Posted on 2004-10-25, sourse: http://pest.cabweb.org/Journals/BNI/Bni25_1/Gennews.htm
Trichoderma in Biological Control: a Taxonomist Reports
Some have estimated that fewer than 10r% of the fungi have been described. Isn't it interesting that almost all of the reports of fungi used in the biological control of diseases caused by other fungi refer to one of about three species of Trichoderma, viz. T. harzianum, T. virens and T. viride? Why Trichoderma and why just these three?
Is Trichoderma a Super Hero of comic book proportions ('Mighty T, Super Fungus'), fighting an unceasing battle against the evil parasites that would deprive us of our daily... chocolate, for example? Will fungi in other groups give effective biological control? Will other species of Trichoderma give effective control of fungus-induced plant diseases? Are all reports based on correct identifications? Are species waiting in nature to be discovered? These are questions that I have been investigating as a research scientist in the Systematic Botany and Mycology Laboratory of the United States Department of Agriculture, Agricultural Research Service (USDA-ARS).
What is a Species of Trichoderma?
In a recent brief article, Christian Kubicek and his collaborators compared DNA sequences of biological control strains identified as T. harzianum. Of the eight strains that they studied, half were reidentified as either T. atroviride or T. asperellum. The low success rate in identifying Trichoderma strains to species is not very surprising. This is a genus that presents few morphological highlights: defining and identifying species is difficult. The 'up side' of the lack of diagnostic characters is that few species have been described. Between its first description early in the 19th century and 1984 no more than nine species were included in the genus Trichoderma. In 1939 G. R. Bisby could recognize only one species, T. viride, and for much of its life as a genus, the vast majority of reports of Trichoderma (primarily in ecology) referred to only that one species. The diversity of activities attributed to T. viride in the older literature is certainly extraordinary!
In 1969 Mein Rifai reviewed Trichoderma and proposed a taxonomy with nine 'species aggregates.' He said of most of these aggregates that they most likely comprised more than one morphologically indistinguishable species. While this was an advance over Bisby's 'single species' taxonomy (which made identification very easy!), it still left the observant user unfulfilled. In order to make an identification, species boundaries had to be very elastic indeed and, given that, there was no confidence that a species name used by different researchers actually referred to the same species. Between 1984 and 1991 John Bissett again took up Trichoderma taxonomy. Critical microscopic observations led him to recognize about 35 species, which he distributed among five sections. Obviously there is a great difference between nine species on the one hand, and 35 on the other. Which taxonomy best reflected the species in nature?
In the mid 1990s DNA sequence analysis was first applied to Trichoderma taxonomy. DNA sequences provided the much-needed independently derived data that would enable a better understanding of species of Trichoderma. A series of papers from Katrin Kuhls and Christian Kubicek and their collaborators established that Bissett's view of Trichoderma was a good approximation: there are certainly more than nine species of Trichoderma, although only some of his morphology-based groupings, or sections, are monophyletic.
Today, DNA sequence analysis is absolutely essential for the description and characterization of Trichoderma species. It is possible that Trichoderma is the only genus for which every species is represented in GenBank, the international database of DNA sequences, by at least one partially sequenced gene and many species are represented by sequences of two or more genes. In truth, it turned out that even Bissett's species concepts were not finely enough drawn. Some of the species that he recognized on the basis of their morphology, such as T. viride and T. koningii, have been divided among two or more species following DNA sequence analysis and a re-evaluation of their respective phenotypes.
Trichoderma entered the modern era of taxonomy (i.e. as defined by DNA sequence analysis) with a small number of species, all of which were represented by correctly identified cultures and at least one gene of each species has been sequenced. Because of this, the likelihood of proposing new species that are the same as previously described species is very low. At least Trichoderma taxonomists will not be accused of being 'name changers' by pathologists. This contrasts to genera such as Fusarium in which hundreds of species have been placed in the genus since the early 19th century; the finding of older names to replace familiar Fusarium names in the literature has been upsetting to some, however necessary the changes. DNA sequence analysis has permitted us to uncover misidentified species: many reports in the literature are based on misidentifications (see, for example, the article mentioned above from Kubicek). While this can lead to confusion in the literature, the end result is an accurately defined species. For example, T. aureoviride is often cited in the biological control and ecology literature. However, Elke Lieckfeldt and her collaborators found that despite these many reports, the species is only found in northern Europe (UK and The Netherlands) and is only known in cultures derived from the ascomycete Hypocrea aureoviridis. In another example, T. viride has long been known as THE species of Trichoderma that has globose and warted conidia and, as such, is easily identified. Lieckfeldt and her collaborators examined cultures identified as T. viride and found a correlation between DNA sequences and the type of warts, which were more or less conspicuous depending on the strain. They separated T. asperellum from T. viride. Currently, the soil fungus T. asperellum is being evaluated in various biological control applications from head-blight of wheat in Russia, caused by a Fusarium, to black pod disease of cocoa ( Theobroma cacao) in Cameroon, caused by Phytophthora megakarya. Pierre Tondje and his collaborators in Cameroon believe that cellulase enzymes produced by the Trichoderma might be responsible for degradation of P. megakarya cell walls.
Trichoderma stromaticum, which is effective in control of witches' broom disease of cocoa caused by the mushroom Crinipellis perniciosa in South America, is the heart of the commercial biocontrol product TRICHOVAB®. This Trichoderma was reported first in the literature as T. viride and then later as T. polysporum. A combined study of morphology and DNA sequence analysis in our lab revealed it to be a new species. Today this species is being applied in areas affected by witches' broom in eastern Brazil, where it reduces inoculum of the pathogen. Indications are that it is becoming established in the area and that sexual reproduction, with possible genetic recombination, is taking place. Prakash Hebbar and Jorge T. de Souza, at USDA-Beltsville and Masterfoods, are investigating this.
The most commonly reported biocontrol Trichoderma is T. harzianum. However, this species was implicated as the cause of the green mould epidemic of commercially grown mushrooms in North America and Europe. The consequences of T. harzianum being a pathogen of such an economically important crop as mushrooms would have been disastrous to biological control. However we demonstrated here at USDA-Beltsville, through study of the morphology and cultural characters of T. harzianum and the mushroom parasite combined with use of DNA sequence analysis, that the mushroom parasite is a morphologically similar but phylogenetically distinct new species of Trichoderma, T. aggressivum. The mushroom parasite can be distinguished reliably from T. harzianum by its greatly diminished ability to grow at 35°C.
The bottom line is that one must view reports of identified species of Trichoderma with some scepticism. While Trichoderma species can be identified using the microscope and cultural characters, the most secure way for most people to identify a species of Trichoderma is through DNA sequences. Keys to the identification of most species of Trichoderma described up to the year 2000 are available in printed form and an interactive key is available at: http://pest.cabweb.org/Journals/BNI/Bni25_1/Gennews.htm
Can We Predict Biological Control Ability?
DNA sequence analysis has permitted us to 'see' the interrelationships of species through the formation of phylogenetic trees. Are phylogenetically homogeneous groups predictive of biological ability?
Christian Kubicek and collaborators found that relatives of T. reesei¸ a strain of which is the industry standard for cellulase production, tended to produce cellulase in higher concentrations than did species outside the T. reesei/ longibrachiatum group. Members of this group are also able to grow and sporulate at 40°C and have been isolated from humans who have compromised immune systems: the warning is that the use of T. longibrachiatum strains in biological control should be considered very carefully with special regard for the possibility that those who prepare or apply the fungus are at high risk of inhaling it. However, in one case a strain of T. longibrachiatum that was reported to have biocontrol ability in Costa Rica was actually T. asperellum.
One of the first volatile antifungal compounds isolated from a Trichoderma species is 6-pentyl-α -pyrone (6PAP). This non-toxic compound has the distinctive coconut odour that characterizes T. viride and its relatives ( Trichoderma sect. Trichoderma). Production of 6PAP has been attributed to T. harzianum, which is not a member of the viride group, but we have never had a culture of true T. harzianum that has the coconut odour. The only isolates in which we have noticed this odour are members of the T. viride group, in confirmation of an observation that was made in 1971 by C. Dennis and J. Webster. Reports from New Zealand that T. harzianum produces 6PAP are in fact based on T. atroviride, a member of Trichoderma sect. Trichoderma. As 6PAP can inhibit oospore formation in isolates of Phytophthora cinnamomi and inhibit conidial germination in Botrytis cinerea, an effort should be made to assay the many members of Trichoderma sect. Trichoderma for enhanced ability to produce 6PAP.
From these preliminary observations, it is possible to predict some biological activity based on phylogenetic relationships. The phylogeny of Trichoderma is being revealed at a great pace. From this phylogenetic framework it is reasonable to ask whether some kinds of biological activity, such as production of certain chitinases or the ability to direct parasitism of another fungus, are phylogenetically based. Unfortunately, there have been as yet no concerted efforts in this direction.
Where Are New Biocontrol Strains and Species Found?
Trichoderma species are found in almost all soils. They have been considered to be at least partially responsible for the control effect of 'suppressive soils', soils on which crops or trees are unaffected by a given pathogen. Control induced by suppressive composts has been attributed in part to elevated levels of some Trichoderma species. But species reported from these habitats are typically identified as one of the usual soil species, viz. T. harzianum or T. hamatum. Whether these species have been correctly identified is, of course, another question but it is 'reasonable' to expect to find these common species in such soils. Several new species of Trichoderma from eastern and southeast Asian soils have recently been described by John Bissett and his collaborators but the biocontrol potential of these new species was not assessed. The high number of new species found in the Asian soils by scientists who were looking for new species was surprising. Reports of Trichoderma species in soils come from all around the world - these can be seen by a quick search of the Internet - and one must wonder how many new (or incorrectly identified) species are found in the normal course of searching for biocontrol fungi in soils.
Another 'source' of new species of Trichoderma is the DNA-based phylogenetic analysis itself. As mentioned above, phylogenetic analysis of the morphological species T. viride revealed that it comprises two or more new species, including the biocontrol species T. asperellum. Similarly, T. koningii is representative of a common morphology in Trichoderma sect. Trichoderma that is shared by at least four species, of which three are undescribed. Were it not for DNA sequence analysis, all would have been made to fit the T. koningii morphological concept. In the light of the sequence analysis, though, T. koningii can be defined in a strict sense by the length/width ratio of its conidia and by its growth rate. One of these T. koningii morphological species is being evaluated for biological control use against frosty pod rot of cocoa, caused by Crinipellis roreri (formerly Moniliophthora roreri and Monilia roreri), in Ecuador but the same species has been found in soil in Brazil, and Germany, and from mushroom compost in Canada (Ontario). It has been isolated as an endophyte from trunks of Theobroma species in Brazil, Ecuador and Brazil and its Hypocrea sexual stage has been found in Cuba, Puerto Rico and the USA ( Kentucky).
Phenotypic and genetic diversity in Trichoderma harzianum has led some to question whether it represents a single species. Priscila Chaverri and her collaborators examined a wide range of isolates using DNA sequences of four genes and found T. harzianumi to be a species complex. While some consistent lineages had developed within the species, there were no consistent geographic, biological or phenotypic characters associated with any of the lineages. She rejected the hypothesis that T. harzianumi comprises more than one species.
A previously unexplored niche is the tissue of healthy cocoa trees where Trichoderma species are found as endophytes. CABI Bioscience scientists Harry Evans and his collaborators are using 'classical biological control' techniques in searching for biocontrol agents in the area of origin of the crop and/or pathogen. The object is to find biocontrol agents that coevolved with the pathogen but that probably did not follow it to new areas. In the case of cocoa, they have isolated endophytic fungi from asymptomatic cocoa and cocoa relatives ( Theobroma and Herrania species) in the upper Amazon region, which is where cocoa is thought to have evolved along with one of its major American pathogens Crinipellis perniciosa and in the western Andes where the second major pathogen, C. roreri, evolved on its original forest host T. gileri. Coevolved antagonists of Phytophthora megakarya have been sought in the Korup National Park of western Cameroon, where the pathogen is thought to have evolved in association with Cola, a relative of cocoa. We have also taken soil samples from the Korup forest in the hope of finding more effective strains of Trichoderma asperellum than are currently being used in Cameroon by Pierre Tondje. Keith Holmes and Harry Evans have isolated endophytes from trunks of Cola species in the Korup National Park. The Trichoderma species found as endophytes of cocoa include well-known species, such as T. harzianumi, as well as a high proportion of new species. In Ecuador, they have found an endophytic strain of T. stromaticum, the species referred to above that is used in control of witches' broom. However, the endophytic strain from Ecuador is more effective, in in - vitro studies, than the one currently used in TRICHOVAB. The finding of Trichoderma - and other soil fungi such as Clonostachys rosea (formerly Gliocladium roseum, a species often used in biological control) - living endophytically within asymptomatic cocoa trees is surprising. The substantial endophyte literature (exclusive of that on the grass endophytes) emphasizes leaves of dicotyledonous plants and not stems, and the fungal endophytes of leaves tend to be species that, outside of the leaf, are found on decaying leaves or as twig, leaf and fruit inhabitants but not soil fungi. We are in the process of describing several new Trichoderma species that are endophytic within trunks of Theobroma species. Keith Holmes from CABI Bioscience was able to reinfect cocoa seedlings with some of them and he could reisolate many from the shoot meristem of the cocoa seedlings. At least one of them, a new species, inhibited radial growth of Crinipellis roreri in vitro. It also persisted on the surface, and within the tissues, of cocoa pods in the field for at least 10 weeks.
One thinks of Trichoderma in biological control simply because most of the fungi used in biological control of fungus-induced plant diseases are species of that genus. Trichoderma species are successful in biological control because they have shown variously an ability to parasitize pathogenic fungi, or out-compete pathogenic fungi for nutrients, or produce compounds that are toxic to pathogenic, or enzymes that lyse the cell walls of pathogenic fungi. They may also enhance plant growth and vigour, enabling the host plant to successfully defend itself against attack. Many aspects of Trichoderma biology and biological control have been reviewed in a two-volume publication edited by Christian Kubicek and Gary Harman.
Recently, Betsy Arnold, Allen Herre and their collaborators studied the leaf endophytes of cocoa in Panama. Typical of endophytes of leaves of dicotyledonous plants, they have isolated many fungi in leaves of cocoa. They have found that the endophytes might be adapted to cocoa in preference to other tree species. Some of these endophytes can be reinoculated into, and reisolated from, cocoa seedlings. None of the isolates is a Trichoderma species but some, unnamed species, can protect seedlings from infection by Phytophthora.
Stem and leaf endophytes of cocoa, respectively, are represented by ecologically and phylogenetically different fungi. Nonetheless, each ecological group offers exciting prospects for biological control applications not only in cocoa but other crops. Moreover, the interactions of endophytic fungi with their host plants may offer insights into how plants defend themselves against attack by fungal pathogens.
Trichoderma species are effective in biological control of fungus-induced plant disease. A search of the Internet will show literally hundreds of examples. New species will be found as different niches are explored and also as the phylogenetic species concept comes into greater use, which is certain to happen as more people utilize DNA sequences and the incredible GenBank database. The study of endophytic fungi in stems and leaves from a biological control perspective, especially when combined with exploration in areas of diversity of hosts and their pathogens, holds the promise of finding new or more effective biocontrol agents and not just in the genus Trichoderma. The study of the interaction between host plants and their endophytes, especially at a molecular level, will certainly give new insights into the resistance of plants to diseases causing fungi. It's all very exciting at this point!
Selected Further Reading
Although this news section does not habitually include references, in this instance many of them are very recent and many readers are likely to clamour for them.
Arnold , A.E., Mejia, L.C., Kyllo, D., Rojas, E.I., Maynard, Z., Robbins, N. & Herre, E.A. (2003) Fungal endophytes limit pathogen damage in a tropical tree. Proceedings of the National Academy of Sciences, USA, 100: 15649-19654.
Bissett, J., Szakacs, G., Nolan, C. & Druzhinina, I. (2003) New species of Trichoderma from Asia. Canadian Journal of Botany 81: 570-586.
Chaverri, P., Castlebury, L.A., Samuels, G.J. & Geiser, D.M. (2003) Multilocus phylogenetic structure of Trichoderma harzianum/ Hypocrea lixii complex. Molecular Phylogenetics and Evolution 27: 302-313.
Dennis, C. & Webster, J. (1971) Antagonistic properties of species-groups of Trichoderma. II. Production of volatile antibiotics. Transactions of the British Mycological Society 57: 41-48.
Evans, H.C., Holmes, K.A. & Thomas, S.E. (2003) Endophytes and mycoparasites associated with Theobroma gileri. Mycological Progress 2: 149-160.
Gams, W. & Bissett, J. (1998) Morphology and identification of Trichoderma. In: Trichoderma and Gliocladium Vol. 1. Basic biology, taxonomy and genetics, Kubicek, C.P. & Harman, G.E. (Eds.) Taylor & Francis, London, pp. 3-25.
Harman, G.E. & Kubicek, C.P. (Eds.) (1998) Trichoderma and Gliocladium. Vol. 2. Enzymes, biological control and commercial applications Taylor & Francis, London, 393 pp.
Kindermann, J., El-Ayouti, Y., Samuels, G.J. & Kubicek, C.P. (1998) Phylogeny of the genus Trichoderma based on sequence analysis of the internal transcribed spacer region 1 of the rDNA cluster. Fungal Genetics and Biology 24: 298-309.
Kubicek, C.P. & Harman, G.E. (Eds.) (1998) Trichoderma and Gliocladium. Vol. 1. Basic biology, taxonomy and genetics Taylor & Francis, London, 278 pp.
Kuhls, K., Lieckfeldt, E., Samuels, G.J., Börner, T., Meyer, W. & Kubicek, C.P. (1997) Revision of Trichoderma sect. Longibrachiatum including related teleomorphs based on analysis of ribosomal DNA internal transcribed spacer sequences. Mycologia 89: 442-460.
Kullnig-Gradinger, C.M., Szakacs, G., Kubicek, C.P. (2002) Phylogeny and evolution of the genus Trichoderma : a multigene approach. Mycological Research 106: 757-767.
Kullnig, C.M., Krupica, T., Woo, S.L., Mach, R.L., Rey, M., Lorito, M. & Kubicek, C.P. (2001) Confusion abounds over identities of Trichoderma biocontrol isolates. Mycological Research 105: 773-782.
Lieckfeldt, E., Kullnig, M., Kubicek, C.P., Samuels, G.J. & Börner, T. (2001) Trichoderma aureoviride : phylogenetic position and characterization. Mycological Research 105: 313-322.
Lieckfeldt, E., Samuels, G.J. & Nirenberg, H.I. (1999) A morphological and molecular perspective of Trichoderma viride : is it one or two species? Applied and Environmental Biology 65: 2418-2428.
Samuels, G.J., Dodd, S.L., Gams, W., Castlebury, L.A. & Petrini, O. (2002) Trichoderma species associated with the green mold epidemic of commercially grown Agaricus bisporus. Mycologia 94:146-170.
By: Gary J. Samuels, USDA-ARS, Systematic Botany and Mycology Lab., Rm. 304, B-011A, Beltsville, MD 20705, USA