. around the host and chitinase formation (32,92). It is postulated that recognition is the first step in a cascade of antagonistic events that trigger the parasitic response in Tricho- derma spp. These observations were extended by Haran et al. (69), who showed that the expres- sion of the various N-acetylglucosaminidases and endochitinases during mycoparasit- ism can be regulated in a very specific and finely tuned manner that is affected by the host. When strain T. harzianum T-Y antagonized S. rolfsii,theN-acetylglucosaminidase CHIT102 was the first to be induced. As early as 12 h after contact, its activity diminished, and the other N-acetylglucosaminidase, CHIT73, was expressed at high levels. However, when T. harzianum antagonized R. solani, the chitinase expression patterns differed con- siderably. Twelve hours after contact, CHIT 102 activity was elevated, and the activities of three additional endochitinases, at 52 kD (CHIT 52), 42 kD (CHIT 42), and 33 kD (CHIT 33), were detected. As the antagonistic interaction proceeded, CHIT102 activity decreased, whereas the activities of the endochitinases gradually increased. Similarly, Carsolio et al. (79) detected the induction of ech42 gene transcription only 24 h after contact of T. harzianum with B. cinerea. These data suggested that chitinase formation takes place during the later stages of the host–mycoparasite interaction, for example, to T. harzianum in penetration of the host hyphae. Therefore, chitinase induction generally has been regarded as a consequence of, rather than a prerequisite for, mycopara- sitism. Krishnamurthy et al. (93) reported that differential induction of chitinase isoforms in vitro might depend on C sources in the growth medium. Nevertheless, in vivo the differential expression of T. harzianum chitinases may influence the overall antagonistic ability of the fungus against a specific host. The specific and unique role of the 102-kD enzyme in triggering the expression of other chitinolytic enzymes was questioned by Zeilinger et al. (94). To monitor chitinase expression during mycoparasitism of strain T. harzianum P1 (ϭT. atroviride) in situ, strains were constructed containing fusions of the green fluorescent protein to the 5′- regulatory sequences of the Trichoderma nag1 and ech42 genes. Confronting these strains with R. solani led to induction of gene expression before or after physical contact in the cases of genes ech42 and nag1, respectively. Separating the two fungi abolished ech42 expression, indicating that macromolecules are involved in its precontact activation. No ech42 expression was triggered by culture filtrates of R. solani or placement of T. harzia- num on plates previously colonized by R. solani. Instead, high expression occurred upon incubation of T. harzianum with the supernatant of R. solani cell walls digested with culture filtrates or purified CHIT42. The results indicate that ech42 is expressed before contact of T. harzianum with R. solani and its induction is triggered by soluble chitooligo- saccharides produced by constitutive activity of CHIT42 and/or other chitinolytic en- zymes. Therefore, ech42 expression, in contrast to that of nag1, is a relatively early event, taking place prior to physical hyphal contact of the fungus with its host (R. solani). This indicates that this enzyme could be involved in the very early stages of the mycoparasitic process. Furthermore, the involvement of chitinase activity in the induction of ech42 gene expression pre contact has been demonstrated by the effect of the chitinase inhibitor allo- samidin, an actinomycete-derived metabolite. Expression of the 73-kD exochitinase nag1 gene was observed only after contact of Trichoderma spp. with its host and was most active during overgrowth of R. solani. Therefore, different mechanisms of induction may occur for ech42 and nag1, and nag1 gene expression and may depend on products gener- ated by CHIT42 activity. The results support the earlier suggestion by Lora and associates (95) that constitutive chitinases may partially degrade the cell walls of the host, thereby Copyright © 2002 Marcel Dekker, Inc. [...]... Regulating the Production of Enzymes and Secondary Metabolites Involved in Biocontrol Activity of Gram-Negative Bacteria In many Gram-negative bacteria, including plant- growth-promoting pseudomonads, three types of control elements are involved in the production of some secondary metabolites and enzymes that are synthesized at the end of exponential growth or during the stationary phase and are involved in. .. role in biocontrol activity against pests in several ways: (i) by isolating chitinolytic B thuringiensis strains and comparing their activity against pests with that of the parental strain, (ii) by adding chitinolytic enzymes from other sources to B thuringiensis with the aim of increasing its toxicity, (iii) by introducing cloned chitinase genes into a B thuringiensis strain, and (iv) by directly using... consisting of two N-acetyl-β-d-glucosaminidases with apparent molecular masses of 89 and 67 kD and a 58-kD endochitinase Additionally, a 50-kD chitobiosidase was observed in two other strains of E agglomerans tested in this work (157) The chitinolytic activity was induced when the strains were grown in the presence of colloidal chitin as the sole C source; the observed chitinolytic enzymes seemed to be the. .. as callose and lignin Acidic PR proteins, including acidic -1 ,3-glucanases and chitinases, act Copyright © 2002 Marcel Dekker, Inc against fungal and bacterial pathogens at an early stage of the infection process; basic β1,3-glucanases and chitinases may interact with pathogens at a later stage of infection (149) Another group of enzymes includes peroxidases, which play a key role in the plant resistance... membranes Neither gene was induced during the interaction of Trichoderma sp with lectin-coated nylon fibers, even through the latter do induce hyphal coiling and appressorium formation (92) Therefore, the signal involved in triggering the production of these hydrolytic enzymes is independent of the recognition mediated by this lectin– carbohydrate interaction The results showed that induction of prb1 and ech42... activities reached their maxima at 72 h after inoculation, indicating the activation of a general defense response in the plant (152) Besides cell-wall lytic enzymes, a few examples showing the involvement of other enzymes in the biocontrol activity of Trichoderma sp and other fungal antagonists of plant pathogens have been found A xylanase produced by T viride has induced defense responses, including ethylene... treatment of tobacco plants, after loss of the effects of the initial treatment, restored the enhanced sensitivity of the tissues to xylanase The continual presence of ethylene was required to maintain its effects, and the timing of the induction and subsequent loss of ethylene’s effects were closely coordinated at the molecular and whole tissue levels (153) Glucose-oxidase activity may play a role in the. .. mobilization of β-glucans under conditions of C- and energy-source exhaustion, and a physiological role in morphogenetic processes during fungal development and differentiation (105) Glucanases have been suggested as another group of key enzymes involved in the mycoparasitism of Gliocladium and Trichoderma spp against fungal plant pathogens (Table 1) The substrate of these enzymes, -1 ,3-glucan, is one of the. .. the incidence of diseases caused by R solani, S rolfsii, and P ultimum The biocontrol ability of this Pseudomonas sp strain was correlated with the induction of the -1 , 3-glucanase by different fungal cell walls in synthetic medium (183) Strain PF-21 of P fluorescens, isolated from the rhizosphere of rice and producing chitinase and -1 ,3glucanase, was found to be very effective in inhibiting the growth... 3.2.1.6) and endo-1,4β-d-glucanase activity of T harzianum isolate T3 is induced in sphagnum peat moss cultivations and dual culture experiments by the presence of P ultimum Further, P ultimum stimulated the germination of Trichoderma sp conidia Low concentrations of purified 17-kD endo-1, 3- -glucanase and 4 0- and 45-kD cellulases were able to inhibit the germination of encysted zoospores and elongation of . Inc. causediseasesanddamageinagriculturalcrops.Chitinasesalsoplayanimportantphysio- logicalandecologicalroleinecosystemsasrecyclersofchitin,bygeneratingCandN sources.Someproducersofchitinases,includingTrichodermaspp.,arealsosourcesof mycolyticenzymepreparations(51,59,60). ChitinolyticenzymesaredefinedasenzymesthatcleaveabondbetweentheC1 andC4oftwoconsecutiveGlcNAcunits.Onthebasisofaminoacidsequencesimilarities, allchitinaseshavebeengroupedintofamilies18,19 ,and2 0,underthemainclassof glycosylhydrolases.Mostofthemicrobialchitinasesbelongtofamily18(61,62).Even withinthesamefamily,chitinasesshowwidelydifferingpropertieswithrespecttosub- stratespecificity,reactionspecificity,andpHoptimum.Thechitinolyticenzymesaredi- videdintothreeprincipaltypesdependingontheiractiononchitinsubstrates.According tothenomenclaturesuggestedbySahaiandManocha(59),endochitinases(EC3.2.1.14) aredefinedasenzymescatalyzingtherandomhydrolysisof1, 4- linkagesofGlcNAcat internalsitesovertheentirelengthofthechitinmicrofibril.Theproductsofthereaction aresoluble,low-molecular-massmultimersofGlcNAcsuchaschitotetraose,chitotriose, anddiacetylchitobiose.Exochitinases,alsotermedchitobiosidasesorchitin-1, 4- -chito- biosidases(63),catalyzetheprogressivereleaseofdiacetylchitobioseunitsinastepwise fashionasthesoleproductfromthechitinchains,suchthatnomonosaccharidesoroligo- saccharidesareformed. Thethirdtypeofchitinolyticenzymeischitobiasealsotermedashexosaminidase (EC3.2.1.52)orN-acetyl- -1 ,4-d-glucosaminidase(EC3.2.1.30)belongstofamily20 andalsoactsinexosplittingmodeondiacetylchitobioseandhigheranalogsofchitin, includingchitotrioseandchitotetraose,toproduceGlcNAcmonomers.Rapidandspecific methodshavebeendevelopedfordetectionandquantitativeassaysofN-acetyl-β-gluco- saminidase,chitobiosidase,andendochitinaseinsolutionsusingp-nitrophenyl-N-acetyl- β-d-glucosaminide,p-nitrophenyl-β-d-N,N′-diacetylchitotriose,andp-nitrophenyl-β-d- N,N′,N″-triacetylchitotrioseorcolloidalchitinassubstrates,respectively(64).Procedures alsoaredescribedforthedirectassayofthesethreeenzymesaftertheirseparationby sodiumdodecylsulfate(SDS)-polyacrylamidegelelectrophoresis(PAGE)inwhichthe enzymesarevisualizedasfluorescentbandsbyusinganagaroseoverlaycontaining 4- methyl-umbelliferylderivativesofN-acetyl-β-d-glucosaminide,β-d-N,N′-diacetyl- chitobioside,orβ-d-N,N,N″-triacetylchitotriose,respectively(65). AsetofchitinolyticenzymessecretedbyvariousstrainsofT.harzianum(e.g.,TM, T-Y,39.1,CECT2413,P1ϭT.atroviride),whengrownonchitinasthesoleCsource, consistsofN-acetylglucosaminidases,endochitinases,andexochitinases(chitobiosidases). Intotal,10separatedchitinolyticenzymeswerelistedbyLorito(50);onlyonestepin themicroparasiticprocessofT.harzianum,whichisthedissolutionofthecellwallof thetargetfungusbyenzymeactivity,mayinvolvemorethan20separategenesandgene productssynergisticonetoanother(Table1).TwoN-acetylglucosaminidaseswithappar- ent. inhibited in all chitin-containing fungi. The degree of inhibition was proportional to the level of chitin in the cell wall of the target fungus. Combining the two enzymes resulted in a synergistic increase. Inc. 2.Glucanases -1 ,3-glucan,orlaminarin,isapolymerofd-glucoseina -1 ,3configuration,arranged ashelicalcoils.Fungalcellwallscontainmorethan60%laminarin.Whereaschitinis arrangedinregularlyorderedlayers,laminarinfibrilsarearrangedinanamorphicmanner. Therearechemicalbondsbetweenthelaminarinandchitin,andtogethertheyforma complexnetofglucanandGlcNAcoligomers(103).Laminarinishydrolyzedmainlyby - 1,3-glucanases,alsoknownaslaminarinases.Theseenzymes,describedinfungi,bacteria, actinomycetes,algae,mollusks,andhigherplants,arefurtherclassifiedasexo-andendo- β-glucanases.Exo- -1 ,3-glucanases( -1 ,3-glucanglucanohydrolase,[EC3.2.1.58])hy- drolyzelaminarinbysequentiallycleavingglucoseresiduesfromthenonreducingendsof polymersoroligomers.Consequently,thesolehydrolysisproductsareglucosemonomers. Endo- -1 ,3-glucanases( -1 ,3-glucanglucanohydrolase[EC3.2.1.6orEC3.2.1.39]) cleave -1 ,3linkagesatrandomsitesalongthepolysaccharidechain,releasingsmaller oligosaccharides.Bothenzymetypesarenecessaryforthefulldigestionoflaminarin (104).Theseenzymeshaveseveralfunctionsinfungiincludingnutritioninsaprotropism, mobilizationofβ-glucansunderconditionsofC-andenergy-sourceexhaustion,anda physiologicalroleinmorphogeneticprocessesduringfungaldevelopmentanddifferentia- tion(105). Glucanaseshavebeensuggestedasanothergroupofkeyenzymesinvolvedinthe mycoparasitismofGliocladiumandTrichodermaspp.againstfungalplantpathogens(Ta- ble1).Thesubstrateoftheseenzymes, -1 ,3-glucan,isoneofthemajorcomponentsof fungal