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BackgroundThe growing importance of the ubiquitous fungal genus Trichoderma (Hypocreales, Ascomycota) requires understanding of its biology and evolution. Many Trichoderma species are used as biofertilizers and biofungicides and T. reesei is the model organism for industrial production of cellulolytic enzymes. In addition, some highly opportunistic species devastate mushroom farms and can become pathogens of humans. A comparative analysis of the first three whole genomes revealed mycoparasitism as the innate feature of Trichoderma. However, the evolution of these traits is not yet understood.ResultsWe selected 12 most commonly occurring Trichoderma species and studied the evolution of their genome sequences. Trichoderma evolved in the time of the Cretaceous-Palaeogene extinction event 66 (±15) mya, but the formation of extant sections (Longibrachiatum, Trichoderma) or clades (Harzianum/Virens) happened in Oligocene. The evolution of the Harzianum clade and section Trichoderma was accompanied by significant gene gain, but the ancestor of section Longibrachiatum experienced rapid gene loss. The highest number of genes gained encoded ankyrins, HET domain proteins and transcription factors. We also identified the Trichoderma core genome, completely curated its annotation, investigated several gene families in detail and compared the results to those of other fungi. Eighty percent of those genes for which a function could be predicted were also found in other fungi, but only 67% of those without a predictable function.ConclusionsOur study presents a time scaled pattern of genome evolution in 12 Trichoderma species from three phylogenetically distant clades/sections and a comprehensive analysis of their genes. The data offer insights in the evolution of a mycoparasite towards a generalist.

When resources are limited, the hypocrealean fungus Trichoderma guizhouense can overgrow another hypocrealean fungus Fusarium oxysporum, cause sporadic cell death and arrest growth. A transcriptomic analysis of this interaction shows that T. guizhouense undergoes a succession of metabolic stresses while F. oxysporum responded relatively neutrally but used the constitutive expression of several toxin-encoding genes as a protective strategy. Because of these toxins, T. guizhouense cannot approach it is potential host on the substrate surface and attacks F. oxysporum from above. The success of T. guizhouense is secured by the excessive production of hydrogen peroxide (H2 O2 ), which is stored in microscopic bag-like guttation droplets hanging on the contacting hyphae. The deletion of NADPH oxidase nox1 and its regulator, nor1 in T. guizhouense led to a substantial decrease in H2 O2 formation with concomitant loss of antagonistic activity. We envision the role of NOX proteins in the antagonism of T. guizhouense as an example of metabolic exaptation evolved in this fungus because the primary function of these ancient proteins was probably not linked to interfungal relationships. In support of this, F. oxysporum showed almost no transcriptional response to T. guizhouense Δnox1 strain indicating the role of NOX/H2 O2 in signalling and fungal communication.

BackgroundCytochrome P450s form an important group of enzymes involved in xenobiotics degradation and metabolism, both primary and secondary. These enzymes are also useful in industry as biotechnological tools for bioconversion and a few are reported to be involved in pathogenicity. Trichoderma spp. are widely used in industry and agriculture and are known for their biosynthetic potential of a large number of secondary metabolites. For realising the full biosynthetic potential of an organism, it is important to do a genome-wide annotation and cataloguing of these enzymes.ResultsHere, we have studied the genomes of seven species (T. asperellum, T. atroviride, T. citrinoviride, T. longibrachiatum, T. reesei , T. harzianum and T. virens) and identified a total of 477 cytochrome P450s. We present here the classification, evolution and structure as well as predicted function of these proteins. This study would pave the way for functional characterization of these groups of enzymes and will also help in realization of their full economic potential.ConclusionOur CYPome annotation and evolutionary studies of the seven Trichoderma species now provides opportunities for exploration of research-driven strategies to select Trichoderma species for various applications especially in relation to secondary metabolism and degradation of environmental pollutants.

Unlike most other fungi, molds of the genus Trichoderma (Hypocreales, Ascomycota) are aggressive parasites of other fungi and efficient decomposers of plant biomass. Although nutritional shifts are common among hypocrealean fungi, there are no examples of such broad substrate versatility as that observed in Trichoderma. A phylogenomic analysis of 23 hypocrealean fungi (including nine Trichoderma spp. and the related Escovopsis weberi) revealed that the genus Trichoderma has evolved from an ancestor with limited cellulolytic capability that fed on either fungi or arthropods. The evolutionary analysis of Trichoderma genes encoding plant cell wall-degrading carbohydrate-active enzymes and auxiliary proteins (pcwdCAZome, 122 gene families) based on a gene tree / species tree reconciliation demonstrated that the formation of the genus was accompanied by an unprecedented extent of lateral gene transfer (LGT). Nearly one-half of the genes in Trichoderma pcwdCAZome (41%) were obtained via LGT from plant-associated filamentous fungi belonging to different classes of Ascomycota, while no LGT was observed from other potential donors. In addition to the ability to feed on unrelated fungi (such as Basidiomycota), we also showed that Trichoderma is capable of endoparasitism on a broad range of Ascomycota, including extant LGT donors. This phenomenon was not observed in E. weberi and rarely in other mycoparasitic hypocrealean fungi. Thus, our study suggests that LGT is linked to the ability of Trichoderma to parasitize taxonomically related fungi (up to adelphoparasitism in strict sense). This may have allowed primarily mycotrophic Trichoderma fungi to evolve into decomposers of plant biomass.

Biological control is a complex process which requires many mechanisms and a high diversity of biochemical pathways. The species of Trichoderma harzianum are well known for their biocontrol activity against many plant pathogens. To gain new insights into the biocontrol mechanism used by T. harzianum, we sequenced the isolate TR274 genome using Illumina. The assembly was performed using AllPaths-LG with a maximum coverage of 100x. The assembly resulted in 2282 contigs with a N50 of 37033bp. The genome size generated was 40.8 Mb and the GC content was 47.7%, similar to other Trichoderma genomes. Using the JGI Annotation Pipeline we predicted 13,932 genes with a high transcriptome support. CEGMA tests suggested 100% genome completeness and 97.9% of RNA-SEQ reads were mapped to the genome. The phylogenetic comparison using orthologous proteins with all Trichoderma genomes sequenced at JGI, corroborates the Trichoderma (T. asperellum and T. atroviride), Longibrachiatum (T. reesei and T. longibrachiatum) and Pachibasium (T. harzianum and T. virens) section division described previously. The comparison between two Trichoderma harzianum species suggests a high genome similarity but some strain-specific expansions. Analyses of the secondary metabolites, CAZymes, transporters, proteases, transcription factors were performed. The Pachybasium section expanded virtually all categories analyzed compared with the other sections, specially Longibrachiatum section, that shows a clear contraction. These results suggests that these proteins families have an important role in their respective phenotypes. Future analysis will improve the understanding of this complex genus and give some insights about its lifestyle and the interactions with the environment.

Many microorganisms with specialized lifestyles have reduced genomes. This is best understood in beneficial bacterial symbioses, where partner fidelity facilitates loss of genes necessary for living independently. Specialized microbial pathogens may also exhibit gene loss relative to generalists. Here, we demonstrate that Escovopsis weberi, a fungal parasite of the crops of fungus-growing ants, has a reduced genome in terms of both size and gene content relative to closely related but less specialized fungi. Although primary metabolism genes have been retained, the E. weberi genome is depleted in carbohydrate active enzymes, which is consistent with reliance on a host with these functions. E. weberi has also lost genes considered necessary for sexual reproduction. Contrasting these losses, the genome encodes unique secondary metabolite biosynthesis clusters, some of which include genes that exhibit up-regulated expression during host attack. Thus, the specialized nature of the interaction between Escovopsis and ant agriculture is reflected in the parasite's genome.

Trichoderma is a genus of common filamentous fungi that display a remarkable range of lifestyles and interactions with other fungi, animals and plants. Because of their ability to antagonize plant-pathogenic fungi and to stimulate plant growth and defence responses, some Trichoderma strains are used for biological control of plant diseases. In this Review, we discuss recent advances in molecular ecology and genomics which indicate that the interactions of Trichoderma spp. with animals and plants may have evolved as a result of saprotrophy on fungal biomass (mycotrophy) and various forms of parasitism on other fungi (mycoparasitism), combined with broad environmental opportunism.

The common soil fungus Trichoderma (teleomorph Hypocrea, Ascomycota) shows increasing medical importance as an opportunistic human pathogen, particularly in immunocompromised and immunosuppressed patients. Regardless of the disease type and the therapy used, the prognosis for Trichoderma infection is usually poor. Trichoderma longibrachiatum has been identified as the causal agent in the majority of reported Trichoderma mycoses. As T. Iongibrachiatum is very common in environmental samples from all over the world, the relationship between its clinical and wild strains remains unclear. Here we performed a multilocus (ITS1 and 2, tef1, cal1 and chit18-5) phylogenetic analysis of all available clinical isolates (15) and 36 wild-type strains of the fungus including several cultures of its putative teleomorph Hypocrea orientalis. The concordance of gene genealogies recognized T. longibrachiatum and H. orientalis to be different phylogenetic species, which are reproductively isolated from each other. The majority of clinical strains (12) were attributed to T. longibrachiatum but three isolates belonged to H. orientalis, which broadens the phylogenetic span of human opportunists in the genus. Despite their genetic isolation, T. longibrachiatum and H. orientalis were shown to be cosmopolitan sympatric species with no bias towards certain geographical locations. The analysis of haplotype association, incongruence of tree topologies and the split decomposition method supported the conclusion that H. orientalis is sexually recombining whereas strict clonality prevails in T. longibrachiatum. This is a rare case of occurrence of sexual reproduction in opportunistic pathogenic fungi. The discovery of the different reproduction strategies in these two closely related species is medically relevant because it is likely that they would also differ in virulence and/or drug resistance. Genetic identity of environmental and clinical isolates of T. longibrachiatum and H. orientalis suggests the danger of nosocomial infections by Hypocrea/Trichoderma and highlights the need for ecological studies of spore dispersal as source of invasive human mycoses.

Light is a fundamental abiotic factor which stimulates growth and development of the majority of living organisms. In soil saprotrophic fungi, light is primarily known to influence morphogenesis, particularly sexual and asexual spore formation. Here we present a new function of light, the enhancement of mycelial growth. The photostimulated mycelial growth of the soil fungus Hypocrea atroviridis was detected on 17 (out of 95 tested carbon sources) carbohydrates and polyols, which are metabolically related to cellulose and hemicelluloses, and which are mainly available in the upper soil litter layer. This stimulation depends differently on the function of the two blue light receptor proteins BLR-1 and BLR-2, respectively, BLR-1 being responsible for carbon source selectivity and response to permanent light. Evocation of oxidative stress response in darkness imitates the photostimulation on nine of these carbon sources, and this effect was fully dependent on the function of BLR-1. We conclude that light in combination with the availability of litter-specific carbon sources serves as a signal for the fungus to be above ground, thereby stimulating fast growth in order to produce a maximum of propagules in the shortest time. We further deduce that this process involves oxidative stress response and the two blue light receptor proteins BLR-1 and BLR-2, the former playing the major role.

Peptaibols are characteristic linear [alpha]-aminoisobutyrate-containing peptides produced by certain Ascomycetes, especially of the genus Hypocrea/Trichoderma [Hypocrea and Trichoderma are the names for the teleo- and anamorph forms of the same taxon; where known to occur in nature, the teleomorph is used to name the species. To aid the inexperienced reader, both names (the less well known one in parentheses) are given at the first mention of each species.] Here we have investigated whether phylogenetic relationships within Trichoderrna permit a prediction of the peptaibol production profiles. To this end, representative strains from a third (28) of the known species of Trichoderma, identified by the sequences of diagnostic genes and covering most clades of the established multilocus phylogeny of Trichoderma/Hypocrea, were investigated by intact-cell MALDI-TOF mass spectrometry. Peptaibols were detected in all strains, and some strains were found to produce up to five peptide families of different sizes. Comparison of the data with phylogenies derived from rRNA spacer regions (ITS1 and 2) and RNA polymerase subunit B (rpb2) gene sequences did not show a strict correlation with the types and sequences of the peptaibols produced, but the production of some groups of peptaibols appears to be found only in some clades or sections of the genus, which could be used for more targeted screening of novel compounds of this type. In an analysis of peptaibol structures, we have defined conserved key positions and have further identified and compared sequences of the corresponding adenylate domains within non-ribosomal peptide synthetases producing trichovirins, paracelsins and atroviridins. These phylogenies are not concordant with those of their producers Hypocrea virens, Hypocrea jecorina and Hypocrea atroviridis as obtained from ITS1 and 2, and rpb2, respectively, and therefore hint at a complex history of peptaibol diversity.

To identify carbon sources that trigger [beta]-N-acetylglucosaminidase (NAGase) formation in Hypocrea atroviridis (anamorph Trichoderma atroviride), a screening system was designed that consists of a combination of Biolog Phenotype MicroArray plates, which contain 95 different carbon sources, and specific enzyme activity measurements using a chromogenic substrate. The results revealed growth-dependent kinetics of NAGase formation and it was shown that NAGase activities were enhanced on carbon sources sharing certain structural properties, especially on [alpha]-glucans (e.g. glycogen, dextrin and maltotriose) and oligosaccharides containing galactose. Enzyme activities were assessed in the wild-type and a H. atroviridis [DELTA]nag1 strain to investigate the influence of the two NAGases, Nag1 and Nag2, on total NAGase activity. Reduction of NAGase levels in the Anagl strain in comparison to the wild-type was strongly carbon-source and growth-phase dependent, indicating the distinct physiological roles of the two proteins. The transcript abundance of nag1 and nag2 was increased on carbon sources with elevated NAGase activity, indicating transcriptional regulation of these genes. The screening method for the identification of carbon sources that induce enzymes or a gene of interest, as presented in this paper, can be adapted for other purposes if appropriate enzyme or reporter assays are available.

A study was conducted to investigate the global carbon metabolism of Hypocera jecorina per se and to compare it to genetically modified strains obtained after classical mutagenesis or DNA-mediated transformation. The data obtained demonstrate that the wild-type isolates and transformed strains had the highest variation in growth patterns on individual carbon sources and cellulase mutants were relatively similar to their parental strains.