Trichoderma: a review of biology and systematics of the genus

Mycol. Res. 100 (8): 923-935 (1996)

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Printed in Great Britain

CENTENARY REVIEW

Trichoderma: a review of biology and systematics of the genus

GARY J. SAMUELS United States Department of Agriculture, Agriculture Research Service, Systematic Botany and Mycology Laboratory, Room 304, B-OI1A, BARC-W, Beltsville, MD 20705-2350, USA.

Species of Trichoderma, a genus of hyphomycetes, are ubiquitous in the environment, but especially in soils. They have been used or encountered in many human activities, including commercial applications in production of enzymes and biological control of plant disease. They are the cause of disease in commercially produced mushrooms, and have been identified as causal agents of disease in immunosuppressed humans. Knowledge about what constitutes a species of Trichoderma, or about interspecific relationships, has not kept pace with the expanding number of applications or frequency of encounter of Trichoderma by biotechnologists, plant pathologists and medical personnel. This review presesents an overview of the interaction between humans and Trichoderma, and a more intensive review of knowledge of systematics and taxonomy of the genus.

Two-hundred years ago, when it was first described, mycologists mistook Trichoderma Pers.: Fr. for a Gasteromycete (Persoon, 1794). It was only half a century later that the true nature of the genus was realized, but movement beyond the point of recognizing the class affinities of Trichoderma to development of a species monograph has been slow. While the only comprehensive taxonomic monograph for the genus is admitted by its author to be artificial (Rifai, 1969 b), researchers in applied fields have been attributing to Trichoderma species novel biological properties and activities. Between the years of 1992 and 1995 alone, approximately 550 articles that cited the genus were catalogued in the United States Department of Agriculture database AGRICOLA. I will not review all those papers here, but refer the reader to the excellent reviews available for the fields of biological control (Papavizas, 1985; Chet, 1987, 1993; Lumsden, 1992) and enzymology (Kubicek et aL 1990). A comprehensive bibliography for Trichoderma up to 1980 is found in Domsch, Gams & Anderson (1980). In the present review I will present some of the major advances in our knowledge of the biology of Trichoderma, followed by a more intense review of the steps taken to understand the systematics of the genus.

BIOLOGY Trichoderma has not yet, given us any 'wonder drugs' such as penicillin, but the ability of some species to produce enzymes and/or to attack or inhibit other fungi has attracted major research efforts in several areas, including biological control of plant disease, and enzyme production, as well as in studies of genetic control and manipulation in filamentous fungi. At a time in human history when we acknowledge the need to appreciate the economic, if not aesthetic, value of our biological diversity, Trichoderma stands as a readily exploitable

source. Whether and how this source can be utilized will depend upon understanding the biology of the species. Trichoderma is a genus of filamentous deuteromycetes. Its members are generally found in all soils including forest humus layer (Wardle, Parkinson & Waller, 1993) as well as in agricultural and orchard soils (see Chet, 1987; Roiger, Jeffers & CaldwelL 1991 and references therein). Davet (1979) and Smith, Wilcox & Harman (1990) have described media selective for Trichoderma. Trichoderma species are rarely reported to occur on living plants and have not been found as endophytes of living plants (Petrini, 1986 & pers. comm.). Danielson & Davey (1973) reported that individual species showed preference for soil temperature and moisture content of forest soils. Widden & Aribtol (1980) found a seasonality to species distribution, although the seasonal occurrence of individual species may be influenced by competition provided by other Trichoderma species. Trichoderma viride Pers. does seem to be adapted to cooler climates (see Roiger et al., 1991, and references). Trichoderma species may be sensitive to some environmental pollution, as is indicated by the low rate of recovery of T. viride from coniferous forests that had been subjected to alkaline dust for a period of 25 years; the high pH (6.6) of the humus layer was blamed (Fritze & Baath, 1993). While Trichoderma species are generally considered to be aggressive competitors, Wardle et al. (1993) found that the ability to compete was species dependent. Thus, Mucor hiemalis Wehmer displaced T. harzianum Rifai from the forest soiL but was itself induced to produce more conidia at the expense of biomass by T. polysporum (Link: Fr.) Rifai in forest humus. Some niche differentiation was suggested, as both of the species mixtures had higher biomass of the fungi after 47 days than the monocultures of each species. Physiological activities of Trichoderma species have been

Trichoderma: a review of biology and systematics of the genus both beneficial and harmful to human enterprise. The ability of Trichoderma reesei E. G. Simmons to break down cellulosic materials through the production of cellulase enzymes has lead to their commercial exploitation (see Reese & Mandels, 1989; El Gogary et al., 1990; Kubicek et aI., 1990; Cuevas, Culiat & Manaligod, 1991; Bedford & Classen, 1993) in areas as diverse as clothes-washing detergent. animal feed and fuel production. Cellulases from T. reesei also have the potential of bleaching kraft pulp, thereby providing an alternative to conventional bleaching with chlorine (Buchert et al., 1994). The effluent from chlorine bleaching contains environmentally problematiC chlorinated hydrocarbons. Cellulolytic enzymes produced by T. viride and T. reesei help the sugar industry to dispose of sugar by-products, molasses and sugar beet pulp by transforming the sugars to protein, that can be fed to ruminants (Nigam, 1994). Katayama & Matsumura (1993) have found that T. harzianum possesses an oxidative system that is capable of degrading the organochlorine pesticide endosulfan. Chitinolytic enzymes from T. harzianum and T. virens O. H. Miller, Giddens & A. A. Foster) Arx (formerly known as Gliocladium virens J. H. Miller, Giddens & A. A. Foster) are thought to be responsible for degradation of fungus cell walls and, thereby, effective in biological control of the fungal pathogen Botrytis cinerea Pers.: Fe. (Cruz et al., 1992; Di Pietro et al., 1993; Lorito et al., 1993, 1994). On the other hand, those same enzymes are responsible for failure of the shi'itake (Lentinula edodes (Berk.) Pegler) crop in Japan (Komatsu, 1976; Tokimoto, 1982) through disruption of the mushroom mycelium. Trichoderma harzianum and T. virens (mostly under the name Gliocladium virens) are the most commonly cited species in biological control (see reviews in Papavizas, 1985; Chet, 1987). Trichoderma harzianum, alone or in combination with other Trichoderma species or chemical adjuvants, has been used in control of several diseases. Some of these include: Rhizoctonia damping-off in radish (Lifshitz, Lifshitz & Baker, 1985), com and soybean (Kommedahl et a!" 1981); greymould on tomato (Migheli et al., 1994), grapes and strawberry (Elad ef al., 1995; Harman et aI., 1995); Colletotrichum storage rot of apple (Tronsmo & Hjeljord, 1995); cucumber fruit rot caused by Rhizoctonia solani J. G. Kuhn (Lewis & Papavizas, 1980). take-all disease in wheat (Ghisalberti & Sivasithamparam, 1991), and sclerotinia sclerotiorum (Lib.) de Bary in pea (Knudsen & Eschen, 1991) and 5. minor Jagger in lettuce drop (Vannacci et al., 1991); and a wilt-complex, predominantly caused by Sclerotium rolfsii Sacc., Rhizoctonia solani and Fusarium oxysporum Schldl. in lentil and chickpeas (Mukhopadhyay, 1995). In addition to a direct role of T. harzianum in individual applications, it is effective in restoring suppressiveness to heat-treated media amended with composted hardwood bark (Nelson & Hoitink, 1983). Soils amended with T. harzianum can be disease suppressive, have greener turf, probably by enhanced root growth, and reduced brown patch (Rhizoctonia soIani). dollar spot (Sderotinia homeocarpa F. T. Benn.), and Pythium blight (Lo, Nelson & Harman, 1995). Trichoderma harzianum also produces volatile chemicals that arrest wood rot induced by basidiomycetes (Morrell. 1990). Various Trichoderma species gave control of Phytophthora crown rot of apple seedlings in Wisconsin (Roiger, Jeffers &

924 Caldwell, 1991). Trichoderma virens and T. longibrachiatum Rifai were useful in control of groundnut root and stem rot diseases caused by Rhizoctonia solani in India (Sreenivasaprasad & Manibushanrao, 1993). Trichoderma harzianum, T. parceramosum Bissett, and T. viride showed in vitro antagonism against Cryphonectria parasitica (Murrill) M. E. Barr, the cause of chestnut blight (Arisan-Atac, Heidenreich & Kubicek, 1995). Trichoderma species, especially T. viride, when used in combination with fumigants, were positively correlated with a decline in viability of Phellinus weirii (Murrill) R. W. Gilb. in live roots of Douglas-fir (Pseudotsuga menziesii) (Nelson, Theis & McWilliams, 1995). The authors also suggested the use of Trichoderma strains in combination with lower levels of fumigant for control of root rot in individual trees of high value. Trichoderma species have been used in commercial preparations for biological control of fungal-induced plant diseases (Lumsden, Lewis & Locke, 1993). Trichoderma harzianum is the active ingredient in TRICHODEX, which is used against postharvest rot of apple, and is combined with T. polysporum in the product BINAB-T, which is used in control of wound decay and wood rot (Ricard, 1981). Trichoderma harzianum AG2, prepared from protoplast fusion. protects against a wide range of soil-borne plant diseases (Harman, 1990). GlioGard has T. virens as its active ingredient and is used to prevent damping-off in seedlings caused by Pythium and Rhiwctonia species (Lumsden & Locke, 1989). Unidentified species of Trichoderma are active in two products used in control of deadarm disease of vines and stone fruit in New Zealand (Trichoject and Tricho Minidowels; Hunt & Gale, 1995). No single mode of action of Trichoderma species against fungal plant parasites is known; for a review of this subject see Chet (1987). Control of Rhizoctonia solani and Pythium ultimum by Trichoderma species, including T. harzianum, may be effected through direct penetration of host hyphae (Dennis & Webster, 1971; Benhamou & Chet, 1993). Inbar & Chet (1992) have found that mycelium of T. harzianum is preferentially attached to nylon fibres that had been coated with a plant lectin (concanavalin A) or a lectin derived from the cell wall of Rhiwctonia solani. Extracellular enzymes. including j3-1,3-glucanase, chitinase and cellulase (Elad, Chet & Henis, 1979; Cruz et aI., 1993; Lorito et al., 1994; Harman et aI., 1995) are effective in disrupting the mycelium of the pathogen. On the other hand, mycoparasitism is not the primary mechanism of biocontrol of Pyfhium ultimum and R. solani by T. virens (Howell. 1987; Lumsden et al., 1992). Howell (1987) showed that mutants of strains of T. virens normally parasitic on R. solani had lost their mycoparasitic ability but retained the antibiotic capability of the parent strains. Later Howell & Stipanovic (1991) divided strains of T. virens into two groups on the basis of antifungal antibiotic production. One group. designated as 'Q' strains, produced gliotoxin and were effective against Rhizoctonia solani but inactive against Pythium ultimum Trow. The other group, designated as 'P' strains. produced gliovirin and were strongly active against P. ultimum but were inactive against R. solani. Inhibitory volatile substances were also suggested as the means of biocontrol of Botrytis cinerea mould of snap beans by T. hamatum (Nelson & Powelson. 1988). Ghisalberti &

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G. J. Samuels Rowland (1993) found several antifungal metabolites produced by T. harzianum to suppress growth of take-all (Gaeumannomycesgraminis (Sacc.) Arx & D. L. Olivier). Faull, Graeme-Cook & Pilkington (1994) studied production of an isonitrile antibiotic by a uv-induced mutant of T. harzianum and its activity against soil-borne plant pathogens (Faull & Scarseletti, 1994). Antifungal antibiotics produced by Trichoderma species are reviewed by Ghisalberti & Sivasithamparan (1991). While Trichoderma species are generally considered either useful to humans or at least ecologically neutraL this is not always the case. Taylor (1986) reported that members of Trichoderma and Gliocladium produce some of the most toxic substances known. These include trichothecenes (Adams & Hanson, 1972; Corley, Miller-Wideman & Durley, 1994) and 'pentaibols', polypeptides containing a high proportion of aaminoisobutyric acid that exhibit toxicity to experimental animals (see Scott, 1991). Trichoderma species are not generally known to affect human health, but there are scattered reports indicating pathogenicity to humans. Trichoderma viride and T. Iongibrachiafum, respectively, have been involved in complications of renal dialysis (Loeppke ef al., 1983; Tanis ef aI., 1995). A liver transplant recipient developed infection of a perihepatic haematoma due to Trichoderma viride, and despite surgical removal and treatment with amphotericin B, abundant quantities of the Trichoderma were recovered from the haematoma (Jacobs ef aI., 1992). More recently Trichoderma species (T. Iongibrachiafum, T. pseudokoningii Rifai) were identified as the cause of death in immunosuppressed humans in the U.s.A. (D. A. McGough, pers. comm.). Trichoderma species are not involved in plant parasitism or in postharvest crop loss. However, because of its mycoparasitic abilities, T. harzianum does cause serious losses in commercial mushroom production. This includes shi'itake and Agaricus mushrooms in Japan (Komatsu, 1976) and other parts of the world (Muthumeenakshi ef al., 1994; Speranzini ef al., 1995; P. Romaine, pers. comm.). Most Trichoderma strains are not encountered in association with sexual stages, and are considered to be strictly mitotic, clonal fungi. The apparent lack of sexuality is a barrier to understanding interrelationships within and among Trichoderma species. Moreover, the absence of outcrossing denies a means of genetic manipulation which has caused dismay for authors such as Papavizas (1985). One of the most important developments in Trichoderma biology has been the discovery of the ability to fuse protoplasts of genetically diverse strains or even species. Improved biological control and industrial strains have been found among the nonparental progeny resulting from the fusion of isolated protoplasts (Pe'er & Chet, 1990; Stasz & Harman, 1990; Harman & Hayes, 1993; Meza ef al., 1995); and genetic linkage data concerning enzyme markers have been obtained from protoplast fusions in T. reesei (Bawa & Sandhu, 1994). Stasz & Harman (1990) could not explain the resultant variation in progeny of crosses involving T. viride, T. hamafum (Bonord) Bainier, and T. koningii Oudem. in terms of classical parasexuality. However, Bawa & Sandhu (1994) used auxotrophic and cellulolytic characterization to demonstrate the presence of haploid parasexual progeny. Strains identified as T. harzianum have also been improved for

biocontrol applications through transformation with bacterial DNA, and the transformants have remained stable in the environment (Sivan & Harman, 1991; Migheli ef al., 1994). Meza ef al. (1995) transferred a benomyl resistance marker between strains of T. reesei, and selected progeny of protoplast fusion were higher in cellulolytic ability than either parent. Shin & Cho (1993) obtained recombinant, interspecific fusion progeny when protoplasts of biological control strains of T. virens and T. harzianum were crossed. The ability to generate protoplasts has also provided an opportunity to separate chromosomes and to localize genes on individual chromosomes, as has been done for T. reesei (Gilly & Sands, 1991; Mantyla ef al., 1992). Sexual reproduction is known in Trichoderma in the sense that the only known teleomorphs of Trichoderma are species of Hypocrea Fr. and closely related genera, members of the ascomycete order Hypocreales. There are few documented cases of Hypocrea species undergoing the sexual cycle in artificial culture; the best known is Hypocrea spinulosa Fuckel (as Chromocrea spinulosa (Fuckel) Petch). In this species sexual reproduction is under control of one locus and two alleles, but half of the progeny appear to be self-fertile owing to unidirectional shifting to the compatible mating type in some mycelial nuclei (Mathieson, 1952; Perkins, 1987). A similar phenomenon was suggested by Canham (1969) for H. cifrina (Fr.) Fr. var. cifrina and var. americana Canham, and by Samuels & Lodge (1996) for H. poronioidea Moller. In the case of H. poronioidea, however, Samuels & Lodge (1996) were not able to induce crossing between the self fertile and self sterile strains and suggested that half of the progeny of meiosis were sexually incompetent. It should be added that Samuels & Lodge (1996) did not use auxotrophic mutants or other genetic markers, so that the possibility of crossing between self fertile and self sterile strains cannot be excluded. On the other hand, Samuels, Petrini & Manguin (1994) observed a normal bipolar segregation of mating type in asci of H. jecorina Berk. & Broome. Rehner (pers. comm.), using amplified fragment length polymorphisms of nuclear rONA, demonstrated the ability of widely geographically separated strains of H. jecorina to undergo meiotic recombination. Hypocrea jecorina, the teleomorph of T. reesei (Kuhls ef al., 1996a), represents an excellent model system for classical genetic studies in Trichoderma.

SYSTEMA TICS Toward a concept of genus While it is difficult to define, there is a general concept of a 'basic' Trichoderma morphology (see for example Rifai, 1969; Bissett, 1984, 1991a-c, 1992; Fig. 4): rapid growth; abundant powdery, green conidia; and ill-defined conidiophores. That this morphology has been unmistakable is attested to by the paucity of generic synonyms: only four taxonomic synonyms were uncovered by Rifai (1969). The most persistent of the generic synonyms was Pachybasium Sacco (1885). Pachybasium was proposed for species of Verficillium Nees that have sterile elongations on the conidiophores (Fig. 2), but Bainier (1906) soon recognized Pachybasium to be synonymous with Trichoderma. Within

Trichoderma: a review of biology and systematics of the genus

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Figs 1-5. Trichoderma and Hypocrea. Fig. 1. Tolypocladium niveum, ex neotype (ARSEF 3280). Fig. 2. Trichoderma polysporum-aggr. anamorph of Hypocrea sp. (G]S 98-121). Fig. 3. Trichoderma minutisporum Bissett, anamorph of Hypocrea sp. (CBS 901.72). Fig. 4. Trichoderma viride anamorph of Hypocrea rufa (G]S 90-97). Fig. 5. Hypocrea pulvinata anamorph (G]S 91-220). Scale bars = 10 11m.

Trichoderma, the Pachybasium-types can be distinguished from Trichoderma sensu stricto in having white conidia produced

a genus, Bissett (1991a, b) accepted the section Pachybasium in

from doliform phialides that are held in botryose clusters, and often with a sterile or terminally fertile extension (Fig. 2). Although most authors, including Hughes (1958) in his critical evaluation of hyphomycete genera, did not accept Pachybasium, a few new species were described in the genus as recently as 1961. While Pachybasium is not now recognized as

The morphological concept of Trichoderma is not completely settled. Although the Trichoderma morphology is usually unmistakable, there is morphological intergradation with other hyphomycete genera. Carmichael et ai. (1980) questioned whether Toiypocladium W. Cams could be a synonym of Trichoderma, possibly because of a similarity in arrangement of

Trichoderma.

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Figs 6-9. Trichoderma and Gliocladium. Fig. 6. Trichoderma virens. ex type culture. Fig. 7. Gliocladium viride (J. P. Jones Gl 114). Fig. 8. Gliocladium prnicillioides, anamorph of Sphaerostilbella aureonitens (GJS 83-286). Fig. 9. Trichoderma anamorph of H. flavovirens (GJS 95-154). Scale bars = 10 11m.

phialides of T. inflatum W. Gams (= To. niveum (0. Rostr.) Bissett; Fig. 1) to that of Trichoderma polysporum (Link:Fr.) Rifai (Fig. 2). However. the morphological comparison is only superficial as To. niveum is now known to be the anamorph of Cordyceps facis Kobayashi & Shimizu (Hodge & Krasnoff, 1995; Hodge, Krasnoff & Humber, 1996), a member of the Clavicipitales.

The inclusion of Gliocladium virens (Fig. 6), one of the most frequently cited of the biological control fungi, in Trichoderma by Arx (1987) has only recently been accepted following DNA sequence analysis (Rehner & Samuels, 1994, 1995). The penicillus of phialides, combined with conidia held in slime, all on a more or less discrete conidiophore (Fig. 6) are generally characteristic of Gliocladium Corda. Trichoderma virens is not

Trichoderma: a review of biology and systematics of the genus the only Trichoderma to have a generalized Gliocladium-type conidiophore, which is found among anamorphs of Hypocrea species such as H. gelatinosa (Webster, 1964; Bissett, 1991b) and various other Hypocrea species (Doi, 1972; e.g. H. flavovirens Berk. & Broome, Fig. 9). When viewed in light of the different teleomorphs and biologies, the morphological similarity between T. virens and the type species of Gliocladium, G. penicillioides Corda (Fig. 8) is not indicative of close phylogenetic relationship. Gliocladium penicillioides, and similar Gliocladium species, are anamorphs of species of Sphaerostilbella (Seifert, 1985). Sphaerostilbella Henn. species occur on basidiomata of members of the Aphyllophorales and the genus is more closely related to Hypomyces Tul. than it is to Hypocrea (Rehner & Samuels, 1995). Further, many unrelated hypocreaceous anamorphs are classified in Gliocladium because they have penicillately arranged phialides (e.g. G. roseum Bainier, which is a Cionostachys Corda; see illustrations in Domsch et al., 1980). Bissett (1991 b) included T. virens in Trichoderma sect. Pachybasium Bissett on the basis of morphological comparison to other species that he included in that section. From the point of view of teleomorphs, T. virens is probably closely related to T. aureoviride Rifai, the anamorph of H. aureoviridis Plowr. & Cooke, a species that is morphologically and anatomically similar to H. gelatinosa Tode: Fr. Rehner & Samuels (1994) compared various hypocrealean ascomycetes that have Gliocladium anamorphs using sequences of large subunit nuclear ribosomal DNA and found T. virens, G. viride, G. roseum, and G. penicillioides to be widely dispersed in the Hypocreales. Trichoderma virens was arranged among Hypocrea species, and can thus be regarded as a species of Hypocrea. The consequence of this finding for Trichoderma taxonomy is that the morphological stereotype of Trichoderma has to be modified to accept this stereotypical Gliocladium morphology. In the case of G. virens, however, it is not too difficult to rationalize the Gliocladium morphology within Trichoderma because (i) of the intergrading Hypocrea anamorphs and (ii) the branching pattern of G. virens can be seen to be a modification of a more typical Trichoderma, (iii) the formation of typical Hypocrea-like chlamydospores (Figs 5, 6), and (iv) the formation of green conidia. In his subdivision of Trichoderma into sections, Bissett (1991 b) tended to the philosophy that Hypocrea anamorphs are, a priori, species of Trichoderma. However true that may be in phylogenetic terms, inclusion of all Hypocrea anamorphs in Trichoderma may require an unacceptable expansion of the morphological concept of the genus. Gliocladium viride Matr. (formerly known as G. deliquescens Sopp, Fig. 7) is the anamorph of Hypocrea Iutea (Tode: Fr.) Petch, although the species is found commonly in its anamorph form from soil isolations. Hypocrea lutea is morphologically, biologically and genetically similar to H. gelatinosa (Rehner & Samuels, 1994, 1995). Although this Gliocladium-like anamorph is not difficult to distinguish from G. penicillioides, it does not bear much morphological similarity to more typical Trichoderma species. While acknowledging that G. viride behaves, genetically, like a Hypocrea species, and thus like any other Trichoderma species, it represents the diagnosticians worst nightmare: it would be impossible to determine this species as a Trichoderma;

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in any current taxonomic treatment it would be placed in Gliocladium. For a general discussion of the integration of anamorph fungi into the ascomycetes see Cams (1995). Bissett (1991 a) expanded the morphological concept of Trichoderma by including anamorphs of species of Hypocrea that have effused stromata in sect. Hypocreanum Bissett. These Hypocrea species include H. pulvinata Fuckel (Rifai & Webster, 1966b; Fig. 5), H. citrina (Rifai & Webster, 1966b; Canham, 1969), and others. Their anamorphs have colourless conidia borne in conspicuous clear and uncoloured liquid on more or less verticillately branched, or unbranched and then Acremonium- or Verticillium-like conidiophores. Bissett (1991a) envisioned a progression of forms among the Hypocrea anamorphs that ranges from these Acremonium-or Verticilliumlike to more typical Trichoderma conidiophores (e.g. H. rufa (Pers.: Fr.) Fr., Fig. 4). Preliminary results of sequence analysis of rDNA (Rehner. peTS. comm.) place these Hypocrea species with effused stromata within the larger genus Hypocrea. However, care should be taken before accepting the placement of the anamorphs of these fungi in Trichoderma, as it is at least possible that rather than being Trichoderma anamorphs, they are synanamorphs or even spermatia. Hypocrea poronioidea, a Hypocrea species with a discrete, turbinate stroma, produces both a typical Trichoderma anamorph as well as a synanamorph that is morphologically similar to the anamorphs of H. pulvinata. In H. poronioidea, however, these colourless conidia apparently only form on incipient stromata, and this suggests that they have a sexual function (Samuels & Lodge, 1996). Furthermore, on the basis of rDNA sequence similarity, H. poronioidea is basal to the species of Hypocrea that have effused stromata, and closer to them than it is to other Hypocrea species (Rehner, pers. comm.). Conceivably the formation of spermatia, or at least Acremonium-like anamorphs, is an apomorphy for this group. Hypocrea poronioidea, hypothesized as being primitive, retains the ability to form Trichoderma conidia as well as the Acremonium-like conidia whereas the ability to form Trichoderma is lost among the more derived members.

Toward a concept of species As is usually the case, species of Trichoderma have been defined on the basis of their morphology. Morphological characters used in species recognition in Trichoderma have been outlined by Rifai (1969b) and Bissett (1984, 1991a-c, 1992). Unfortunately, some of the richest characters for species recognition in hyphomycetes in general are either not variable enough, or are difficult to describe in Trichoderma. Conidial size and shape, an extremely useful character in other genera, are of limited value in Trichoderma. Conidia of most species are less than 5 I.lm long and wide. Conidia may be globose, subglobose, ellipsoidal or oblong, and the shape is useful in recognizing groups of species, but within groups its value is diminished. Conidia may be some shade of green or greenish yellow, or colourless. The differences in shade of green, which may range from a deep green to nearly grey, may be taxonomically significant, but difficult to interpret and communicate. Conidial ornamentation, which may be smooth,

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warted or tuberculate, is certainly a useful species character but may require scanning electron microscopy to demonstrate. There are usually no definable conidiophores in Trichoderma (see, for example, Figs 2-4). The conidia tend to aggregate into pulvinate masses, the aggregates formed of intertwined hyphae bearing phialides and often appearing to be attached to the substrate at one or a few points. An ontogenetic study of such aggregates could be fascinating, and could also help in defining a conidiophore. For example, do aggregates originate with a single hypha that proliferates7 The width of the hyphae within the aggregate is significant. Bissett (1991 b) has made extensive taxonomic use of fertile or sterile elongations of conidiophores (Fig. 2). The arrangement of phialides on hyphae within aggregates is characteristic but is virtually impossible to define and communicate (compare Figs 2-4). Phialide shape is characteristic of a species but, again, is difficult to define. Perhaps new mathematical definitions can be developed that would reflect form of phialides and patterns of branching more accurately than the available words. Seifert, Wingfield & Wingfield (1995) have suggested that branching in Trichoderma resembles 'iterated functional systems' that are derived from mathematical formulae. Chlamydospores in Trichoderma tend to be globose to subglobose, terminal or intercalary in hyphae, smooth, green, and less than 15 ~m in diameter (Figs 5-7). While these chlamydospores are characteristic of Trichoderma and Hypocrea, their form is not diagnostic of species but their presence may be (Samuels, Doi & Rogerson 1990; Bissett 1991 b). Synanamorphs, apart from chlamydospores, are rare in Trichoderma. Hypocrea suIawesensis Yoshim. Doi (Samuels et al., 1990) produces solitary' macroconidia' at the tips of branches of conidiophores of its Trichoderma anamorph. In H. poronioidea (Samuels & Lodge, 1996), hyaline conidia held in drops of colourless liquid at the tips of sparingly branched conidiophores form in addition to a typical Trichoderma anamorph. As was discussed above, these hyaline conidia may be spermatia. Characteristics of colonies grown on agar media are subtle. Most species grow rapidly and conidiate readily on common media. Unlike comparable genera such as Penicillium Link or Fusarium Link, where cultures present many diagnostic characters, in Trichoderma there is virtually no aerial mycelium and most diffusable pigmentation is typically in shades of yellow. Yellow pigment is a characteristic for some species of sect. Longibrachiatum. Trichoderma colonies often show zonation in conidial development; Schrufer & Lysek (1990) demonstrated that rhythmic growth and sporulation are heterogeneous in populations. They also reported a dependence on light for induction of sporulation. Whether conidial production occurs in aggregates, or predominantly on 'mononematous' conidiophores diffused throughout the colony, may be a species character. It has been my observation that a relatively weak medium such as cornmeal agar with 2 % dextrose permits better observation of conidial aggregation when compared to media such as potato dextrose or oatmeal agar, where conidial production is generally much more profuse. However, diffusable pigment forms more reliably on potato dextrose agar than cornmeal agar with dextrose or oatmeal agar. Some cultures develop sweet odours that are

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sometimes reminiscent of coconut. The coconut odour produced by some strains of T. viride and T. harzianum has been attributed to an antifungaL unsaturated pyrone, 6pentyl-a-pyrone (Serrano-Carreon et aI., 1992; see also Claydon et ai., 1977). Rifai (1969) concluded, not surprisingly, that anamorph characters alone might not provide a useful taxonomy of Trichoderma. He took the attitude that there is no real way to define a biological species in morphological terms, that the morphological characters were continuously variable, and that there is no way of knowing the degree of variation tolerable within an individual species. One way to chart variation within a biological species of Trichoderma is to use anamorphs of known Hypocrea species, so that there would be no doubt that all strains, derived from ascospores as they are, would represent only one species. Because, prior to 1969, the number of Hypocrea species that had been grown in pure culture was smalL Rifai refrained from proposing narrow species concepts in Trichoderma. He adopted instead the concept of 'aggregate' species, which he defined as ' aggregations of morphologically very similar and often hardly separable species.' Of these aggregates, five were based on Hypocrea species, viz. T. aureoviride Rifai (H. aureoviridis), T. hamatum Rifai (H. semiorbis (Hook.) Berk.); T. piluIiferum Webster & Rifai (H. piIuIifera J. Webster & Rifai), T. pseudokoningii (H. d. schweinitzii (Fr.) Sacc.), and T. viride (H. rufa). Although he considered that his work was preliminary and not to be taken as a complete taxonomic treatment, it has become the primary source of species identification for Trichoderma. Bissett (1984, 1991a-c, 1992), like Rifai, adopted a morphological approach to taxonomy of Trichoderma. He essentially elevated each of Rifai's aggregate species to sectional level (Bissett, 1991a) and proposed one new section for Acremonium-like or Verticillium-like anamorphs of the Hypocrea species, such as H. puIvinata, that have effused stromata. Doi, Abe & Sugiyama (1987) added an additional section, sect. Saturnisporum for species that have warted, wrinkled or nearly winged conidia. Bissett has published revisions of sections Longibrachiatum (Bissett, 1984) and Pachybasium (Bissett, 1991 b) and recognized several presumably biological species in each. In the publications of Rifai (1969) and Bissett (1984, 1991 a, b) we have taxonomies that reflect differing taxonomic philosophies. Bissett has perceived noncontinuous morphological characteristics of biological species where Rifai saw a continuum within a few basic morphologies. Is one system any more reflective of reality than the other? Independently derived, macromolecular, data have been applied in tests of the morphotaxonomic hypotheses.

Macromolecular approaches to Trichoderma taxonomy The first efforts at macromolecular characterization of Trichoderma strains and species were undertaken not by professional taxonomists, but by frustrated users of the existing taxonomy. Stasz et aI. (1988, p. 170) plaintively remarked that, ' ... methods are lacking to differentiate among strains for patent purposes, or to determine the variability and abundance of strains in natural ecosystems'. Morphology alone in Trichoderma has not led to a satisfactory taxonomy or,

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Trichoderma: a review of biology and systematics of the genus at any rate, to a taxonomy that has been useful to many users. Characters derived from nucleic acids and enzymes are attractive because, with cladistic analysis, they seem to offer the possibility of greater objectivity than do traditionally observed and analyzed data. Whether these techniques increase taxonomic objectivity may be debated, but the additional characters are certainly welcome. Macromolecular analyses based on enzymes and nucleic acids have shown that at least some of the aggregate species are phylogenetically based (e.g. Okuda, Fujiwara & Fujiwara, 1982; Stasz et aI., 1989; Leuchtmann, Petrini & Samuels, 1996) at the same time confirming genetic diversity of the individual aggregates (Zamir & Chet, 1985; Stasz et aI., 1989; Meyer, 1991; Meyer et al., 1992; Fujimori & Okuda, 1994; Muthumeenakshi ef al., 1994; Samuels ef al., 1994; Zimand ef aL 1994; Kuhls et al., 1996a, b; Leuchtmann ef aI., 1996). Stasz et al. (1989) evaluated five aggregate species of Trichoderma using enzyme polymorphisms. While they concluded that morphological species are not characterized by specific alleles at single loci, or specific patterns of alleles at multiple loci, they demonstrated what they called 'core groups' of morphological species. Despite individual strains that gave widely divergent allozyme patterns, the core groups coincided well with Rifai's aggregate species T. pseudokoningii, T. koningii, T. hamafum, and T. viride. Strains used under the name of T. harzianum aggregate fell into one of two clusters. These results indicate that, in part. there is a genetic basis for the morphological aggregate species, and also confirm Rifai's own contention that each aggregate is genetically heterogeneous. Meyer & Plaskowitz (1989) observed two types of conidial ornamentation among twelve strains referable to T. viride, a member of sect. Trichoderma. Meyer (1991) later found that mitochondrial DNA restriction fragment data distinguished between those two groups and he suggested that each group, characterized by conidial ornamentation and mtDNA type, could represent a distinct species. This is an interesting correlation that should be confirmed by the study of more strains if only because T. viride is the type species of the genus. Okuda et al. (1982) found that the aggregate species T. hamatum, T. harzianum, T. koningii, and T. viride could be subdivided on the basis of production of isonitrile antibiotics. There was some correspondence between the respective groups and phenotypical characters of the strains. Zamir & Chet (1985) divided twenty-three strains of the T. harzianum-aggregate among five different types according to their enzyme profiles. Fujimori & Okuda (1994) and Muthumeenakshi et al. (1994), also working with the T. harzianum-aggregate, used various molecular techniques to distinguish, respectively, two and three groups within the aggregate. Muthumeenakshi et ai. (1994) found three distinct types of ITS-I, and strains characterized by ITS type 2 were aggressive antagonists in commercial mushroom production. In this regard it is interesting that Stasz et al. (1989), using isozyme analysis, also found two enzyme profiles in T. harzianum. Fujimori & Okuda (1994) successfully used RAPD's to identify duplicate Trichoderma strains in microbial screening. Kuhls, Lieckfeldt & Borner (1995) used RAPD's of T. reesei to

determine that T. 'todica' (ATCC 36936), an unpublished strain patented for the production of antiviral antibiotics, is actually T. parceramosum, a member of sect. Longibrachiafum Bissett. Schlick et ai. (1994) used DNA fingerprinting to identify patent strains of T. harzianum. They also found that strains that had been produced by gamma irradiation had the same ITS-1 and ITS-2 sequences as the parent strains. Zimand et al. (1994) used the RAPD procedure to identify biocontrol strains of Trichoderma species. Bissett (1984) elevated the T. Iongibrachiatum-aggregate species to status of section and included the species T. citrinoviride, T. Iongibrachiafum, T. parceramosum, T. pseudokoningii, and later the anamorph of H. schweinitzii (Bissett, 1991c). Samuels et ai. (1994), using isozymes, and Kuhls et ai. (1996a, b), using a variety of DNA techniques, confirmed that sect. Longibrachiatum is distinct from other sections. Bissett (1984) included the species T. pseudokoningii, which Rifai (1969) regarded as an aggregate species, in sect. Longibrachiatum. Trichoderma pseudokoningii was originally isolated from ascospores of Hypocrea d. schweinifzii in eastern Australia, and Leuchtmann et a/. (1996) found that T. pseudokoningii is similar to other Hypocrea strains found in New Zealand. They also concluded that T. pseudokoningii, a species commonly reported in the literature, is most likely limited in distribution to Australia and New Zealand and has probably been widely misinterpreted (e.g. Bissett, 1984). Trichoderma sect. Longibrachiafum includes T. reesei, a species that is well known for cellulase production (Reese & Mandels, 1989) and that accounted for about half of the approximately 550 articles that cited Trichoderma in the USDA AGRICOLA data base for 1992-1995. Bissett (1984) synonymized T. reesei with T. Iongibrachiafum on morphological grounds. Meyer et al. (1992), using DNA-fingerprint analysis, and Samuels ef ai. (1994), combining morphometries and isoenzyme profiles, distinguished between T. reesei and T. Iongibrachiatum. In another isozyme study (Leuchtmann et al., 1996), using a much larger set of strains than was used by Samuels et al. (1994), T. reesei and H. jecorina were closely joined to each other and formed a group that was distinct from all other species in sect. Longibrachiatum, including other associated Hypocrea teleomorphs. Kuhls et al. (1996a), using rDNA sequence analysis and PCR-fingerprints, observed T. reesei to behave exactly as any strain of H. jecorina, concluded that T. reesei is the anamorph of H. jecorina, differing in minor phenotypic characters and in its inability to undergo sexual reproduction (Samuels et aI., 1994). Doi et al. (1987) proposed the new sect. Saturnisporum for two species, T. ghanense Y. Doi, Y. Abe & Sugiy. and T. safurnisporum HammilL species that have conspicuously warted, sometimes alate, conidia. Branching patterns of both species are similar to branching in sect. Longibrachiafum and, like sect. Longibrachiatum, both have ellipsoidal conidia. Kuhls et al. (1996b) and Turner et ai. (1996) used nucleic acid analyses to place, respectively, T. safurnisporum and T. ghanense in sect. Longibrachiatum. The teleomorphs of Trichoderma

Trichoderma has all the essential characteristics of anamorphs of the ascomycete order Hypocreales (Samuels & Seifert.

G. J. Samuels 1987): brightly to lightly coloured conidia, conidiophores and colonies, and conidia formed from phialides. By the time the first volume of the Transactions of the British Mycological Society was published in 1902, the specific link between T. viride and H. ntfa was an accepted fact (Smith, 1902), thus establishing the generic link between Hypocrea and Trichoderma. Berkeley, as early as 1860, suspected a link between l. viride and some unnamed ascomycete when he enigmatically noted that the Trichoderma species was' probably not autonomous.' In that same year L.-R. Tulasne (Tulasne, 1860) proved that l. viride and the ascomycete Hypocrea rufa are expressions of one life cycle. The brothers Tulasne (Tulasne & Tulasne, 1865) illustration of T. viride is remarkably accurate in the representation of the phialides and their disposition on the conidiophore. The observation of the relationship between H. rufa and l. viride, later proven by single ascospore cultures by Brefeld (1891), represented a major advance in our understanding of the interrelationship between deuteromycetes and ascomycetes. The only proven teleomorphs for Trichoderma have been closely related genera, viz. Hypocrea with fewer in Podostroma P. Karst. and sarawakus Boedijn. Our knowledge of all of these genera is slowly developing, but a picture is emerging wherein species of Podostroma and sarawakus that have Trichoderma anamorphs could be placed in Hypocrea. Since 1860 many Hypocrea species have been grown in pure culture and their Trichoderma anamorphs described (see list in Doi & Doi, 1979). Because only members of the Hypocreales have been proven to have Trichoderma anamorphs, there is no doubt that Trichoderma is a genus of that order. It is, however, somewhat more difficult to assign Trichoderma to a single teleomorph genus because the three, albeit closely related, ascomycete genera have Trichoderma anamorphs. Ribosomal DNA sequences (Rehner & Samuels, 1994; Kuhls et al., 1996a), PCRfingerprints (Kuhls et al., 1996a, b; Turner et al., 1996) and isozyme studies (Samuels et al., 1994; Leuchtmann et al., 1996) have integrated individual Trichoderma species, not known to reproduce sexually, among Hypocrea species. Bisby (1939), unable to distinguish the Trichoderma anamorphs of H. rufa and H. gelatinosa, referred to both as l. viride. Because, in his estimation, the anamorphs were the same, he considered the green-spored H. gelatinosa to be a growth form of the white-spored H. ntfa. Bisby can, perhaps, be forgiven for confusing the anamorphs of H. rufa and H. gelatinosa because, in Bisby's time, several species of Hypocrea could have been confused with H. gelatinosa, and all of them have a Trichoderma anamorph that could have been confused with l. viride (for example H. f/avovirens, Fig. 9). In 1964 the anamorph of true H. gelatinosa was recognized to be a Trichoderma with slimy conidia (Webster, 1964) similar to l. virens. Ten years later Webster and Rifai published a series of papers detailing connections between Hypocrea species and their Trichoderma anamorphs (Webster, 1964; Rifai & Webster, 1966a, b; Webster & Rifai, 1968). In addition to H. gelatinosa, this series established the current holomorphic concept of the common species H. pulvinata and H. ntfa, and culminated in the publication of Rifai's revision of Trichoderma in 1969. The work of Y. Doi and his collaborators, which includes

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Japanese, Asian Pacific and tropical American species, is the most comprehensive study of Hypocrea available (Doi & Doi, 1979, 1986; Doi, 1966 - 1968, 1971 - 1973a, b, 1975, 1976, 1978, 1980; Doi, Doi & Toyozawa, 1984; Doi & Yamatoya, 1989). Unfortunately, Doi did not publish keys to either teleomorphs or anamorphs. Domsch et al. (1980) assigned many of the anamorphs described in Doi's work to Rifai's aggregate species, but any aggregate could be linked to more than one Hypocrea species. Doi (1972), in his revision of Japanese Hypocrea, recognized two subgenera. He further divided one of those subgenera into sections, subsections, and series based on stromal anatomy. While there was broad consistency within the subsections and series as to the type of anamorph, anamorphs in sect. Hypocrea subsect. Hypocrea, which includes the type species H. rufa, could be assigned to one of four of Rifai's aggregate species. Thus, at least in the case of one subsection, the anamorphs and teleomorphs are telling different phylogenetic stories because it is not possible to predict from most Hypocrea species the aggregate species of the Trichoderma anamorph. Hypocrea perithecia do not form in cultures of Trichoderma strains isolated from natural substrates; however, Trichoderma anamorphs occasionally develop in cultures derived from Hypocrea ascospores. One can question whether Trichoderma strains encountered in the absence of a teleomorph are parts of Hypocrea life cycles. Some of the aggregate species described by Rifai (I969) are Hypocrea species in the sense that they are based on ascospore isolations from Hypocrea species (see above). However, given the difficulty of understanding what constitutes a species of Trichoderma, it is still unclear whether Trichoderma strains encountered in nature are actually Hypocrea anamorphs. As was discussed above, T. reesei, while apparently incapable of sexual reproduction and culturally distinct from H. jecorina, behaves exactly like any strain of H. jecorina (Kuhls et ai., 1996a). However, l. reesei is known only from its original isolation and the mutants that have been derived from it (EI Gogary et al., 1990). Leuchtmann et al. (1996), Kuhls et ai. (1996 b), and Turner et al. (1996) studied the relationship between T. citrinoviride (sect. Longibrachiatum) and H. schweinitzii (Fr.) Sacc. Unlike l. reesei, l. citrinoviride is a common and cosmopolitan species. In each study, four strains of H. schweinitzii, three from eastern USA and one from France, were interspersed with strains of l. citrinoviride, some HypocrealTrichoderma pairs or clusters more closely interrelated than individual pairs or clusters were related to other individual pairs or clusters. There also remained several l. citrinoviride strains that clustered singly or alone, but not with Hypocrea strains. Unfortunately because H. schweinitzii has not been induced to reproduce sexually in vitro, no comment can be made about interfertility among the various Hypocrea and Trichoderma strains or whether the l. citrinoviride strains are sexually competent. There is little doubt, however, that H. schweinitzii is the teleomorph of T. citrinoviride. Thus, Hypocrea teleomorphs have been proven for at least two commonly occurring species of Trichoderma, viz. l. citrinoviride and T. viride. Bissett (1991 c) illustrated an anamorph for H. schweinitzii that was based on a New Zealand collection (CBS 243.63). New Zealand H. schweinitzii strains

Trichoderma: a review of biology and systematics of the genus are genetically diverse, comprising at least two populations (Kuhls et al., 1996b; Leuchtmann et al., 1996), both of which may be distinct species and different from typicaL north temperate H. schweinitzii.

How many species of Trichoderma are there 7 Just how many species are included in Trichoderma has been a continuing matter of uncertainty. The genus was originally proposed by Persoon in 1794 with four species, of which only one, T. uiride, remains. In all of Saccardo's Sylloge fungorum only 27 species of Trichoderma were included, and prior to 1980 there were fewer than fifty described species. The only key to Trichoderma prior to 1939 was that of Gilman & Abbott (1927), who recognized Pachybasium with one species, and distinguished four species of Trichoderma on the basis of colony characters, and conidial shape and pigmentation. Bisby (1939) considered that colony characters and conidial shape and ornamentation were too variable to define species, and concluded that there was only one species, T. uiride. He noted that while a small number of isolates might exhibit characteristic colonies or morphology, apparent differences were blurred on examination of many more cultures. Between 1939 and 1969 Trichoderma was basically seen to include only one species. Obviously, literature about the biology of Trichoderma from that period should be viewed carefully. Today there are about seventy-five described species of Trichoderma. Doi & Doi (1986) listed the species described before 1986. How many species are there really? The answer to this question will depend upon the operational definition of a species. If the example of T. reesei given above is representative, then there could be an endless number of morphologically distinct clonal species that are genetically very close to other morphospecies. If one assumes that the majority of Hypocrea represent 'good' species of Trichoderma, and if only half of more than 200 described species of Hypocrea are 'good' species, then there must be at least 100 biological species of Trichoderma. But this would be an underestimation as it does not account for the many Hypocrea species still to be described. The state of our knowledge of Hypocrea taxonomy is not much further advanced than it is for Trichoderma itself. An idea of the described us unknown species diversity in Hypocrea is seen in Doi's (1972) monograph of the genus for Japan. Of the fifty-two taxa (including forms) that he accepted, 31 (approximately 60%) were described as new. The majority of these had Trichoderma anamorphs; while many could be assigned at least to one of the aggregate species, it is evident that there is more morphological variation than is accounted for in Rifai's aggregates. So, while Domsch et al. (1980) correctly noted that each of Rifai's nine species aggregates could be connected with more than one teleomorph species, much of the phenotypical diversity in Trichoderma remains undescribed. THE FUTURE?

Trichoderma has been shown to be a valuable source for the commercial production of enzymes, for example, that can be

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helpful in recycling cellulosic waste materials while producing useful by-products. Trichoderma species are also a valuable tool for the biological control of plant pathogens, thus helping to reduce the need for polluting chemicals. A comprehensive taxonomic scheme is urgently required to provide rapid identifications, to distinguish deleterious strains, and to adequately and safely survey the biodiversity, and realize the commercial potentiaL of Trichoderma. Emerging evidence has shown that some of Rifai's (1969) aggregate species, such as T. longibrachiatum, are monophyletic. Other aggregates, such as T. harzianum have been proven to be perhaps even more diverse than Rifai had envisioned. There has only been limited testing of more narrowly circumscribed morphological species, but at least in sect. Longibrachiatum the species proposed by Bissett (1984) have been supported. We still do not know what a species of Trichoderma is. Development of a species concept in Trichoderma requires a combination of phenotypic and genetic information derived from repeated collection of Hypocrea and Trichoderma strains. It may be possible to use phenotypic characters in the identification of many species, but it must always be borne in mind that strains that look different, and that in more 'naIve times,' before the development of isozyme and nucleic acid analyses, might have been described as distinct taxa, are not necessarily genetically distinct. This is certainly the case with T. reesei and H. jecorina. We cannot even begin to guess at the number of so-called Trichoderma species that are nothing more than clonal lines, genetically like but phenotypically distinct from other lines. A challenge for the future will be to understand how speciation occurs in Hypocrea and Trichoderma. I am indebted to Ms Kathie Hodge for letting me see her unpublished manuscript on Cordyceps facis, and for providing the illustration of Tolypocladium inflatum. Dr S. A. Rehner allowed me access to his unpublished data on Hypocrea and Trichoderma. Drs M. E. Palm, C. T. Rogerson and A. Y. Rossman read various drafts of this work and provided helpful comments.

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(Accepted 26 February 1996)

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