![39. Kubicek, C. P., Baker, S., Gamauf, C., Kenerley, C. M.
& Druzhinina, I. S. Purifying selection and
birth‑and‑death evolution in the class II hydrophobin
gene families of the ascomycete Trichoderma/
Hypocrea. BMC Evol. Biol. 8, 4 (2008).
40. Papapostolou, I. & Georgiou, C. D. Superoxide
radical induces sclerotial differentiation in filamentous
phytopathogenic fungi: a superoxide dismutase
mimetics study. Microbiology 156, 960–966 (2010).
41. Aliferis, K. A. & Jabaji, S. Metabolite composition and
bioactivity of Rhizoctonia solani sclerotial exudates.
J. Agric. Food Chem. 58, 7604–7615 (2010).
42. Ruocco, M. et al. Identification of a new biocontrol
gene in Trichoderma atroviride: the role of an ABC
transporter membrane pump in the interaction with
different plant-pathogenic fungi. Mol. Plant Microbe
Interact. 22, 291–301 (2009).
43. Latgé, J. P. The cell wall: a carbohydrate armour for
the fungal cell. Mol. Microbiol. 66, 279–290 (2007).
44. Seidl, V. Chitinases of filamentous fungi: a large group
of diverse proteins with multiple physiological
functions. Fungal Biol. Rev. 22, 36–42 (2008).
This article describes the diversity of chitinases
from Hypocrea/Trichoderma spp. and other
filamentous fungi.
45. Limón M. C. et al. Increased antifungal and chitinase
specific activities of Trichoderma harzianum CECT
2413 by addition of a cellulose binding domain.
Appl. Microbiol. Biotechnol. 64, 675–685 (2004).
46. Ihrmark, K. et al. Comparative molecular evolution of
Trichoderma chitinases in response to mycoparasitic
interactions. Evol. Bioinform. Online 6, 1–26 (2010).
This paper provides evidence for positive selection
of some chitinases in mycoparasitic Hypocrea/
Trichoderma spp.
47. Montero, M., Sanz, L., Rey, M., Llobell, A. & Monte, E.
Cloning and characterization of bgn16·3, coding for
a β‑1,6‑glucanase expressed during Trichoderma
harzianum mycoparasitism. J. Appl. Microbiol.
103, 1291–1300 (2007).
48. Djonovic, S., Pozo, M. J. & Kenerley, C. M. Tvbgn3,
a β‑1,6‑glucanase from the biocontrol fungus
Trichoderma virens, is involved in mycoparasitism
and control of Pythium ultimum. Appl. Environ.
Microbiol. 72, 7661–7670 (2006).
49. Casas-Flores, S. E. & Herrera-Estrella, A. in The
Mycota, Environmental and Microbial Relationships
2nd edn Vol. 4, 159–187 (Springer Berlin‑Heidelberg,
New York, 2007).
50. Bird, A. F. & Bird, J. The Structure of Nematodes.
(Academic Press, San Diego & London, 1991).
51. Sharon, E. et al. Biological control of the root-knot
nematode Meloidogyne javanica by Trichoderma
harzianum. Phytopathology 91, 687–693 (2001).
52. Suarez, B., Rey, M., Castillo, P., Monte, E. & Llobell, A.
Isolation and characterization of PRA1, a trypsin-like
protease from the biocontrol agent Trichoderma
harzianum CECT 2413 displaying nematicidal activity.
Appl. Microbiol. Biotechnol. 65, 46–55 (2004).
53. Chen, L. L. et al. Characterization and gene cloning of
a novel serine protease with nematicidal activity from
Trichoderma pseudokoningii SMF2. FEMS Microbiol.
Lett. 299, 135–142 (2009).
54. Kredics, L. et al. Clinical importance of the genus
Trichoderma. A review. Acta Microbiol. Immunol.
Hung. 50, 105–117 (2003).
55. Kratzer, C., Tobudic, S., Schmoll, M., Graninger, W. &
Georgopoulos, A. In vitro activity and synergism of
amphotericin B, azoles and cationic antimicrobials
against the emerging pathogen Trichoderma spp.
J. Antimicrob. Chemother. 58, 1058–1061 (2006).
56. Seibel, C. et al. PR4.5: Pathogenesis related gene
expression in the opportunistic fungal pathogen
Trichoderma longibrachiatum. 9th European
Conference in Fungal Genetics — Meeting Abstracts
[online], http://www.fgsc.net/ecfg9/ecfg_9_poster_
session_2.htm (2008).
57. Mulaw, T. B., Kubicek, C. P. & Druzhinina, I. S. The
rhizosphere of Coffea arabica in its native highland
forests of Ethiopia is associated with a distinguished
diversity of Trichoderma. Diversity 2, 527–549 (2010).
58. Migheli, Q. et al. Soils of a Mediterranean hot spot
of biodiversity and endemism (Sardinia, Tyrrhenian
Islands) are inhabited by pan-European, invasive
species of Hypocrea/Trichoderma. Environ. Microbiol.
11, 35–46 (2009).
59. Calvet, C., Pera, J. & Barea, J. M. Growth response
of marigold (Tagetes erecta L.) to inoculation with
Glomus mosseae, Trichoderma aureoviride and
Pythium ultimum in a peat-perlite mixture. Plant Soil
148, 1–6 (1993).
60. Datnoff, L. E., Nemec, S. & Pernezny, K. Biological
control of fusarium crown and root rot of tomato in
Florida using Trichoderma harzianum and Glomus
intraradices. Biol. Control 5, 427–431 (1995).
61. McAllister, C. B., García-Romera, I., Godeas, A. &
Ocampo, J. A. Interactions between Trichoderma
koningii, Fusarium solani and Glomus mosseae:
effects on plant growth, arbuscular mycorrhizas
and the saprophyte inoculants. Soil Biol. Biochem.
26, 1363–1367 (1994).
62. Nemec, S., Datnoff, L. E., Strandberg, J. Efficacy of
biocontrol agents in planting mixes to colonize plant
roots and control root diseases of vegetables and
citrus. Crop Prot. 15, 735–742 (1996).
63. Siddiqui, Z. A. & Mohmood, I. Biological control of
Heterodera cajani and Fusarium udum on pigeonpea
by Glomus mosseae, Trichoderma harzianum, and
Verticillium chlamydosporium. Isr. J. Plant Sci.
44, 49–56 (1996).
64. Green, H., Larsen, J., Olsson, P. A., Jensen, D. F. &
Jakobsen, I. I. Suppression of the biocontrol agent
Trichoderma harzianum by mycelium of the arbuscular
mycorrhizal fungus Glomus intraradices in root-free
soil. Appl. Environ. Microbiol. 65, 1428–1434
(1999).
65. Werner, A. & Zadworny, M. In vitro evidence of
mycoparasitism of the ectomycorrhizal fungus
Laccaria laccata against Mucor hiemalis in the
rhizosphere of Pinus sylvestris. Mycorrhiza 13,
41–47 (2003).
66. Moran-Diez, E. et al. The ThPG1
endopolygalacturonase is required for the
Trichoderma harzianum–plant beneficial interaction.
Mol. Plant Microbe Interact. 22, 1021–1031 (2009).
67. Nehls, U., Göhringer, F., Wittulsky, S. & Dietz, S.
Fungal carbohydrate support in the ectomycorrhizal
symbiosis: a review. Plant Biol. (Stuttg.) 12, 292–301
(2010).
68. Vargas, W. A., Mandawe, J. C. & Kenerley, C. M.
Plant-derived sucrose is a key element in the
symbiotic association between Trichoderma virens
and maize plants. Plant Physiol. 151, 792–808
(2009).
69. Vargas, W. A., Crutcher, F. K. & Kenerley, C. M.
Functional characterization of a plant-like sucrose
transporter from the beneficial fungus Trichoderma
virens. Regulation of the symbiotic association with
plants by sucrose metabolism inside the fungal cells.
New Phytol. 189, 777–789 (2011).
This paper shows that H. virens can use
plant-derived sucrose.
70. Jones, J. D. & Dangl, J. L. The plant immune system.
Nature 444, 323–329 (2006).
71. Shoresh, M., Harman, G. E. & Mastouri, F. Induced
systemic resistance and plant responses to fungal
biocontrol agents. Annu. Rev. Phytopathol. 48,
21–43 (2010).
This review summarizes how fungi that are used
as biocontrol agents stimulate the plant response.
72. Yedidia, I. I., Benhamou, N. & Chet, I. Induction of
defense responses in cucumber plants (Cucumis
sativus L.) by the biocontrol agent Trichoderma
harzianum. Appl. Environ. Microbiol. 65, 1061–1070
(1999).
73. Segarra, G. et al. Proteome, salicylic acid, and
jasmonic acid changes in cucumber plants inoculated
with Trichoderma asperellum strain T34. Proteomics
7, 3943–3952 (2007).
74. Dean, J. F. & Anderson, J. D. Ethylene
biosynthesis-inducing xylanase: II. purification
and physical characterization of the enzyme produced
by Trichoderma viride. Plant Physiol. 95, 316–323
(1991).
75. Hanson, L. E. & Howell, C. R. Elicitors of plant defense
responses from biocontrol strains of Trichoderma
virens. Phytopathol. 94, 171–176 (2004).
76. Enkerli, J., Felix, G. & Boller, T. The enzymatic activity
of fungal xylanase is not necessary for its elicitor
activity. Plant Physiol. 121, 391–397 (1999).
77. Sharon, A., Fuchs, Y. & Anderson, J. D. The elicitation
of ethylene biosynthesis by a Trichoderma xylanase is
not related to the cell wall degradation activity of the
enzyme. Plant Physiol. 102, 1325–1329 (1993).
78. Ron, M. & Avni, A. The receptor for the fungal elicitor
ethylene-inducing xylanase is a member of a
resistance-like gene family in tomato. Plant Cell
16, 1604–1615 (2004).
79. Bar, M. & Avni, A. EHD2 inhibits ligand-induced
endocytosis and signaling of the leucine-rich repeat
receptor-like protein LeEix2. Plant J. 59, 600–611
(2009).
80. Bailey, B. A., Korcak, R. F. & Anderson, J. D.
Alterations in Nicotiana tabacum L. cv xanthi cell
membrane function following treatment with an
ethylene biosynthesis-inducing endoxylanase. Plant
Physiol. 100, 749–755 (1992).
81. Viterbo, A., Wiest, A., Brotman, Y., Chet, I. &
Kenerley, C. M. The 18mer peptaibols from
Trichoderma virens elicit plant defence responses.
Mol. Plant Pathol. 8, 737–746 (2007).
This work demonstrates that peptaibols can induce
a plant response.
82. Saloheimo, M. et al. Swollenin, a Trichoderma reesei
protein with sequence similarity to the plant
expansins, exhibits disruption activity on cellulosic
materials. Eur. J. Biochem. 269, 4202–4211 (2002).
83. Brotman, Y., Briff, E., Viterbo, A. & Chet, I. Role of
swollenin, an expansin-like protein from Trichoderma,
in plant root colonization. Plant Physiol. 147,
779–789 (2008).
84. Guo, W. et al. Soybean β‑expansin gene GmEXPB2
intrinsically involved in root system achitecture
responses to abiotic stresses. Plant J. 66, 541–552
(2011).
85. Djonovic, S. et al. A proteinaceous elicitor Sm1 from
the beneficial fungus Trichoderma virens is required
for induced systemic resistance in maize. Plant
Physiol. 145, 875–889 (2007).
This article describes the stimulation of ISR by
a small cysteine-rich protein from H. virens.
86. Seidl, V., Marchetti, M., Schandl, R., Allmaier, G. &
Kubicek, C. P. EPL1, the major secreted protein of
Hypocrea atroviridis on glucose, is a member of a
strongly conserved protein family comprising plant
defense response elicitors. FEBS J. 273, 4346–4359
(2006).
87. Vargas, W. A., Djonovic, S., Sukno, S. A. &
Kenerley, C. M. Dimerization controls the activity
of fungal elicitors that trigger systemic resistance in
plants. J. Biol. Chem. 283, 19804–19815 (2008).
88. Martin, F. et al. The genome of Laccaria bicolor
provides insights into mycorrhizal symbiosis. Nature
452, 88–92 (2008).
89. Contreras-Cornejo, H. A., Macias-Rodriguez, L.,
Cortes-Penagos, C. & Lopez-Bucio, J. Trichoderma
virens, a plant beneficial fungus, enhances biomass
production and promotes lateral root growth through
an auxin-dependent mechanism in Arabidopsis. Plant
Physiol. 149, 1579–1592 (2009).
90. Wang, K., Li, H. & Ecker, J. Ethylene biosynthesis and
signaling networks. Plant Cell 14, S131–S151 (2002).
91. Viterbo, A., Landau, U., Kim, S., Chernin, L. & Chet, I.
Characterization of ACC deaminase from the biocontrol
and plant growth-promoting agent Trichoderma
asperellum T203. FEMS Microbiol. Lett. 305, 42–48
(2010).
92. Glick, B. R. Modulation of plant ethylene levels by the
bacterial enzyme ACC deaminase. FEMS Microbiol.
Lett. 251, 1–7 (2005).
93. Piotrowski, M. & Volmer, J. J. Cyanide metabolism
in higher plants: cyanoalanine hydratase is a NIT4
homolog. Plant Mol. Biol. 61, 111–122 (2006).
94. Bae, H. et al. The beneficial endophyte Trichoderma
hamatum isolate DIS 219b promotes growth and
delays the onset of the drought response in
Theobroma cacao. J. Exp. Bot. 60, 3279–3295
(2009).
This paper provides evidence for the beneficial
effects of T. hamatum, an endophytic species,
to the plant.
95. Chaverri, P., Gazis, R. O. & Samuels, G. J. Trichoderma
amazonicum, a new endophytic species on Hevea
brasiliensis and H. guianensis from the Amazon basin.
Mycologia 103, 139–151 (2011).
96. Samuels, G. J. et al. The Trichoderma koningii
aggregate species. Stud. Mycol. 56, 67–133 (2006).
97. Samuels, G. J. & Ismaiel, A. Trichoderma evansii and
T. lieckfeldtiae: two new T. hamatum-like species.
Mycologia 101, 142–152 (2009).
98. Rodriguez, R. J., White, J. F. Jr, Arnold, A. E. &
Redman, R. S. Fungal endophytes: diversity and
functional roles. New Phytol. 182, 314–330 (2009).
99. De Jaeger, N., Declerck, S. & de la Providencia, I. E.
Mycoparasitism of arbuscular mycorrhizal fungi:
a pathway for the entry of saprotrophic fungi into
roots. FEMS Microbiol. Ecol. 73, 312–322 (2010).
This study shows that mycoparasites can use
mycorrhizal fungi to become endophytes.
100. McNeill, J. et al. International Code of Botanical
Nomenclature (Vienna Code) Adopted By the
Seventeenth International Botanical Congress Vienna,
Austria (Gantner, Ruggell, Liechtenstein, 2005).
REVIEWS
758 | OCTOBER 2011 | VOLUME 9 www.nature.com/reviews/micro
© 2011 Macmillan Publishers Limited. All rights reserved](https://image.slidesharecdn.com/trichodermareview-211021162444/85/Trichoderma-review-10-320.jpg)
Trichoderma review
- 1. Speciesofthefilamentous-ascomycetegenus Trichoderma are among the most commonly isolated saprotrophic fungi. They are frequently found in soil and growing on wood, bark, other fungi and innumerable other sub- strates, demonstrating their high opportunistic potential and their adaptability to various ecological conditions1–4 . The nomenclature of these fungi (BOX 1) is complicated because of their pleiomorphism — that is, some of them can exist in two morphologically and physiologi- cally different stages. The sexual (teleomorphic) stage is known by the generic name Hypocrea, whereas the asexual (anamorphic or mitosporic) stage is called Trichoderma; here, we refer to the genus collectively as Hypocrea/Trichoderma. Although several common species have lost their ability to reproduce sexually and have become clonal species (or agamospecies; for example, Trichoderma longibrachiatum, Trichoderma harzianum and Trichoderma parareesei)5–8 , the majority of the genetic diversity of the genus is represented by sexual forms3,4,9,10 , and some species are isolated equally frequently as both anamorphs and teleomorphs. Most fruiting bodies of Hypocrea spp. are found associated with specific basidiomycete fungi; for exam- ple, Hypocrea estonica and Hypocrea parestonica always grow on Hymenochaete spp., Hypocrea fomiticola is found on Fomes fomentarius, and Hypocrea pulvinata grows on Fomitopsis pinicola and Piptoporus betulinus (FIG. 1). Mycoparasitic species of Hypocrea/Trichoderma can degrade and grow within the resting structures (sclerotia) that are produced by a wide variety of plant- pathogenic fungi, such as Sclerotinia spp., Typhula spp., Macrophomina phaseolina and Verticillium dahliae11 . These data support the hypothesis of Rossman et al.12 that Hypocrea spp. and some other members of the Hypocreales evolved as biotrophic associates (that is, parasites, in a broad sense of the word) of wood-rotting fungi and later on explored the wood as an optional eco- logical niche. Some species such as Hypocrea jecorina/ Trichoderma reesei13 , an important industrial producer of cellulolytic and hemicellulolytic enzymes14 , may have switched to living on the pre-degraded wood rather than the host fungus itself13 . Thus, the ability to antagonize, parasitize or even kill other fungi seems to be widespread among Hypocrea/Trichoderma spp. This property was the reason why researchers and industry started testing and using Hypocrea/Trichoderma strains for the antago- nization and eventual killing of plant pathogens15,16 (that is, for biological control, or biocontrol). Although the genus Hypocrea/Trichoderma contains many species3,4,9,10 , most research on mycoparasitism has been performed with only a few of these species, such as T. cf. harzianum sensu lato, Hypocrea atroviridis/ Trichoderma atroviride, Hypocrea virens/Trichoderma virens, Trichoderma asperellum and Trichoderma asperelloides15,16 . In the course of these studies, it was observed that Hypocrea/Trichoderma strains that are used for biocontrol can establish themselves in the plant rhizosphere, stimulate plant growth and elicit plant defence reactions against pathogens15–17 . Moreover, some Hypocrea/Trichoderma strains were isolated as endophytes (that is, as colonizers of intercellular plant compartments)18 . *Area Gene Technology and Applied Biochemistry, Institute of Chemical Engineering, Vienna University of Technology, 1060 Vienna, Austria. ‡ Laboratorio Nacional de Genómica para la Biodiversidad, Cinvestav Campus Guanajuato, 36821, Irapuato, Guanajuato, Mexico. § Department of Biology, Technion - Israel Institute of Technology, Haifa 32000, Israel. || Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas 77843, USA. ¶ Spanish-Portuguese Centre for Agricultural Research (CIALE), Department of Microbiology and Genetics, University of Salamanca, Salamanca 370007, Spain. # Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400085, India. **Department of Energy Joint Genome Institute, Walnut Creek, California 94598, USA. ‡‡ Present address: Central Institute for Cotton Research, Shankarnagar, Nagpur 440010, India. Correspondence to C.P.K. e-mail: ckubicek@mail.zserv. tuwien.ac.at doi:10.1038/nrmicro2637 Corrected online 24 October 2011 Trichoderma: the genomics of opportunistic success Irina S. Druzhinina*,Verena Seidl-Seiboth*, Alfredo Herrera-Estrella‡ , Benjamin A. Horwitz§ , Charles M. Kenerley|| , Enrique Monte¶ , Prasun K. Mukherjee#‡‡ , Susanne Zeilinger*, Igor V. Grigoriev** and Christian P. Kubicek* Abstract|Trichodermaisagenusofcommonfilamentousfungithatdisplayaremarkablerange of lifestyles and interactions with other fungi, animals and plants. Because of their ability to antagonize plant-pathogenic fungi and to stimulateplantgrowthanddefenceresponses, someTrichodermastrainsareusedforbiologicalcontrol of plant diseases. In this Review, we discuss recent advances in molecular ecology and genomicswhichindicatethatthe interactionsofTrichodermaspp.with animalsandplantsmayhave evolved as a result of saprotrophy on fungal biomass (mycotrophy) and various forms of parasitismonotherfungi (mycoparasitism),combinedwithbroadenvironmentalopportunism. REVIEWS NATURE REVIEWS | MICROBIOLOGY VOLUME 9 | OCTOBER 2011 | 749 © 2011 Macmillan Publishers Limited. All rights reserved
- 2. Nature Reviews | Microbiology a c e f d b FS T T FS FS H H H H H H FS FS FS Figure 1 | Mycotrophy of Hypocrea/Trichoderma spp. Examples of Hypocrea/ Trichoderma spp. growing on various fungal substrates (FS). Hypocrea (H) is the sexual stage (teleomorph) for the species, and in some cases the corresponding asexual stage (anamorph; Trichoderma (T)) is present. a | Hypocrea thelephoricola growing on Steccherinumochraceum.b | HypocrealixiiandaTrichodermasp.growingonaPhellinus sp. c | Hypocrea protopulvinata growing on a Fomitopsis sp. d | Hypocrea sulphurea growing on an Exidia sp. e | Hypocrea parestonica and Trichoderma parestonicum growing on a Hymenochaete sp. f | Hypocrea pulvinata growing on Piptoporus betulinus. Images courtesy of Walter M. Jaklitsch, University of Vienna, Austria. Saprotrophic Using extracellular digestion of dead or decayed organic matter as a food source. Opportunistic Able to rapidly adapt to occupy a newly arising ecological niche. Biotrophic Relying on another living organism for nutrition. This includes the broad spectrum of parasitic, mutualistic and commensalistic interactions. Parasites Organisms that take part in inter-species biotrophic interactions in which the parasites benefit at the expense of the other organisms in the interaction (the hosts). The recent sequencing of the genomes of two species that are widely used in biocontrol, H. atroviridis and H. virens13 , and the advent of associated ‘omic’ technol- ogies in Hypocrea/Trichoderma research19,20 have shed new light on the ecology of the genus and the evolu- tion of its traits. In this Review, we summarize recent insights from the genomic analyses of H. atroviridis and H. virens and emphasize that mycotrophy in a broad sense (including mycoparasitism) seems to be a wide- spread property within the genus and a key to a better understanding of the broad spectrum of opportunis- tic interactions with other organisms such as animals and plants. A mosaic of mycotrophic interactions The direct interactions between Hypocrea/Trichoderma spp. and other fungi are conventionally described as necrotrophic hyperparasitism or mycoparasitism17 . This view is supported by a recent survey of >1,100 Hypocrea/Trichoderma strains from 75 molecularly defined species, which showed that all the species tested possess mycoparasitic potential against three causative agents of plant diseases: Alternaria alternata, Botryotinia fuckeliana (anamorph Botrytis cinerea) and Sclerotinia sclerotiorum (I.S.D., unpublished observations). However, because Hypocrea/Trichoderma spp. can also feed on dead fungal biomass, the lifestyle of the genus may be better defined as mycotrophic rather than myco- parasitic, to include both biotrophic and saprotrophic nutritional strategies. Sensing the presence of the prey. Genomic sequencing of three Hypocrea/Trichoderma spp. (that is, H. jecorina, H. virens and H. atroviridis)13 and the application of tran- scriptomics19,20 have recently provided several important insights into the molecular physiology of mycotrophy. Many genes that encode proteases and oligopeptide transporters are expressed before and during contact with the prey in different Hypocrea/Trichoderma spp.20,21 . Most of these proteases belong to the subtilisin-like ser- ine protease group, and genes encoding these enzymes are significantly over-represented in expressed sequence tags (ESTs) derived from T. cf. harzianum CECT 2413 grown under biocontrol conditions21 . An abundance of genes encoding subtilisin-like serine proteases was also observed in an analysis of the ESTs that accumu- lated at the onset of contact between H. atroviridis and its fungal prey species Thanatephorus cucumeris (ana morph Rhizoctonia solani) and S. sclerotiorum20 . Strains overexpressing one of these proteases (encoded by the gene prb1) from H. atroviridis exhibited enhanced myco- parasitic activity22 . The actions of any of these proteases on the prey fungus may release oligopeptides that may then be bound by receptors on H. atroviridis which sense nitrogen starvation20 . Such a mechanism would be remi- niscent of that found in nematophagous fungi, in which trapping of the prey is induced by oligopeptides from the nematode23 (FIG. 2). It has been suggested that the class IV G protein-coupled receptors (GPCRs) that are present Box 1 | Nomenclature of the genus Hypocrea/Trichoderma According to the International Code of Botanical Nomenclature (ICBN; article 59)100 , which also applies to fungi for historical reasons, the teleomorph (sexual stage) name of a fungus should be used for species for which a complete (holomorphic) life cycle has been described. The anamorph name should be used for confirmed agamospecies (clonal species) or when no sexual stage is known. In this Review, when the whole genus of Trichoderma and Hypocrea spp. is considered, the term Hypocrea/Trichoderma is applied; both teleomorph (Hypocrea) and anamorph (Trichoderma) names are used at first mention for those species for which the complete life cycle is known, and only the teleomorph name is used thereafter. We acknowledge that the modern trend is to abolish the use of the name Hypocrea in favour of Trichoderma for the holomorph, and this practice will become valid after 1 January 2013 (according to the decision made at the International Botanical Congress in July 2011). REVIEWS 750 | OCTOBER 2011 | VOLUME 9 www.nature.com/reviews/micro © 2011 Macmillan Publishers Limited. All rights reserved
- 3. Papilla-like structure Distressed hyphae Proteases Peptides and small molecules ROS and secondary metabolites Detoxification and stress response Plant-pathogenic fungus Hypocrea/Trichoderma sp. Cell wall hydrolases and secondary metabolites G proteins Gpr1 Nitrogen-sensing receptor Gene regulation MAPK TFs Nature Reviews | Microbiology Healthy hypha Figure 2 | Mycoparasitism of Hypocrea/Trichoderma spp. within the soil community. Hypocrea/Trichoderma spp. recognize a plant-pathogenic fungus (a prey) via small molecules that are released by the pathogen; some of these molecules may be peptides that are released by the action of proteases secreted by the Hypocrea/Trichoderma sp. before contact. These molecules may bind to G protein-coupled receptors (such as Gpr1) or nitrogen-sensing receptors on the surface of the Hypocrea/Trichoderma sp. hyphae, thereby eliciting a signalling cascade comprising G proteins and mitogen-activated protein kinases (MAPKs), which may ultimately modulate the activities of as-yet-unknown transcription factors (TFs). These factors then enhance the constitutive expression of genes that encode enzymes for the biosynthesis of secondary metabolites and for cell wall lysis. Lectins from the pathogenic fungus and proteins harbouring cellulose-binding modules from hyphae of Hypocrea/Trichoderma spp. may collaborate in the attachment of the predator to the prey. At the same time, the plant-pathogenic prey responds by forming secondary metabolites and reactive oxygen species (ROS) that elicit a stress response and detoxification in Hypocrea/Trichoderma spp. Hemicellulolytic Relating to the degradation of plant hemicelluloses such as xylans and pectins. Nematophagous Pertaining to fungi: specialized in trapping and digesting nematodes. G protein-coupled receptors (Guanine-nucleotide-binding protein-coupled receptors). Receptors that possess seven transmembrane helices, bind an extracellular signalling molecule and transmit this binding by activating a Gα subunit. in H. atroviridis20 act as sensors for these oligopeptides13 . H. atroviridis, H. virens and H. jecorina each have two paralogues that are members of the class IV GPCRs13 . There may be further GPCRs involved in sensing the prey. For example, Gpr1 (protein identification number 160995 in the JGI T. atroviride v2.0 genome of the Joint Genome Institute (JGI) Genome Portal), a member of the cyclic AMP receptor-like GPCRs, is required for mycoparasitism in H. atroviridis24 . Further signal transduction from any of these receptors occurs via a conserved G protein signalling cascade (FIG. 2) that comprises three Gα subunits, one Gβ subunit and one Gγ subunit. H. atroviridis loss-of-function mutants for the Gα subunit Tga1 displayed a complete loss of myco- parasitic overgrowth on three hosts, a strong reduction of chitinase activities and decreased production of the antifungal compound 6‑pentyl pyrone25,26 . By contrast, the deletion of tgaA (a tga1 homologue) in H. virens resulted in only a somewhat reduced mycoparasitic activity on Athelia rolfsii (anamorph Sclerotium rolfsii)27 . Mitogen-activated protein kinase (MAPK) pathways represent one of the most prominent signal transduc- tion systems in fungi28 . The Hypocrea/Trichoderma spp. genomes harbour genes that encode three MAPKs: the so-called pathogenicity MAPK (TmkA; also known as Tvk1 and Tmk1), the cell integrity kinase (TmkB) and the osmoregulatory MAPK (Hog1)28 . Deletion of tmkA in a ‘P’ strain of H. virens (‘P’ strains produce gliovirin and are effective against Pythium spp.) resulted in a loss of antagonism against S. rolfsii but not R. solani29,30 . By contrast, deletion of tmkA in an H. virens ‘Q’ strain (‘Q’ strains secrete copious amounts of gliotoxin and are effective against R. solani) resulted in further improved biocontrol against both R. solani and Pythium ultimum31 . The different secondary metabolite profiles of the ‘P’ and ‘Q’ strains may explain these different results, and more complete information about the genome of ‘P’ strains will help test this hypothesis. The deletion of the tmk1 homologue in H. atroviridis resulted in reduced myco- parasitism against R. solani and increased production of chitinase and antifungal compounds32 . The roles of the other two MAPKs, TmkB and Hog1, are less well under- stood because mutants for these genes are characterized by poor growth, which precludes successful antagonism. Forexample,H. virensmutantsforTmkBweredefectivein mycoparasitism on S. rolfsii33 , and H. atroviridis mutants REVIEWS NATURE REVIEWS | MICROBIOLOGY VOLUME 9 | OCTOBER 2011 | 751 © 2011 Macmillan Publishers Limited. All rights reserved
- 4. Nature Reviews | Microbiology a b c d e f Dead hypha of Neurospora crassa N. crassa N. crassa Hypocrea atroviridis H. atroviridis H. atroviridis Figure 3 | Mycotrophy of Hypocrea atroviridis/Trichoderma atroviridae. a,b | Hypocrea atroviridis/Trichoderma atroviridae grows around, enwraps and attaches to a dead hyphal fragment of the fungus Neurospora crassa (part a is a brightfield image, and part b is a confocal image). The membrane-sensitive red dye FM4‑64 stains the membranes of intact hyphae and the complete hyphal compartment of dead hyphal fragments (because the cell wall and the plasma membrane are then permeable). The scale bars represent 20 μm. c | Attachment of H. atroviridis to an N. crassa strain that expressed cytosolic GFP, showing the formation of papilla-like structures (arrow). Membranes were stained with FM4‑64. The scale bar represents 20 μm. d | An H. atroviridis hypha grows towards and around an N. crassa hypha. Membranes were stained with FM4‑64, and an N. crassa strain expressing both cytosolic GFP and nucleus-specific histone H1–GFP was used. The scale bar represents 10 μm. e,f | Brightfield differential interference contrast images of H. atroviridis growing in coils around its own hyphae. The scale bars represent 50 μm. Images courtesy of N. D. Read (University of Edinburgh, UK), V.S.‑S. and C.P.K. Lectins Sugar-binding proteins that are highly specific for the respective sugar moiety and have a role in the recognition of cells and proteins. for Hog1 (a protein which is involved in tolerance to osmotic and oxidative stress) showed no mycoparasitic ability34 . Attachment to the prey hypha. Although mycotrophy requires only an attachment to the fungal substrate, mycoparasitism typically requires coiling around the prey mycelium and formation of helix-shaped hyphae17,18 , and this phenomenon is dependent on the recognition of lectins from the fungal prey35 (FIGS 2,3). However, plant lectins induce coiling to a similar extent, suggesting that lectins are not determinants of specificity in the attachment of Hypocrea/Trichoderma spp. to their prey species25 . Furthermore, coiling is not stringently correlated with mycoparasitism, as hyphae of some Hypocrea/Trichoderma spp. can coil around themselves in the absence of prey36 . Indeed, spiral or helical hyphal elongations are diagnostic characteristics for many spe- cies, for example Trichoderma spirale and Trichoderma helicum (see Trichoderma Online). Mycoparasitic attack by Hypocrea/Trichoderma spp. is often proceded by their growth alongside the host hyphae and by their formation of papilla-like structures (FIG. 3), events that are independent of the prey spe- cies25,37 . Degradation of the cell wall and penetration of the lumen occur at points where papilla-like structures are formed17,18,37 . These structures are similar to those induced in T. cf. harzianum by tomato (Solanum lyco- persicum)37 and analogous to the appressorium of plant- pathogenic fungi. In the rice blast fungus Magnaporthe grisea, glycerol that is generated from storage lipids serves to build up the turgor needed for the mechanical pressure that enables penetration of the plant cell wall38 . The papilla-like structures of Hypocrea/Trichoderma spp. may also build up glycerol for a similar purpose, as the transcription of genes involved in lipid catabolism and osmoregulation increases during the contact stage of mycoparasitism in H. atroviridis20 . Contact with and binding to a potential prey is not restricted to parasite hyphae: spores of H. atroviridis adhere to the hyphae of P. ultimum before germinating on them36 . The mechanism of conidial affinity for the host mycelium is unknown but could involve hydro- phobins; these are small amphiphilic proteins contain- ing eight cysteine residues, and Hypocrea/Trichoderma spp. have the highest number of these proteins among the ascomycetes (as deduced from genomic sequences)39 . Defence responses of Hypocrea/Trichoderma spp. Another event that is common to Hypocrea/Trichoderma spp. is the induction of genes for the heat shock response, the oxidative stress response and detoxification pro- cesses (such as those encoding ABC efflux transporters and the pleiotropic and multidrug resistance transport- ers) in the presence of the prey19,20 (FIG. 2). The fungal prey R. solani uses radical oxygen species as signalling molecules during sclerotia formation40 and excretes antifungal metabolites41 , and both radical oxygen spe- cies and antifungal metabolites may elicit the stress response that is observed in Hypocrea/Trichoderma spp. An H. atroviridis knockout for one of the genes encoding an ABC transporter (Abc2) resulted in decreased biocon- trol of R. solani, thus providing support for the role of detoxification in mycoparasitism42 . Killing the prey. The final death of the prey results from the synergistic actions of antifungal secondary metabolites (BOX 2) and cell wall-hydrolytic enzymes REVIEWS 752 | OCTOBER 2011 | VOLUME 9 www.nature.com/reviews/micro © 2011 Macmillan Publishers Limited. All rights reserved
- 5. Appressorium A flattened hyphal pressing structure from which an infection peg emerges that enters the host. Predation An inter-organism association in which one organism affects another adversely and itself benefits from the interaction. A predator ultimately kills its prey and consumes all or part of the prey organism. that are secreted by Hypocrea/Trichoderma spp. The importance of these molecules to the lifestyle of the mycoparasites is reflected in the Hypocrea/ Trichoderma spp. genomes, which contain a large number of genes encoding enzymes for the synthesis of these molecules13 . As an example, H. virens has the largest number (28) of nonribosomal-peptide syn- thetases known for any fungus. In addition, ortholo- gous genes that are shared between H. atroviridis and H. virens (but are not present in H. jecorina) seem to encode proteins for the synthesis of secondary metabo- lites13 and may thus represent the machinery for the synthesis of as-yet-unknown antifungal compounds. The cell wall accounts for approximately 30% of the dry weight of the fungal cell and consists mainly of chi- tin, β‑1,3‑glucans, α‑1,3‑glucans and α‑1,4‑glucans43 . Interestingly, H. atroviridis and H. virens have many chitinases (29 and 36, respectively)13,44 . Enhancing chi- tinase activity by adding a cellulose-binding module to the chitinases Chit33 and Chit42 increased the myco- parasitic ability of T. cf. harzianum45 . The addition of cellulose-binding modules enabled these chitinases to bind more tightly to insoluble chitin substrates. Some chitinases from Hypocrea/Trichoderma spp. have evolved under positive selection46 , which is typical of a co- evolutionary arms race between host and pathogen. However, the deletion of certain chitinase genes in some Hypocrea/Trichoderma spp. did not result in the loss of mycoparasitism or biocontrol abilities15,18 , probably because of gene redundancy. Hypocrea/Trichoderma spp. also contain an expanded set of chitosanases of the GH family 75; these proteins hydrolyse chitosan, a partially deacetylated form of chitin13 . The second most abundant polymer in fungal cell walls is β‑1,3‑glucan43 with β‑1,6‑branches, which is hydrolysed by β‑1,3‑glucanases; genes encoding this type of enzyme seem to be over-represented in the genomes of Hypocrea/Trichoderma spp. when com- pared with the genomes of other related fungi13 . β‑1,6‑glucanases have been detected in the area of interaction between Hypocrea/Trichoderma spp. and their prey species. Overexpression of the β‑1,6‑glucanase Bgn16.3 in T. cf. harzianum CECT 2413 resulted in a more efficient biocontrol strain for inhibition of the growth of B. cinerea, R. solani and Phytophthora citroph- thora47 . In addition, T. cf. harzianum and H. virens strains overproducing β‑1,6‑glucanases exhibited more efficient biocontrol of R. solani, B. cinerea46 and P. ultimum48 . Animals as targets of an opportunist Some of the traits that seem to have evolved in Hypocrea/ Trichoderma spp. in relation to mycotrophy may have functioned as pre-adaptations to allow parasitism or predation on animals. For example, several Hypocrea/ Trichoderma spp. successfully antagonize and kill plant- parasitic nematodes that occur in the rhizosphere49 . Commercially relevant nematode pests in agriculture, such as the root-knot nematodes (Meloidogyne spp.) and the cyst nematodes (Heterodera spp. and Globodera spp.), cannot be controlled by crop rotation owing to their broad host range49 . However, it is remarkable that differ- ent Hypocrea/Trichoderma spp. such as T. cf. harzianum can protect plants against the attack of Meloidogyne incognita by colonizing the eggs and second- stage juveniles of the nematode49 . This parasitism of nematode eggs requires penetration of the eggshell, which is formed by several layers (including a thick chi- tinous layer) that are considered to be a major barrier for infection50 . Thus, the rich arsenal of chitinases in Hypocrea/Trichoderma spp. may provide an advantage for opportunistic nematophagy. In addition, the high number of subtilisin-like S8 proteases in Hypocrea/ Trichoderma spp. may be important for penetration of the adult nematode cuticle, which is composed of colla- gen-like and keratin-like proteins (FIG. 4). Subtilisins and chemotrypsins have been cloned from several Hypocrea/ Trichoderma spp.51–53 , and the H. atroviridis alkaline sub- tilisin Prb1 and the T. cf. harzianum chemotrypsin-like protease Pra1, which both have important roles in myco- parasitism20,50 , also contribute to the ability of the fungi to penetrate nematode eggs51,52 . Some Hypocrea/Trichoderma spp. can cause invasive mycoses inmammals, including immunocompromised humans54 . Although these fungi are not a major threat to humans, they nevertheless pose therapeutic chal- lenges because of their resistance to most antifungal agents55 . This remarkable resistance may be the result of the adaptation of Hypocrea/Trichoderma spp. to combat the defence metabolites that are produced by prey fungi. To date, only two closely related species, T. longibrachia- tum and Hypocrea orientalis, have been shown to infect Box 2 | Secondary metabolites produced by Hypocrea/Trichoderma spp. Nonribosomal peptides Nonribosomal peptides are synthesized by large modular enzymes that are known as nonribosomal peptide synthetases (NRPSs). Peptaibols are 11–25 amino acid linear nonribosomal peptides that are rich in α‑aminoisobutyric acid and bear an acetylated amino terminus and a carboxy‑terminal amino alcohol101 . They are amphipathic in nature and have antibiotic properties because of their ability to self-assemble and to form voltage-dependent ion channels in membranes. They act synergistically with cell wall hydrolases to antagonize other fungi by preventing resynthesis of the cell wall, and thus potentially have a role in mycotrophy102 . Another nonribosomal peptide, gliotoxin, is produced by Hypocrea virens/Trichoderma virens ‘Q’ strains, which give very effective disease control of cotton seedling disease103,104 . However, there are contradictory reports on the role of gliotoxin in mycotrophy under controlled conditions105–107 . Polyketides Polyketides are synthesized by polyketide synthases (PKSs). There are several NRPS–PKS hybrid enzymes encoded in the genomes of Hypocrea atroviridis/ Trichoderma atroviride, Hypocrea jecorina/Trichoderma reesei and H. virens13 , but their roles remain unknown. Isoprenoid-derived metabolites H. virens produces the fungistatic and anticancer steroid viridin, which can be reduced to viridiol, a compound with herbicidal properties108 . A gene cluster that is putatively involved in viridin biosynthesis has been identified in H. virens109 . In addition, Trichoderma arundinaceum and Trichoderma brevicompactum produce the trichothecenes harzianum A and trichodermin, respectively, the latter being highly fungitoxic and phytotoxic and formed by a cascade of reactions in which trichodiene synthase (Tri5) catalyses the first step110 . Pyrones 6‑pentyl‑2H‑pyran‑2‑one (6‑PP) is a volatile component (with a coconut aroma) that has antifungal activity and is produced by H. atroviridis111 . REVIEWS NATURE REVIEWS | MICROBIOLOGY VOLUME 9 | OCTOBER 2011 | 753 © 2011 Macmillan Publishers Limited. All rights reserved
- 6. Nature Reviews | Microbiology Hypocrea/ Trichoderma sp. Detoxification Ethylene IAA Nitrilase Xylanases AAC deaminase Proteases Peptaibols Cerato- platanins and other SSCPs Hydrophobins and swollenin Growth stimulation Soil nematode Sucrose Plant root Induced systemic resistance Mucigel Hydroxyperoxide lyase, peroxidase and phenylalanine ammonia lyase Soil-borne plant- pathogenic fungus Figure 4 | Interactions of Hypocrea/Trichoderma spp. with other organisms in the rhizosphere. Hyphae of Hypocrea/Trichoderma spp. release several components that trigger systemic resistance in the plant. Only the effects that occur in the rhizosphere and are triggered by a known Hypocrea/Trichoderma spp. component are shown (for an update on further positive effects, such as resistance to abiotic plant stresses, enhancement of photosynthetic efficiency and improved nitrogen usage, see REF. 71). Peptaibols and the cerato-platanin Sm1 (also known as Epl1 in some species) induce a systemic resistance in the plants, culminating in the synthesis of plant hydroperoxide lyase, peroxidase and phenylalanine ammonia lyase (which induces lignification). Furthermore, the xylanase Eix elicits plant defence responses, probably acting as a microorganism-associated molecular pattern. The 1‑aminocyclopropane‑1‑carboxylic- acid (AAC) deaminase inhibits ethylene formation by the plant, and this leads to enhanced root growth; a constitutively secreted nitrilase might aid in the formation of the auxin 3‑indole acetic acid (IAA). Attachment of Hypocrea/Trichoderma spp. to the plant roots requires hydrophobins and swollenin. Finally, Hypocrea/Trichoderma spp. benefit from the plant roots by receiving sucrose as a carbon source, enabling faster fungal growth. The nematophagy of Hypocrea/Trichoderma spp. probably involves chitinases and subtilisin-like S8 proteases. SSCPs, small secreted cysteine-rich proteins. Mycoses Fungal infections of animals or humans. Mycorrhizal fungi A group of fungi that establish symbiotic or weakly parasitic associations with the roots of vascular plants. immunocompromised patients7 . However, it should be notedthatwhereasT. longibrachiatumisessentiallyclonal, H. orientalis forms a worldwide recombining popula- tion8 ; this may be relevant for antifungal therapy, as genes encoding factors for antibiotic resistance and virulence could be exchanged during sexual reproduction in H. ori- entalis. Clinical isolates of both species have identical hap- lotypes to common environmental strains, indicating that there is a threat of nosocomial infections, as virtually any strain of these species could cause invasive mycoses. There have been few attempts towards an under- standing of the mechanisms by which particular mem- bers of the Hypocrea/Trichoderma genus infect human cells. All the infecting species can grow at 37 °C, but not all Hypocrea/Trichoderma strains that can grow at 37 °C are opportunistic human pathogens. When T. longibra- chiatum is incubated with lung cell cultures, the human cells rapidly start to sediment and lose their adhesive properties, suggesting that proteases and/or secondary metabolites from the fungus are acting on these cells. No such effect was observed for H. jecorina, which was used as a non-pathogenic control56 . In the rhizosphere Why the rhizosphere? Therhizosphereisamongthe com- mon ecological niches for Hypocrea/Trichoderma spp. and provides opportunities for both biotrophy and sapro trophy on plant root exudates. This is illustrated by the fact that the highest species richness of this fungal genus in a single habitat has been found in the rhizosphere of the coffee plant Coffea arabica in Ethiopian highland for- ests57 ; by contrast, a similar survey in non-rhizosphere soil on Sardinia (Italy) showed remarkably poor diver- sity58 . The affinity of Hypocrea/Trichoderma spp. for the rhizosphere can be explained by two of their nutritional preferences. First, the roots of 92% of land plants are colonized by mycorrhizal fungi that are potential prey for a mycotroph. However, the interactions between mycor rhizal fungi and Hypocrea/Trichoderma spp. are still poorly understood59–64 : whereas some studies sug- gest a synergism between the two types of fungi, others have observed that Hypocrea/Trichoderma spp. attack arbuscular mycorrhizal fungi and suppress their colo- nization of plant roots. Moreover, a reduction in the population density of Hypocrea/Trichoderma spp. due to arbuscular mycorrhizal fungi has been noted64 . Few studies have focused on the interaction of Hypocrea/ Trichoderma spp. with ectomycorrhizal fungi65 . Second, the plant roots, and especially the root tips, are covered by a gel-like slimy capsule (called the mucigel) com- posed of highly hydrated polysaccharides such as pectins and hemicelluloses (particularly rhamnogalacturo- nans and arabinoxylans) that are secreted from the outermost cells of the root cap. These components are easily degradable targets for the hemicellulases of Hypocrea/Trichoderma spp., which may have evolved for the utilization of polysaccharides that are released from pre-degraded wood by potential fungal prey. Indeed, successful establishment of T. cf. harzianum CECT 2413 in the tomato rhizosphere requires an endopolygalacturonase66 . Monosaccharides and disaccharides excreted by plant roots into the rhizosphere provide an important carbon substrate for mycorrhizae67 , and sucrose has a similar role for the establishment of H. virens in the rhizo sphere68 . As the genomes of H. atroviridis, H. virens and H. jeco- rina contain genes encoding intracellular (but not extra cellular) invertases, sucrose must be taken up by a sucrose permease before being hydrolysed. H. virens contains a highly specific sucrose transporter that is induced in the early stages of root colonization and has biochemi- cal properties that are similar to plant sucrose carriers69 , suggesting that sucrose is actively transfered from plant to fungus. In addition, the genomes of H. atroviridis and H. virens encode a large number of major facilitator REVIEWS 754 | OCTOBER 2011 | VOLUME 9 www.nature.com/reviews/micro © 2011 Macmillan Publishers Limited. All rights reserved
- 7. Induced systemic resistance A process by which plants respond to a non-pathogenic microorganism, with a signalling cascade that is dependent on jasmonate and ethylene. This response leads to a long-lasting ability to mount a faster and stronger broad-spectrum defence when challenged by a pathogen. Both pathogen-associated molecular pattern-triggered immunity and effector-triggered immunity can lead to induced systemic resistance. Ethylene A gaseous, unsaturated hydrocarbon that acts as a plant hormone to promote growth and development and as an inhibiting stress factor. Callose A β‑1,3‑linked polysaccharide of the plant cell wall; this polysaccharide is formed in response to wounding (including infections by pathogens). Systemic acquired resistance A plant defence mechanism that is usually induced by exposure to a pathogen and confers long-lasting protection against a broad spectrum of microorganisms. It involves the production of the signal molecule salicylic acid, which then leads to the accumulation of pathogenesis-related proteins that are thought to contribute to resistance. solute transporters13 , the roles for which in the acquisi- tion of other root exudates remain unknown. In sum- mary, the presence of fungal prey and the availability of root-derived nutrients may have been major attractors for the ancestors of Hypocrea/Trichoderma spp. to establish themselves in the rhizosphere and to develop interactions with plant roots. Dialogue with the plant. Like fungi and animals, plants respond to the presence of other organisms by activat- ing potential defence mechanisms. This is best under- stood for various plant-pathogenic species that elicit a two-branched innate immune defence70 . The first stage generally recognizes and responds to pathogen- associated molecular patterns (PAMPs) or micro- organism-associated molecular patterns (MAMPs) — molecules that are commonly found in microorgan- isms — and is known as PAMP-triggered immunity (PTI), whereas the second stage responds to viru- lence factors from the pathogen and is called effector- triggered immunity (ETI). As do other microorganisms that are not plant pathogens, Hypocrea/Trichoderma spp. trigger induced systemic resistance (ISR) (FIG. 4), which culminates in the accumulation of components of the associated jasmonate and ethylene signalling pathways, such as hydroperoxide lyase, peroxidase and phenylalanine ammonia lyase (which induces lig- nification)71 . For example, the action of fungal endo pectinases on the mucigel releases oligogalacturonides that activate plant defence mechanisms66 . As a result of recognizing MAMPs and/or molecules released during the initial stages of the interaction, the plant deposits more callose and cellulose in its cell walls and releases phenolic compounds, both of which actions prevent fur- ther colonization, as observed during the early stages of root colonization by T. asperelloides on Cucumis sativus (cucumber)72,73 . As Hypocrea/Trichoderma spp. are not plant pathogens, they are not expected to elicit the second stage of the plant innate immune response. However, systemic acquired resistance (SAR), normally associated with the second stage of the plant immune response, is induced by T. asperellum in cucumber plants in a concentration-dependent manner and may occur in the early stages of the fungal interactions with roots73 . It must be noted that these effects have been studied in only a few Hypocrea/Trichoderma species and strains that are particularly effective in the stimulation of plant defences, namely T. asperelloides T203 (formerly classified as T. asperellum T203), H. virens and the protoplast fusion hybrid T. “harzianum” T‑22. Several classes of molecules from Hypocrea/ Trichoderma spp., such as xylanases, peptaibols, swol- lenin and cerato-platanins, act as MAMPs (FIG. 4). The endoxylanase Eix (also known as Xyn2) from ‘T. viride’ ATCC 52438 was the first Hypocrea/Trichoderma spp. protein known to elicit ethylene formation in tobacco (Nicotiana tabacum) and tomato74 . Unfortunately, the species identity of this strain has never been re-assessed by molecular methods and thus must be considered as uncertain. An effective biocontrol strain of H. virens secretes an endoxylanase that is identical to Eix75 , and genes encoding homologous enzymes are found in the genomes of H. jecorina (protein identifier 123818 in the JGI T. reesei v2.0 genome) and H. virens (protein identi- fier 72838 in the JGI T. virens Gv29‑8 v2.0 genome). Remarkably, the catalytic activity of Eix is not required for eliciting the plant defence responses76,77 ; thus, the enzyme itself, and not its reaction product, must be acting as a MAMP. In fact, to elicit the plant response, Eix binds to plant Eix receptor 2 (Eix2; also known as LeEix), a member of a superfamily of plant leucine-rich repeat receptor-like proteins that also carry a signal for receptor-mediated endocytosis, which is essential for the proper induction of defence responses78,79 . In addition, binding of Eix to the plant receptors causes alterations in membrane function that are required for eliciting the plant defence response80 . Blocking the synthesis of peptaibols (a group of non- ribosomal peptides; see BOX 2) in H. virens by disrupting the gene encoding the peptaibol synthase Tex1 results in strains that do not induce ISR in cucumber, although this can be overcome by the addition of peptaibol mixtures81 . The mechanism by which peptaibols induce ISR is not known but may be related to the ability of these peptides to alter membrane function, as described for Eix (REF. 80). Swollenin is a protein that carries a cellulose-binding module and can disrupt the crystalline cellulose struc- ture of plant cell walls82 . It contributes to root coloniza- tion in T. asperellum and induces local defence responses but not ISR83 . Swollenin has sequence similarity to expansins, which are plant proteins that facilitate expan- sion of the plant cell wall in roots and root hairs84 , and Hypocrea/Trichoderma spp. may take advantage of a swollenin-induced increase in root surface area when establishing themselves in the plant rhizosphere. Cerato-platanins are small secreted proteins that are characterized by four cysteines which form two disul- phide bonds. The H. virens cerato-platanin Sm1 (also known as Epl1) induces ISR in maize (Zea mays) and cot- ton (Gossypium hirsutum)85 , and the orthologue of Sm1 in H. atroviridis (Epl1)is one of the major proteins that is constitutively secreted by the fungus86 . Glycosylation of Sm1 maintains the protein in a monomeric form, which elicits ISR87 . Deglycosylation leads to the forma- tion of an Sm1 dimer, which does not elicit ISR. It has been suggested that the plant may alter the aggregation state of Sm1 by deglycosylation and ultimately affect its ability to induce defence responses. H. jecorina, H. virens and H. atroviridis have three paralogues of sm1 each, whereas most other fungi from related genera have only one, suggesting that cerato-platanins may be important for Hypocrea/Trichoderma spp. Other small secreted cysteine-rich proteins are encoded in the Hypocrea/ Trichoderma spp. genomes13 and may have a role in root colonization, similar to that described for the small secreted proteins of the ectomycorrhizal basidiomycete Laccaria bicolor, which accumulate in the hyphae that colonize the plant root88 . Promotion of plant growth. At least in some cases, the association of Hypocrea/Trichoderma spp. with roots can promote plant growth (FIG. 4). For example, H. virens REVIEWS NATURE REVIEWS | MICROBIOLOGY VOLUME 9 | OCTOBER 2011 | 755 © 2011 Macmillan Publishers Limited. All rights reserved
- 8. increases the root system biomass and the lateral-root growth rate of Arabidopsis thaliana. Auxin-mediated response pathways may have a role in mediating these effects, as plant mutants with defects in these pathways show reduced effects of Hypocrea/Trichoderma spp. associations89 . However, the promotion of plant growth may also be mediated by a decrease in the levels of the plant hormone ethylene90 . T. asperelloides T203 possesses an α‑1‑aminocyclopropane‑1‑carboxylate (ACC) deaminase gene (acc1) that encodes an enzyme which cleaves ACC, a key intermediate in ethylene bio- synthesis, andis expressed during the interaction with roots of Brassica napus (oilseed rape)91 . A knockout for this gene reduced the ability of the fungus to promote root elongation. Because a sustained high level of ethyl- ene inhibits root elongation, the Acc1 enzyme provides a mechanism for facilitating the formation of longer roots. Similar enzymes have been described in bacteria that promote plant growth92 . In addition, the Hypocrea/Trichoderma spp. genomes contain many genes that encode nitrilases, as compared with other ascomycetes13 . These nitrilases may have a role either in hydrolysing β‑cyano‑l-alanine, a metabolite whichisformedfromcyanidereleasedduringthefinalstep ofethylenebiosynthesis,orinconvertingtheplantmetabo lite indole‑3‑acetonitrile to indole‑3‑acetic acid (IAA), a hormone that promotes the growth of plant roots93 . Endophytism Endophytic biotrophy (that is, symptomless growth inside plant tissue) is common among bacteria and fungi. These microorganisms offer a wide range of benefits to the host, including stimulation of plant growth, a delay totheonsetofdroughtstressandthepreventionofattacks by pathogens94 . Only a few Hypocrea/Trichoderma spp. have been isolated as endophytes (TABLE 1), although it is likely that many other species can behave as faculta- tive endophytes. Almost all the isolated endophytes have been classified as new taxa and — with the excep- tion of Hypocrea koningiopsis/Trichoderma koningiopsis, Hypocrea stilbohypoxyli/Trichoderma stilbohypoxyli and Hypocrea stromatica/Trichoderma stromaticum — have no known teleomorphs. A phylogenetic analysis places them in a terminal position within their clades, suggesting that the development of endophytism in the genus Hypocrea/Trichoderma was evolutionarily recent95–97 . Some species, such as Trichoderma hamatum, are detected both as endophytes and as common inhabit- ants of the soil and the rhizosphere, and such a character- istic is known for many other opportunistic fungal genera aswell98 .Itisthereforeunclearwhetheranyobligate endo- phytic Hypocrea/Trichoderma spp. exist. Interestingly, the mycelium of arbuscular mycorrhizae on the outer side of the colonized roots of potato (Solanum tuberosum) can be used by Hypocrea/Trichoderma spp. to enter into the plant roots99 , which suggests that traits related to myco trophy facilitate the evolution of endophytism. No genomes from Hypocrea/Trichoderma strains that were isolated as endophytes have yet been sequenced. Conclusions The recent advent of genomic and transcriptomic data, combined with insights into the molecular ecology and population genetics of Hypocrea/Trichoderma spp., has provided a wealth of information that allows a deeper understanding of this important fungal genus. Until recently, it was commonly thought that most Trichoderma spp. were asexual soil fungi. This was in part due to difficulties in mating these organisms under labora tory conditions. However, the application of population genetics has now shown that many of these Trichoderma spp. that were previously believed to be asexual in fact display an evolutionary history of sexual recombination, and only four have been proved to be clonal (strictly asexual)5–8 . These conclusions are also reflected in the results from diversity surveys on the genus Hypocrea/Trichoderma, which showed that, out of ~150 species characterized by genetic markers, the main fraction comprises holomorphic species that grow on decaying wood or on basidiomycetes3,4 . Mycotrophy is thus widespread within the genus. Comparativeanalysisofthegenomesfrom H. jecorina, H. virens and H. atroviridis further expanded this finding toconcludethatmycotrophyisinfactaveryancienttraitof the genus: a phylogenetic analysis of 100 orthologues and syntenicproteinsfromthethreespecies(rootedagainstthe related genera Chaetomium and Gibberella) and a genus phylogeny based on the nucleotide sequence of the gene encoding the RNA polymerase β-subunit revealed that H. atroviridis has an ancestral position within the genus13 . However,thegeneinventoryofH. atroviridis includessev- eral amplified gene families with roles in competition and antagonism13 , thus indicating a genetic predisposition of the species for mycotrophy. All these data suggest that mycotrophy is a basic prop- erty of the genus Hypocrea/Trichoderma and still the Table 1 | Endophytic Hypocrea/Trichoderma spp. Species Putatively obligate? Host plant Location Trichoderma amazonicum95 Yes Hevea spp. Peru Trichoderma caribbaeum var. aequatoriale96 Yes Theobroma spp. Tropical America Trichoderma evansii97 Yes Lophira alata Cameroun Cola verticillata Cameroun Theobroma gileri Peru Trichoderma hamatum94 No Theobroma cacao Ecuador Hypocrea koningiopsis/ Trichoderma koningiopsis96 No Theobroma spp. Not available Trichoderma martiale112 Yes T. cacao Brazil Trichoderma ovalisporum96 Yes Banisteriopsis caarpi Ecuador Trichoderma paucisporum113 Yes T. cacao Ecuador Trichoderma scalesiae114 Yes Scalesia pedunculata Galapagos Islands Hypocrea stilbohypoxyli/ Trichoderma stilbohypoxyli96 No Fagus spp. United Kingdom Trichoderma taxi115 Yes Taxus mairei China Trichodermatheobromicola113 Yes T. cacao Peru REVIEWS 756 | OCTOBER 2011 | VOLUME 9 www.nature.com/reviews/micro © 2011 Macmillan Publishers Limited. All rights reserved
- 9. major lifestyle for many of its species. However, several species seem to have evolved further towards new eco- logical niches or to have developed special traits, probably facilitated by the presence of genes that enable effective competition and opportunism13 . For example, T. longi- brachiatum can colonize immunocompromised humans, H. jecorina specializes on colonizing dead wood, and other species have so far been isolated only as endophytes; all these species occupy terminal positions in the phylo- genetic trees6,7,95–97 , which suggests that they are the most recent taxa of the genus. The presence of potential fungal prey and plant root- derived nutrients in the rhizosphere may have attracted Hypocrea/Trichoderma spp. ancestors towards coloniz- ing plant roots. Moreover, mycotrophy-related traits (such as the presence of certain proteases, chitinases and secondary metabolites) may have facilitated the evolu- tion of further positive interactions between Hypocrea/ Trichoderma spp. and plants. We should note, however, that no known components or mechanisms deployed by Hypocrea/Trichoderma strains seem to have evolved spe- cifically for this process, as most of these components are involved in other cellular functions (such as nutrition or competition) or have orthologues in other fungi that have not been described as taking part in communication with the plant13 . Further studies of the interactions between plants, the rhizosphere, mycorrhizae and Hypocrea/ Trichoderma spp. are needed for a better understanding of these processes. Finally, large-scale genome-sequencing projects of additional Hypocrea/Trichoderma spp. (such as T. harzi- anum, T. asperellum and T. longibrachiatum) are currently being undertaken by the US Department of Energy’s JGI Fungal Genetics Program and will enable a more comprehensive molecular-level analysis of the ecological diversity of the genus. This will not only help us to under- stand the molecular basis of the opportunistic nature and environmental successes of Hypocrea/Trichoderma spp. but also improve the use of these fungi in biotechnology, agriculture and other areas. 1. Klein, D. & Eveleigh, D. E. In: Trichoderma and Gliocladium Vol.1 (eds Kubicek, C. P. & Harman, G. E.), 57–69 (Taylor and Francis, London, 1998). 2. Brotman, Y., Kapuganti, J. G. & Viterbo, A. Trichoderma. Curr. Biol. 20, R390–R391 (2010). 3. Jaklitsch, W. M. European species of Hypocrea Part I. The green-spored species. Stud. Mycol. 63, 1–91 (2009). 4. Jaklitsch, W. M. European species of Hypocrea Part II: species with hyaline ascospores. Fungal Divers. 48, 1–247 (2011). Together with reference 3, this provides an excellent survey of the diversity and ecology of Hypocrea/Trichoderma spp. 5. Druzhinina, I. S., Kubicek, C. P., Komoń-Zelazowska, M., Mulaw, T. B. & Bissett, J. The Trichoderma harzianum demon: complex speciation history resulting in coexistence of hypothetical biological species, recent agamospecies and numerous relict lineages. BMC Evol. Biol. 10, 94 (2010). This paper highlights the diversity and evolution of the T. cf. harzianum species complex. 6. Druzhinina, I. S., Komoń-Zelazowska, M., Atanasova, L., Seidl, V. & Kubicek, C. P. Evolution and ecophysiology of the industrial producer Hypocrea jecorina (anamorph Trichoderma reesei) and a new sympatric agamospecies related to it. PLoS ONE 5, e9191 (2010). 7. Druzhinina, I. S. et al. Alternative reproductive strategies of Hypocrea orientalis and genetically close but clonal Trichoderma longibrachiatum, both capable of causing invasive mycoses of humans. Microbiology 154, 3447–3459 (2008). This work shows that T. longibrachiatum and H. orientalis are two different species with different reproduction strategies, and that both species can cause invasive mycoses. 8. Samuels, G. J., Ismaiel, A., Bon, M. C., De Respinis, S. & Petrini, O. Trichoderma asperellum sensu lato consists of two cryptic species. Mycologia 102, 944–966 (2010). 9. Kubicek, C. P., Komon-Zelazowska, M. & Druzhinina, I. S. Fungal genus Hypocrea/Trichoderma: from barcodes to biodiversity. J. Zhejiang Univ. Sci. B 9, 753–763 (2008). 10. Druzhinina, I. S., Kopchinskiy, A. & Kubicek, C. P. The first 100 Trichoderma species characterized by molecular data. Mycoscience 47, 55–64 (2006). 11. Elad, Y., Barak, R. & Chet, I. Parasitism of sclerotia of Sclerotium rolfsii by Trichoderma harzianum. Soil Biol. Biochem. 16, 381–386 (1984). 12. Rossmann, A. Y., Samuels, G. J., Rogerson, C. T. & Lowen, R. Genera of Bionectriaceae, Hypocreaceae and Nectriaceae (Hyprocrealses, Ascomycetes). Stud. Mycol. 42, 1–83 (1999). 13. Kubicek, C. P. et al. Comparative genome sequence analysis underscores mycoparasitism as the ancestral life style of Trichoderma. Genome Biol. 12, R40 (2011). A comparative analysis of the genomes of H. jecorina, H. virens and H. atroviridis, highlighting the gene repertoire that is related to mycoparasitism. 14. Martinez, D. et al. Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina). Nature Biotech. 26, 553–560 (2008). 15. Benítez, T., Rincón, A. M., Limón, M. C. & Codón, A. C. Biocontrol mechanisms of Trichoderma strains. Int. Microbiol. 7, 249–260 (2004). 16. Howell, C. R. Mechanisms employed by Trichoderma species in the biological control of plant diseases: the history and evolution of current concepts. Plant Dis. 87, 4–10 (2003). 17. Harman, G. E. Multifunctional fungal plant symbionts: new tools to enhance plant growth and productivity. New Phytol. 189, 647–649 (2011). 18. Harman, G. E., Howell, C. R., Viterbo, A., Chet, I. & Lorito, M. Trichoderma species—opportunistic, avirulent plant symbionts. Nature Rev. Microbiol. 2, 43–56 (2004). 19. Lorito, M., Woo, S. L., Harman, G. E. & Monte, E. Translational research on Trichoderma: from ‘omics to the field. Annu. Rev. Phytopathol. 48, 395–417 (2010). 20. Seidl, V. et al. Transcriptomic response of the mycoparasitic fungus Trichoderma atroviride to the presence of a fungal prey. BMC Genomics 10, 567 (2009). This study identifies genes that are upregulated in H. atroviridis during its antagonistic interaction with plant-pathogenic fungi. 21. Suárez, M. B., Vizcaíno, J. A., Llobell, A. & Monte, E. Characterization of genes encoding novel peptidases in the biocontrol fungus Trichoderma harzianum CECT 2413 using the TrichoEST functional genomics approach. Curr. Genet. 51, 331–342 (2007). 22. Flores, A., Chet, I. & Herrera-Estrella, A. Improved biocontrol activity of Trichoderma harzianum by over-expression of the proteinase-encoding gene prb1. Curr. Genet. 31, 30–37 (1997). 23. Dijksterhuis, J., Veenhuis, M., Harder, W. & Nordbring-Hertz, B. Nematophagous fungi: physiological aspects and structure–function relationships. Adv. Microb. Physiol. 36, 111–143 (1994). 24. Omann, M. et al. A cAMP receptor-like GPCR is involved in Trichoderma atroviride mycoparasitism. IOBC/WPRS Bull. 43, 105–108 (2009). 25. Rocha-Ramirez, V. et al. Trichoderma atroviride G.‑protein α‑subunit gene tga1 is involved in mycoparasitic coiling and conidiation. Eukaryot. Cell 1, 594–605 (2002). 26. Reithner, B. et al. The G protein α subunit Tga1 of Trichoderma atroviride is involved in chitinase formation and differential production of antifungal metabolites. Fungal Genet. Biol. 42, 749–760 (2004). 27. Mukherjee, P. K., Latha, J., Hadar, R. & Horwitz, B. A. Role of two G‑protein alpha subunits, TgaA and TgaB, in the antagonism of plant pathogens by Trichoderma virens. Appl. Environ. Microbiol. 70, 542–549 (2004). 28. Schmoll, M. The information highways of a biotechnological workhorse—signal transduction in Hypocrea jecorina. BMC Genomics 9, 430 (2008). 29. Mukherjee, P., Latha, J., Hadar, R. & Horwitz, B. A. TmkA, a mitogen-activated protein kinase of Trichoderma virens, is involved in biocontrol properties and repression of conidiation in the dark. Eukaryot. Cell 2, 446–455 (2003). 30. Viterbo, A., Harel, M., Horwitz, B. A., Chet, I. & Mukherjee, P. K. Trichoderma mitogen-activated protein kinase signaling is involved in induction of plant systemic resistance. Appl. Environ. Microbiol. 71, 6241–6246 (2005). 31. Mendoza-Mendoza, A. et al. The MAP kinase TVK1 regulates conidiation, hydrophobicity and the expression of genes encoding cell wall proteins in the fungus Trichoderma virens. Microbiology 153, 2137–2147 (2007). 32. Reithner, B. et al. Signaling via the Trichoderma atroviride mitogen-activated protein kinase Tmk1 differentially affects mycoparasitism and plant protection. Fungal Genet. Biol. 44, 1123–1133 (2007). 33. Kumar, A. et al. Overlapping and distinct functions of two Trichoderma virens MAP kinases in cell-wall integrity, antagonistic properties and repression of conidiation. Biochem. Biophys. Res. Commun. 398, 765–770 (2010). 34. Delgado-Jarana, J., Sousa, S., González, F., Rey, M. & Llobell, A. ThHog1 controls the hyperosmotic response in Trichoderma harzianum. Microbiology 162, 1687–1700 (2006). 35. Inbar, J. & Chet, I. The role of lectins in recognition and adhesion of the mycoparasitic fungus Trichoderma spp. to its host. Adv. Exp. Med. Biol. 408, 229–231 (1996). 36. Lu, Z. et al. In vivo study of Trichoderma‑pathogen‑plant interactions, using constitutive and inducible green fluorescent protein reporter systems. Appl. Environ. Microbiol. 70, 3073–3081 (2004). This investigation monitors the antagonism of H. atroviridis against plant-pathogenic fungi directly in soil. 37. Chacón, M. R. et al. Microscopic and transcriptome analyses of early colonization of tomato roots by Trichoderma harzianum. Int. Microbiol. 10, 19–27 (2007). 38. de Jong, J. C., McCormack, B. J., Smirnoff, N. & Talbot, N. J. Glycerol generates turgor in rice blast. Nature 389, 244–245 (1997). REVIEWS NATURE REVIEWS | MICROBIOLOGY VOLUME 9 | OCTOBER 2011 | 757 © 2011 Macmillan Publishers Limited. All rights reserved
- 10. 39. Kubicek, C. P., Baker, S., Gamauf, C., Kenerley, C. M. & Druzhinina, I. S. Purifying selection and birth‑and‑death evolution in the class II hydrophobin gene families of the ascomycete Trichoderma/ Hypocrea. BMC Evol. Biol. 8, 4 (2008). 40. Papapostolou, I. & Georgiou, C. D. Superoxide radical induces sclerotial differentiation in filamentous phytopathogenic fungi: a superoxide dismutase mimetics study. Microbiology 156, 960–966 (2010). 41. Aliferis, K. A. & Jabaji, S. Metabolite composition and bioactivity of Rhizoctonia solani sclerotial exudates. J. Agric. Food Chem. 58, 7604–7615 (2010). 42. Ruocco, M. et al. Identification of a new biocontrol gene in Trichoderma atroviride: the role of an ABC transporter membrane pump in the interaction with different plant-pathogenic fungi. Mol. Plant Microbe Interact. 22, 291–301 (2009). 43. Latgé, J. P. The cell wall: a carbohydrate armour for the fungal cell. Mol. Microbiol. 66, 279–290 (2007). 44. Seidl, V. Chitinases of filamentous fungi: a large group of diverse proteins with multiple physiological functions. Fungal Biol. Rev. 22, 36–42 (2008). This article describes the diversity of chitinases from Hypocrea/Trichoderma spp. and other filamentous fungi. 45. Limón M. C. et al. Increased antifungal and chitinase specific activities of Trichoderma harzianum CECT 2413 by addition of a cellulose binding domain. Appl. Microbiol. Biotechnol. 64, 675–685 (2004). 46. Ihrmark, K. et al. Comparative molecular evolution of Trichoderma chitinases in response to mycoparasitic interactions. Evol. Bioinform. Online 6, 1–26 (2010). This paper provides evidence for positive selection of some chitinases in mycoparasitic Hypocrea/ Trichoderma spp. 47. Montero, M., Sanz, L., Rey, M., Llobell, A. & Monte, E. Cloning and characterization of bgn16·3, coding for a β‑1,6‑glucanase expressed during Trichoderma harzianum mycoparasitism. J. Appl. Microbiol. 103, 1291–1300 (2007). 48. Djonovic, S., Pozo, M. J. & Kenerley, C. M. Tvbgn3, a β‑1,6‑glucanase from the biocontrol fungus Trichoderma virens, is involved in mycoparasitism and control of Pythium ultimum. Appl. Environ. Microbiol. 72, 7661–7670 (2006). 49. Casas-Flores, S. E. & Herrera-Estrella, A. in The Mycota, Environmental and Microbial Relationships 2nd edn Vol. 4, 159–187 (Springer Berlin‑Heidelberg, New York, 2007). 50. Bird, A. F. & Bird, J. The Structure of Nematodes. (Academic Press, San Diego & London, 1991). 51. Sharon, E. et al. Biological control of the root-knot nematode Meloidogyne javanica by Trichoderma harzianum. Phytopathology 91, 687–693 (2001). 52. Suarez, B., Rey, M., Castillo, P., Monte, E. & Llobell, A. Isolation and characterization of PRA1, a trypsin-like protease from the biocontrol agent Trichoderma harzianum CECT 2413 displaying nematicidal activity. Appl. Microbiol. Biotechnol. 65, 46–55 (2004). 53. Chen, L. L. et al. Characterization and gene cloning of a novel serine protease with nematicidal activity from Trichoderma pseudokoningii SMF2. FEMS Microbiol. Lett. 299, 135–142 (2009). 54. Kredics, L. et al. Clinical importance of the genus Trichoderma. A review. Acta Microbiol. Immunol. Hung. 50, 105–117 (2003). 55. Kratzer, C., Tobudic, S., Schmoll, M., Graninger, W. & Georgopoulos, A. In vitro activity and synergism of amphotericin B, azoles and cationic antimicrobials against the emerging pathogen Trichoderma spp. J. Antimicrob. Chemother. 58, 1058–1061 (2006). 56. Seibel, C. et al. PR4.5: Pathogenesis related gene expression in the opportunistic fungal pathogen Trichoderma longibrachiatum. 9th European Conference in Fungal Genetics — Meeting Abstracts [online], http://www.fgsc.net/ecfg9/ecfg_9_poster_ session_2.htm (2008). 57. Mulaw, T. B., Kubicek, C. P. & Druzhinina, I. S. The rhizosphere of Coffea arabica in its native highland forests of Ethiopia is associated with a distinguished diversity of Trichoderma. Diversity 2, 527–549 (2010). 58. Migheli, Q. et al. Soils of a Mediterranean hot spot of biodiversity and endemism (Sardinia, Tyrrhenian Islands) are inhabited by pan-European, invasive species of Hypocrea/Trichoderma. Environ. Microbiol. 11, 35–46 (2009). 59. Calvet, C., Pera, J. & Barea, J. M. Growth response of marigold (Tagetes erecta L.) to inoculation with Glomus mosseae, Trichoderma aureoviride and Pythium ultimum in a peat-perlite mixture. Plant Soil 148, 1–6 (1993). 60. Datnoff, L. E., Nemec, S. & Pernezny, K. Biological control of fusarium crown and root rot of tomato in Florida using Trichoderma harzianum and Glomus intraradices. Biol. Control 5, 427–431 (1995). 61. McAllister, C. B., García-Romera, I., Godeas, A. & Ocampo, J. A. Interactions between Trichoderma koningii, Fusarium solani and Glomus mosseae: effects on plant growth, arbuscular mycorrhizas and the saprophyte inoculants. Soil Biol. Biochem. 26, 1363–1367 (1994). 62. Nemec, S., Datnoff, L. E., Strandberg, J. Efficacy of biocontrol agents in planting mixes to colonize plant roots and control root diseases of vegetables and citrus. Crop Prot. 15, 735–742 (1996). 63. Siddiqui, Z. A. & Mohmood, I. Biological control of Heterodera cajani and Fusarium udum on pigeonpea by Glomus mosseae, Trichoderma harzianum, and Verticillium chlamydosporium. Isr. J. Plant Sci. 44, 49–56 (1996). 64. Green, H., Larsen, J., Olsson, P. A., Jensen, D. F. & Jakobsen, I. I. Suppression of the biocontrol agent Trichoderma harzianum by mycelium of the arbuscular mycorrhizal fungus Glomus intraradices in root-free soil. Appl. Environ. Microbiol. 65, 1428–1434 (1999). 65. Werner, A. & Zadworny, M. In vitro evidence of mycoparasitism of the ectomycorrhizal fungus Laccaria laccata against Mucor hiemalis in the rhizosphere of Pinus sylvestris. Mycorrhiza 13, 41–47 (2003). 66. Moran-Diez, E. et al. The ThPG1 endopolygalacturonase is required for the Trichoderma harzianum–plant beneficial interaction. Mol. Plant Microbe Interact. 22, 1021–1031 (2009). 67. Nehls, U., Göhringer, F., Wittulsky, S. & Dietz, S. Fungal carbohydrate support in the ectomycorrhizal symbiosis: a review. Plant Biol. (Stuttg.) 12, 292–301 (2010). 68. Vargas, W. A., Mandawe, J. C. & Kenerley, C. M. Plant-derived sucrose is a key element in the symbiotic association between Trichoderma virens and maize plants. Plant Physiol. 151, 792–808 (2009). 69. Vargas, W. A., Crutcher, F. K. & Kenerley, C. M. Functional characterization of a plant-like sucrose transporter from the beneficial fungus Trichoderma virens. Regulation of the symbiotic association with plants by sucrose metabolism inside the fungal cells. New Phytol. 189, 777–789 (2011). This paper shows that H. virens can use plant-derived sucrose. 70. Jones, J. D. & Dangl, J. L. The plant immune system. Nature 444, 323–329 (2006). 71. Shoresh, M., Harman, G. E. & Mastouri, F. Induced systemic resistance and plant responses to fungal biocontrol agents. Annu. Rev. Phytopathol. 48, 21–43 (2010). This review summarizes how fungi that are used as biocontrol agents stimulate the plant response. 72. Yedidia, I. I., Benhamou, N. & Chet, I. Induction of defense responses in cucumber plants (Cucumis sativus L.) by the biocontrol agent Trichoderma harzianum. Appl. Environ. Microbiol. 65, 1061–1070 (1999). 73. Segarra, G. et al. Proteome, salicylic acid, and jasmonic acid changes in cucumber plants inoculated with Trichoderma asperellum strain T34. Proteomics 7, 3943–3952 (2007). 74. Dean, J. F. & Anderson, J. D. Ethylene biosynthesis-inducing xylanase: II. purification and physical characterization of the enzyme produced by Trichoderma viride. Plant Physiol. 95, 316–323 (1991). 75. Hanson, L. E. & Howell, C. R. Elicitors of plant defense responses from biocontrol strains of Trichoderma virens. Phytopathol. 94, 171–176 (2004). 76. Enkerli, J., Felix, G. & Boller, T. The enzymatic activity of fungal xylanase is not necessary for its elicitor activity. Plant Physiol. 121, 391–397 (1999). 77. Sharon, A., Fuchs, Y. & Anderson, J. D. The elicitation of ethylene biosynthesis by a Trichoderma xylanase is not related to the cell wall degradation activity of the enzyme. Plant Physiol. 102, 1325–1329 (1993). 78. Ron, M. & Avni, A. The receptor for the fungal elicitor ethylene-inducing xylanase is a member of a resistance-like gene family in tomato. Plant Cell 16, 1604–1615 (2004). 79. Bar, M. & Avni, A. EHD2 inhibits ligand-induced endocytosis and signaling of the leucine-rich repeat receptor-like protein LeEix2. Plant J. 59, 600–611 (2009). 80. Bailey, B. A., Korcak, R. F. & Anderson, J. D. Alterations in Nicotiana tabacum L. cv xanthi cell membrane function following treatment with an ethylene biosynthesis-inducing endoxylanase. Plant Physiol. 100, 749–755 (1992). 81. Viterbo, A., Wiest, A., Brotman, Y., Chet, I. & Kenerley, C. M. The 18mer peptaibols from Trichoderma virens elicit plant defence responses. Mol. Plant Pathol. 8, 737–746 (2007). This work demonstrates that peptaibols can induce a plant response. 82. Saloheimo, M. et al. Swollenin, a Trichoderma reesei protein with sequence similarity to the plant expansins, exhibits disruption activity on cellulosic materials. Eur. J. Biochem. 269, 4202–4211 (2002). 83. Brotman, Y., Briff, E., Viterbo, A. & Chet, I. Role of swollenin, an expansin-like protein from Trichoderma, in plant root colonization. Plant Physiol. 147, 779–789 (2008). 84. Guo, W. et al. Soybean β‑expansin gene GmEXPB2 intrinsically involved in root system achitecture responses to abiotic stresses. Plant J. 66, 541–552 (2011). 85. Djonovic, S. et al. A proteinaceous elicitor Sm1 from the beneficial fungus Trichoderma virens is required for induced systemic resistance in maize. Plant Physiol. 145, 875–889 (2007). This article describes the stimulation of ISR by a small cysteine-rich protein from H. virens. 86. Seidl, V., Marchetti, M., Schandl, R., Allmaier, G. & Kubicek, C. P. EPL1, the major secreted protein of Hypocrea atroviridis on glucose, is a member of a strongly conserved protein family comprising plant defense response elicitors. FEBS J. 273, 4346–4359 (2006). 87. Vargas, W. A., Djonovic, S., Sukno, S. A. & Kenerley, C. M. Dimerization controls the activity of fungal elicitors that trigger systemic resistance in plants. J. Biol. Chem. 283, 19804–19815 (2008). 88. Martin, F. et al. The genome of Laccaria bicolor provides insights into mycorrhizal symbiosis. Nature 452, 88–92 (2008). 89. Contreras-Cornejo, H. A., Macias-Rodriguez, L., Cortes-Penagos, C. & Lopez-Bucio, J. Trichoderma virens, a plant beneficial fungus, enhances biomass production and promotes lateral root growth through an auxin-dependent mechanism in Arabidopsis. Plant Physiol. 149, 1579–1592 (2009). 90. Wang, K., Li, H. & Ecker, J. Ethylene biosynthesis and signaling networks. Plant Cell 14, S131–S151 (2002). 91. Viterbo, A., Landau, U., Kim, S., Chernin, L. & Chet, I. Characterization of ACC deaminase from the biocontrol and plant growth-promoting agent Trichoderma asperellum T203. FEMS Microbiol. Lett. 305, 42–48 (2010). 92. Glick, B. R. Modulation of plant ethylene levels by the bacterial enzyme ACC deaminase. FEMS Microbiol. Lett. 251, 1–7 (2005). 93. Piotrowski, M. & Volmer, J. J. Cyanide metabolism in higher plants: cyanoalanine hydratase is a NIT4 homolog. Plant Mol. Biol. 61, 111–122 (2006). 94. Bae, H. et al. The beneficial endophyte Trichoderma hamatum isolate DIS 219b promotes growth and delays the onset of the drought response in Theobroma cacao. J. Exp. Bot. 60, 3279–3295 (2009). This paper provides evidence for the beneficial effects of T. hamatum, an endophytic species, to the plant. 95. Chaverri, P., Gazis, R. O. & Samuels, G. J. Trichoderma amazonicum, a new endophytic species on Hevea brasiliensis and H. guianensis from the Amazon basin. Mycologia 103, 139–151 (2011). 96. Samuels, G. J. et al. The Trichoderma koningii aggregate species. Stud. Mycol. 56, 67–133 (2006). 97. Samuels, G. J. & Ismaiel, A. Trichoderma evansii and T. lieckfeldtiae: two new T. hamatum-like species. Mycologia 101, 142–152 (2009). 98. Rodriguez, R. J., White, J. F. Jr, Arnold, A. E. & Redman, R. S. Fungal endophytes: diversity and functional roles. New Phytol. 182, 314–330 (2009). 99. De Jaeger, N., Declerck, S. & de la Providencia, I. E. Mycoparasitism of arbuscular mycorrhizal fungi: a pathway for the entry of saprotrophic fungi into roots. FEMS Microbiol. Ecol. 73, 312–322 (2010). This study shows that mycoparasites can use mycorrhizal fungi to become endophytes. 100. McNeill, J. et al. International Code of Botanical Nomenclature (Vienna Code) Adopted By the Seventeenth International Botanical Congress Vienna, Austria (Gantner, Ruggell, Liechtenstein, 2005). REVIEWS 758 | OCTOBER 2011 | VOLUME 9 www.nature.com/reviews/micro © 2011 Macmillan Publishers Limited. All rights reserved
- 11. 101. Kubicek, C. P., Komoń-Zelazowska, M., Sándor, E. & Druzhinina, I. S. Facts and challenges in the understanding of the biosynthesis of peptaibols by Trichoderma. Chem. Biodivers. 4, 1068–1082 (2007). 102. Lorito, M., Farkas, V., Rebuffat, S., Bodo, B. & Kubicek, C. P. Cell wall synthesis is a major target of mycoparasitic antagonism by Trichoderma harzianum. J. Bacteriol. 178, 6382–6385 (1996). 103. Howell, C. R. Understanding the mechanisms employed by Trichoderma virens to effect biological control of cotton diseases. Phytopathology 96, 178–180 (2006). 104. Lumsden, R. D., Locke, J. C., Adkins, S. T., Walter, J. F. & Ridout, C. J. Isolation and localization of the antibiotic gliotoxin produced by Gliocladium virens from alginate prill in soil and soilless media. Phytopathology 82, 230–235 (1992). 105. Howell, C. R., Stipanovic, R. & Lumsden, R. Antibiotic production by strains of Gliocladium virens and its relation to biocontrol of cotton seedling diseases. Biocontrol. Sci. Technol. 3, 435–441 (1993). 106. Howell, C. R. & Stipanovic, R. D. Mechanisms in the biocontrol of Rhizoctonia solani-induced cotton seedling disease by Gliocladium virens: antibiosis. Phytopathology 85, 469–472 (1995). 107. Howell, C. R. & Puckhaber, L. S. A study of the characteristics of “P” and “Q” strains of Trichoderma virens to account for differences in biological control efficacy against cotton seedling diseases. Biol. Control 33, 217–222 (2005). 108. Jones, R. W. & Hancock, J. G. Conversion of viridin to viridiol by viridin-producing fungi. Can. J. Microbiol. 33, 963–966 (1987). 109. Mukherjee, M., Horwitz, B. A., Sherkhane, P. D., Hadar, R. & Mukherjee, P. K. A secondary metabolite biosynthesis cluster in Trichoderma virens: evidence from analysis of genes underexpressed in a mutant defective in morphogenesis and antibiotic production. Curr. Genet. 50, 193–202 (2006). 110. Tijerino, A. et al. Overexpression of the trichodiene synthase gene tri5 increases trichodermin production and antimicrobial activity in Trichoderma brevicompactum. Fungal Genet. Biol. 48, 285–296 (2011). 111. Serrano-Carreon, L., Hathout, Y., Bensoussan, M. & Belin, J. M. Metabolism of linoleic acid or mevalonate and 6‑pentyl‑α-pyrone biosynthesis by Trichoderma species. Appl. Environ. Microbiol. 59, 2945–2950 (1993). 112. Hanada, R. E. et al. Trichoderma martiale sp. nov., a new endophyte from sapwood of Theobroma cacao with a potential for biological control. Mycol. Res. 112, 1335–1343 (2008). 113. Samuels, G. J. et al. Trichoderma theobromicola and T. paucisporum: two new species isolated from cacao in South America. Mycol. Res. 110, 381–392 (2006). 114. Jaklitsch, W. M., Samuels, G. J., Dodd, S. L., Lu, B. S. & Druzhinina, I. S. Hypocrea rufa/Trichoderma viride: a reassessment, and description of five closely related species with and without warted conidia. Stud. Mycol. 56, 135–177 (2006). 115. Zhang, C. L., Liu, S. P., Lin, F. C., Kubicek, C. P. & Druzhinina, I. S. Trichoderma taxi sp. nov., an endophytic fungus from Chinese yew Taxus mairei. FEMS Microbiol. Lett. 270, 90–96 (2007). Acknowledgments Work in the C.P.K., I.S.D., V.S.-S. and S.Z. laboratory (headed by C.P.K.) was supported by grants from the Austrian Science Foundation (P17895-B06, P20559, T390 and P-19340) and the Vienna Science and Technology Fund (WWTF LS09-036). The work of B.A.H., C.M.K. and P.K.M. was supported in part by grant TB-8031-08 from the Texas Department of Agriculture, USA, and the US–Israel Binational Agricultural Research and Development Fund. Competing interests statement The authors declare no competing financial interests. FURTHER INFORMATION Christian P. Kubicek’s homepage: http://www.vt.tuwien.ac.at/molbio/ International Subcommission on Trichoderma and Hypocrea Taxonomy (ICTF): http://www.isth.info/ JGI Fungal Genetics Program: http://genome.jgi-psf.org/programs/fungi/index.jsf JGI Genome Portal: http://genome.jgi-psf.org/ JGI genome T. atroviride v2.0: http://genome.jgi-psf.org/Triat2/Triat2.home.html JGI genome T. reesei v2.0: http://genome.jgi-psf.org/Trire2/Trire2.home.html JGI genome T. virens Gv29‑8 v2.0: http://genome.jgi-psf.org/ TriviGv29_8_2/TriviGv29_8_2.home.html Trichoderma Online: http://nt.ars-grin.gov/taxadescriptions/ keys/TrichodermaIndex.cfm ALL LINKS ARE ACTIVE IN THE ONLINE PDF REVIEWS NATURE REVIEWS | MICROBIOLOGY VOLUME 9 | OCTOBER 2011 | 759 © 2011 Macmillan Publishers Limited. All rights reserved