- Volume 158, Issue 1, 2012

Species belonging to the genus Trichoderma are free-living fungi common in soil and root ecosystems, and have a broad range of uses in industry and agricultural biotechnology. Some species of the genus are widely used biocontrol agents, and their success is in part due to mycoparasitism, a lifestyle in which one fungus is parasitic on another. In addition Trichoderma species have been found to elicit plant defence responses and to stimulate plant growth. In order to survive and spread, Trichoderma switches from vegetative to reproductive development, and has evolved with several sophisticated molecular mechanisms to this end. Asexual development (conidiation) is induced by light and mechanical injury, although the effects of these inducers are influenced by environmental conditions, such as nutrient status and pH. A current appreciation of the links between the molecular participants is presented in this review. The photoreceptor complex BLR-1/BLR-2, ENVOY, VELVET, and NADPH oxidases have been suggested as key participants in this process. In concert with these elements, conserved signalling pathways, such as those involving heterotrimeric G proteins, mitogen-activated protein kinases (MAPKs) and cAMP-dependent protein kinase A (cAMP-PKA) are involved in this molecular orchestration. Finally, recent comparative and functional genomics analyses allow a comparison of the machinery involved in conidiophore development in model systems with that present in Trichoderma and a model to be proposed for the key factors involved in the development of these structures.

Trichoderma (teleomorph Hypocrea) is a fungal genus found in many ecosystems. Trichoderma spp. can reduce the severity of plant diseases by inhibiting plant pathogens in the soil through their highly potent antagonistic and mycoparasitic activity. Moreover, as revealed by research in recent decades, some Trichoderma strains can interact directly with roots, increasing plant growth potential, resistance to disease and tolerance to abiotic stresses. This mini-review summarizes the main findings concerning the Trichoderma–plant interaction, the molecular dialogue between the two organisms, and the dramatic changes induced by the beneficial fungus in the plant. Efforts to enhance plant resistance and tolerance to a broad range of stresses by expressing Trichoderma genes in the plant genome are also addressed.

Lysis of the prey’s cell wall is one of the key steps during mycoparasitism. Genome analysis of two mycoparasitic Trichoderma species, T. atroviride and T. virens, revealed an expanded arsenal of genes encoding enzymes potentially involved in cell wall hydrolysis. Glycoside hydrolase family 18, which contains all fungal chitinases, is the largest family of carbohydrate-active enzymes in mycoparasitic Trichoderma species. However, in addition to their aggressive functions during mycoparasitism, the roles of chitinases and other cell wall degrading enzymes also include remodelling and recycling of the fungus’s own cell wall. In this review we discuss current knowledge about fungal cell wall degrading enzymes in Trichoderma and how the fungus distinguishes between self- and non-self fungal cell wall degradation. In the past few years, the chitinolytic enzyme machinery of Trichoderma has been used as a model system to address this question. Gene expression profiles of most investigated chitinases indicate an overlap of functions of the respective enzymes and an involvement in both self- and non-self fungal cell wall degradation. Similar sets of enzymes appear to be involved in mycoparasitism, exogenous chitin decomposition and recycling of the fungus’s own cell wall. Thus, we hypothesize that the regulation of self and non-self fungal cell wall degradation is not due to a speciation of individual chitinases. Rather, we hypothesize that it is regulated by substrate accessibility due to cell wall protection in healthy hyphae vs deprotection during mycoparasitic attack, hyphal ageing and autolysis.

Trichoderma spp. are a rich source of secondary metabolites (SMs). The recent publication of the genome sequences of three Trichoderma spp. has revealed a vast repertoire of genes putatively involved in the biosynthesis of SMs, such as non-ribosomal peptides, polyketides, terpenoids and pyrones. Interestingly, the genomes of the mycoparasitic species Trichoderma virens and Trichoderma atroviride are enriched in secondary metabolism-related genes compared with the biomass-degrading Trichoderma reesei: 18 and 18 polyketide synthases compared with 11; 28 and 16 non-ribosomal peptide synthetases compared with 10, respectively. All three species produce a special class of non-ribosomally synthesized peptides known as peptaibols, containing non-proteinogenic amino acids (particularly α-aminoisobutyric acid). In common with other filamentous ascomycetes, Trichoderma spp. may require siderophores (also produced by non-ribosomal peptide synthetases) to grow in iron-poor conditions and to compete with their hosts for available iron. Two generalizations can be made about fungal SM genes: they are often found in clusters, and many are not expressed under standard laboratory conditions. This has made it difficult to identify the compounds. Trichoderma, in particular, interacts with other microbes in the soil and with plant roots in the rhizosphere. A detailed metabolomic–genomic study would eventually unravel the roles of many of these SMs in natural ecosystems. Novel genetic tools developed recently, combined with biological understanding of the function of SMs as toxins or signals, should lead to ‘awakening’ of these ‘silent’ clusters. Knowledge of the SM repertoire should precede application of Trichoderma strains for biocontrol: some metabolites could be toxic to plants and their consumers, and thus should be avoided. Others could be beneficial, antagonizing pathogens or inducing resistance in crop plants.

Trichoderma reesei (Hypocrea jecorina) is an efficient cell factory for protein production that is exploited by the enzyme industry. Yields of over 100 g secreted protein l−1 from industrial fermentations have been reported. In this review we discuss the spectrum of proteins secreted by T. reesei and the studies carried out on its protein secretion system. The major enzymes secreted by T. reesei under production conditions are those degrading plant polysaccharides, the most dominant ones being the major cellulases, as demonstrated by the 2D gel analysis of the secretome. According to genome analysis, T. reesei has fewer genes encoding enzymes involved in plant biomass degradation compared with other fungi with sequenced genomes. We also discuss other T. reesei secreted enzymes and proteins that have been studied, such as proteases, laccase, tyrosinase and hydrophobins. Investigation of the T. reesei secretion pathway has included molecular characterization of the pathway components functioning at different stages of the secretion process as well as analysis of the stress responses caused by impaired folding or trafficking in the pathway or by expression of heterologous proteins. Studies on the transcriptional regulation of the secretory pathway have revealed similarities, but also interesting differences, with other organisms, such as a different induction mechanism of the unfolded protein response and the repression of genes encoding secreted proteins under secretion stress conditions.

The hypersecreting mutant Trichoderma reesei RUT-C30 (ATCC 56765) is one of the most widely used strains of filamentous fungi for the production of cellulolytic enzymes and recombinant proteins, and for academic research. The strain was obtained after three rounds of random mutagenesis of the wild-type QM6a in a screening program focused on high cellulase production and catabolite derepression. Whereas RUT-C30 achieves outstanding levels of protein secretion and high cellulolytic activity in comparison to the wild-type QM6a, recombinant protein production has been less successful. Here, we bring together and discuss the results from biochemical-, microscopic-, genomic-, transcriptomic-, glycomic- and proteomic-based research on the RUT-C30 strain published over the last 30 years.

In this paper, we report on the in situ diversity of the mycotrophic fungus Trichoderma (teleomorph Hypocrea, Ascomycota, Dikarya) revealed by a taxon-specific metagenomic approach. We designed a set of genus-specific internal transcribed spacer (ITS)1 and ITS2 rRNA primers and constructed a clone library containing 411 molecular operational taxonomic units (MOTUs). The overall species composition in the soil of the two distinct ecosystems in the Danube floodplain consisted of 15 known species and two potentially novel taxa. The latter taxa accounted for only 1.5 % of all MOTUs, suggesting that almost no hidden or uncultivable Hypocrea/Trichoderma species are present at least in these temperate forest soils. The species were unevenly distributed in vertical soil profiles although no universal factors controlling the distribution of all of them (chemical soil properties, vegetation type and affinity to rhizosphere) were revealed. In vitro experiments simulating infrageneric interactions between the pairs of species that were detected in the same soil horizon showed a broad spectrum of reactions from very strong competition over neutral coexistence to the pronounced synergism. Our data suggest that only a relatively small portion of Hypocrea/Trichoderma species is adapted to soil as a habitat and that the interaction between these species should be considered in a screening for Hypocrea/Trichoderma as an agent(s) of biological control of pests.

Trichoderma species are ubiquitous soil fungi that hold enormous potential for the development of credible alternatives to agrochemicals and synthetic fertilizers in sustainable crop production. In this paper, we show that substantial improvements in plant productivity can be met by genetic modification of a plant-growth-promoting and biocontrol strain of Trichoderma hamatum, but that these improvements are obtained in the absence of disease pressure only. Using a quantitative monoclonal antibody-based ELISA, we show that an N-acetyl-β-d-glucosaminidase-deficient mutant of T. hamatum, generated by insertional mutagenesis of the corresponding gene, has impaired saprotrophic competitiveness during antagonistic interactions with Rhizoctonia solani in soil. Furthermore, its fitness as a biocontrol agent of the pre-emergence damping-off pathogen Sclerotinia sclerotiorum is significantly reduced, and its ability to promote plant growth is constrained by the presence of both pathogens. This work shows that while gains in T. hamatum-mediated plant-growth-promotion can be met through genetic manipulation of a single beneficial trait, such a modification has negative impacts on other aspects of its biology and ecology that contribute to its success as a saprotrophic competitor and antagonist of soil-borne pathogens. The work has important implications for fungal morphogenesis, demonstrating a clear link between hyphal architecture and secretory potential. Furthermore, it highlights the need for a holistic approach to the development of genetically modified Trichoderma strains for use as crop stimulants and biocontrol agents in plant agriculture.

Fusarium head blight (FHB) is a re-emerging wheat disease that causes extensive damage through direct losses in yield and quality due to the presence of damaged Fusarium kernels and their associated mycotoxins such as the trichothecene deoxynivalenol (DON). Biological control, including the treatment of crop residues with antagonists, in order to reduce pathogen inoculum of FHB, holds considerable promise. Ten Trichoderma isolates, previously selected for their ability to grow in the presence of DON, were preliminarily investigated as potential antagonists against Fusarium culmorum and F. graminearum mycotoxigenic strains in plate confrontation assays. The three Trichoderma isolates showing antibiosis and mycoparasitism were evaluated for their capacity to inhibit DON production by F. graminearum and F. culmorum on two natural substrates. The expression of some chitinase-encoding genes by the two best resulting Trichoderma strains, during interaction with F. culmorum and F. graminearum, was monitored. All investigated genes from chitinase subgroups A, B and the new subgroup C responded to mycoparasitic conditions and were upregulated before contact and/or when in contact with the host. T. gamsii 6085, the best antagonist, was finally used in a competition test against F. culmorum and F. graminearum on natural substrates, using a qPCR approach to evaluate its effect on the pathogen’s growth and DON production in haulms and rice. This test confirmed the ability of T. gamsii 6085 to antagonize the pathogens on rice. On wheat haulms, an extreme oligotrophic environment, T. gamsii 6085 seemed to develop very poorly and the growth of both the pathogens was unaffected by the presence of the antagonist.

Mycoparasitic Trichoderma species are applied as biocontrol agents in agriculture to guard plants against fungal diseases. During mycoparasitism, Trichoderma directly interacts with phytopathogenic fungi, preceded by a specific recognition of the host and resulting in its disarming and killing. In various fungal pathogens, including mycoparasites, signalling via heterotrimeric G proteins plays a major role in regulating pathogenicity-related functions. However, the corresponding receptors involved in the recognition of host-derived signals are largely unknown. Functional characterization of Trichoderma atroviride Gpr1 revealed a prominent role of this seven-transmembrane protein of the cAMP-receptor-like family of fungal G-protein-coupled receptors in the antagonistic interaction with the host fungus and governing of mycoparasitism-related processes. Silencing of gpr1 led to an avirulent phenotype accompanied by an inability to attach to host hyphae. Furthermore, gpr1-silenced transformants were unable to respond to the presence of living host fungi with the expression of chitinase- and protease-encoding genes. Addition of exogenous cAMP was able to restore host attachment in gpr1-silenced transformants but could not restore mycoparasitic overgrowth. A search for downstream targets of the signalling pathway(s) involving Gpr1 resulted in the isolation of genes encoding e.g. a member of the cyclin-like superfamily and a small secreted cysteine-rich protein. Although silencing of gpr1 caused defects similar to those of mutants lacking the Tga3 Gα protein, no direct interaction between Gpr1 and Tga3 was observed in a split-ubiquitin two-hybrid assay.

Trichoderma spp. are widely used as biopesticides and biofertilizers to control diseases and to promote positive physiological responses in plants. In vitro and in vivo assays with Trichoderma harzianum CECT 2413 (T34), Trichoderma virens Gv29-8 (T87) and Trichoderma hamatum IMI 224801 (T7) revealed that these strains affected the growth and development of lateral roots in tomato plants in different ways. The early expression profiles of these Trichoderma strains were studied after 20 h of incubation in the presence of tomato plants, using a high-density oligonucleotide (HDO) microarray, and compared to the profiles in the absence of plants. Out of the total 34 138 Trichoderma probe sets deposited on the microarray, 1077 (3.15 %) showed a significant change of at least 2-fold in expression in the presence of tomato plants. The numbers of probe sets identified in the individual Trichoderma strains were 593 in T. harzianum T34, 336 in T. virens T87 and 94 in T. hamatum T7. Carbohydrate metabolism – the chitin degradation enzymes N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase and chitinase – was the most significantly overrepresented process commonly observed in the three Trichoderma strains in early interactions with tomato plants. Strains T7 and T34, which had similar positive effects on plant development in biological assays, showed a significantly overrepresented hexokinase activity in interaction with tomato. In addition, genes encoding a 40S ribosomal protein and a P23 tumour protein were altered in both these strains.

The Trichoderma harzianum qid74 gene encodes a cysteine-rich cell wall protein that has an important role in adherence to hydrophobic surfaces and cellular protection; this gene was upregulated in Trichoderma high-density oligonucleotide (HDO) microarrays in interaction with tomato roots. Using a collection of qid74-overexpressing and disrupted mutants the role of this gene in cucumber and tomato root architecture was analysed in hydroponic and soil systems under greenhouse conditions. No significant differences were found in the pattern of root colonization and the length of primary roots of cucumber or tomato plants inoculated by T. harzianum CECT 2413 wild-type (wt) strain or any of the qid74 transformants. However, compared to the wt treatment, lateral roots were significantly longer in plants inoculated with the overexpressing transformants, and shorter in those treated with the disruptant strains. Microscopic observations revealed more and longer secondary root hairs in cucumber plants treated with the qid74-overexpressing mutants and fewer and shorter hairs in roots treated with qid74-disrupted transformants, compared to those observed in plants inoculated with the wt strain. qid74-induced modifications in root architecture increased the total absorptive surface, facilitating nutrient uptake and translocation of nutrients in the shoots, resulting in increased plant biomass through an efficient use of NPK and micronutrients.

In the present study we have assessed, by transcriptional and metabolic profiling, the systemic defence response of Arabidopsis thaliana plants to the leaf pathogen Pseudomonas syringae pv. tomato DC3000 (Pst) induced by the beneficial fungus Trichoderma asperelloides T203. Expression analysis (qPCR) of a set of 137 Arabidopsis genes related to Pst defence responses showed that T203 root colonization is not associated with major detectable transcriptomic changes in leaves. However, plants challenged with the bacterial pathogen showed quantitative differences in gene expression when pre-inoculated with T203, supporting priming of the plant by this beneficial fungus. Among the defence-related genes affected by T203, lipid transfer protein (LTP)4, which encodes a member of the lipid transfer pathogenesis-related family, is upregulated, whereas the WRKY40 transcription factor, known to contribute to Arabidopsis susceptibility to bacterial infection, shows reduced expression. On the other hand, root colonization by this beneficial fungus substantially alters the plant metabolic profile, including significant changes in amino acids, polyamines, sugars and citric acid cycle intermediates. This may in part reflect an increased energy supply required for the activation of plant defences and growth promotion effects mediated by Trichoderma species.

Members of the economically important ascomycete genus Trichoderma are ubiquitously distributed around the world. The mycoparasitic lifestyle and plant defence-inducing interactions of Trichoderma spp. make them ideal biocontrol agents. Of the Trichoderma enzymes that produce secondary metabolites, some of which likely play important roles in biocontrol processes, polyketide synthase (PKSs) have garnered less attention than non-ribosomal peptide synthetases such as those that produce peptaibols. We have taken a phylogenomic approach to study the PKS repertoire encoded in the genomes of Trichoderma reesei, Trichoderma atroviride and Trichoderma virens. Our analysis lays a foundation for future research related to PKSs within the genus Trichoderma and in other filamentous fungi.

Trichoderma virens genome harbours genes encoding 22 non-ribosomal peptide synthetases (NRPSs) with at least one complete module (containing adenylation, thiolation and condensation domains) and four PKS/NRPS (polyketide synthase/NRPS) hybrid enzymes. After a primary screen for expression of these 26 genes when mycelia of T. virens are in contact with maize roots, seven genes that are upregulated were selected for further study. Using homologous recombination, loss-of-function mutants in six of these were obtained (the seventh, tex2, was acquired from our previous studies). Plant assays in a hydroponics system revealed that all seven mutants retained the ability to internally colonize maize roots. However, a mutation in one of the PKS/NRPS hybrid genes impaired the ability of T. virens to induce the defence response gene pal (phenylalanine ammonia lyase), suggesting a putative role for the associated metabolite product in induced systemic resistance. Interestingly, the mutant retained its ability to induce another defence response gene aos (allene oxide synthase). We thus provide evidence that a PKS/NRPS hybrid enzyme is involved in Trichoderma–plant interactions resulting in induction of defence responses.

Antibiosis is one of the widespread strategies used by Trichoderma spp. against plant fungal pathogens, the mechanism of which, however, remains poorly understood. Peptaibols are a large family of antimicrobial peptides produced by Trichoderma spp. Our previous study showed that trichokonins, a type of peptaibol from Trichoderma pseudokoningii SMF2, exhibited antibiotic activities against plant fungal pathogens. In this study, we first demonstrated that trichokonin VI (TK VI) induced extensive apoptotic programmed cell death in plant fungal pathogens. For a deeper insight into the apoptotic mechanism involved in the action of TK VI, Fusarium oxysporum was used as a model. Cells of F. oxysporum treated with TK VI showed apoptotic hallmarks, such as exposure of phosphatidylserine, the appearance of reactive oxygen species and fragmentation of nuclear DNA. Moreover, TK VI-treated cells exhibited an accumulation of cytoplasmic vacuoles with loss of the mitochondrial transmembrane potential, and this process was independent of metacaspases. Therefore, TK VI induces metacaspase-independent apoptotic cell death in F. oxysporum. This represents what is believed to be the first report to reveal the antibiotic mechanism of peptaibols against plant fungal pathogens.

The stringent response (SR) is a regulatory mechanism that enables bacteria to adapt to nutrient stress through the production of the alarmone (p)ppGpp. The aim of the current study was to understand how the SR affects the antifungal (AF) activity of Pseudomonas chlororaphis PA23. Two SR mutants were generated, PA23relA and PA23relAspoT, that no longer produced (p)ppGpp. Both mutants exhibited increased inhibition of Sclerotinia sclerotiorum in vitro and elevated pyrrolnitrin (PRN), lipase and protease production. Phenazine (PHZ) levels, on the other hand, remained unchanged. Through transcriptional fusion analysis we discovered that prnA–lacZ (PRN) activity was increased in the SR mutants, whereas phzA–lacZ (PHZ) activity was equal to that of the wild-type. We also examined how the sigma factor RpoS impacts PA23-mediated antagonism. Similar to the SR mutants, an rpoS mutant of PA23, called PA23rpoS, exhibited enhanced AF activity in vitro and increased expression of PRN, protease and lipase. However, PHZ production and expression of phzA–lacZ were dramatically reduced. Consistent with what has been reported for other bacteria, the SR exerted positive control over rpoS expression. In addition, providing rpoS in trans restored the SR phenotype to that of the wild-type. Collectively, our findings indicate that this global stress response impacts production of PA23 AF compounds via regulation of rpoS transcription and has an overall negative influence on S. sclerotiorum antagonism.