True fungi (Fungi) and fungus-like organisms (e.g.Mycetozoa,Oomycota) constitute the second largest group of organisms based on global richness estimates, with around 3 million predicted species. Compared to plants and animals, fungi have simple body plans with often morphologically and ecologically obscure structures. This poses challenges for accurate and precise identifications. Here we provide a conceptual framework for the identification of fungi, encouraging the approach of integrative (polyphasic) taxonomy for species delimitation, i.e. the combination of genealogy (phylogeny), phenotype (including autecology), and reproductive biology (when feasible). This allows objective evaluation of diagnostic characters, either phenotypic or molecular or both. Verification of identifications is crucial but often neglected. Because of clade-specific evolutionary histories, there is currently no single tool for the identification of fungi, although DNA barcoding using the internal transcribed spacer (ITS) remains a first diagnosis, particularly in metabarcoding studies. Secondary DNA barcodes are increasingly implemented for groups where ITS does not provide sufficient precision. Issues of pairwise sequence similarity-based identifications and OTU clustering are discussed, and multiple sequence alignment-based phylogenetic approaches with subsequent verification are recommended as more accurate alternatives. In metabarcoding approaches, the trade-off between speed and accuracy and precision of molecular identifications must be carefully considered. Intragenomic variation of the ITS and other barcoding markers should be properly documented, as phylotype diversity is not necessarily a proxy of species richness. Important strategies to improve molecular identification of fungi are: (1) broadly document intraspecific and intragenomic variation of barcoding markers; (2) substantially expand sequence repositories, focusing on undersampled clades and missing taxa; (3) improve curation of sequence labels in primary repositories and substantially increase the number of sequences based on verified material; (4) link sequence data to digital information of voucher specimens including imagery. In parallel, technological improvements to genome sequencing offer promising alternatives to DNA barcoding in the future. Despite the prevalence of DNA-based fungal taxonomy, phenotype-based approaches remain an important strategy to catalog the global diversity of fungi and establish initial species hypotheses.

Modern taxonomy has developed towards the establishment of global authoritative lists of species that assume the standardized principles of species recognition, at least in a given taxonomic group. However, in fungi, species delimitation is frequently subjective because it depends on the choice of a species concept and the criteria selected by a taxonomist. Contrary to it, identification of fungal species is expected to be accurate and precise because it should predict the properties that are required for applications or that are relevant in pathology. The industrial and plant-beneficial fungi from the genus Trichoderma (Hypocreales) offer a suitable model to address this collision between species delimitation and species identification. A few decades ago, Trichoderma diversity was limited to a few dozen species. The introduction of molecular evolutionary methods resulted in the exponential expansion of Trichoderma taxonomy, with up to 50 new species recognized per year. Here, we have reviewed the genus-wide taxonomy of Trichoderma and compiled a complete inventory of all Trichoderma species and DNA barcoding material deposited in public databases (the inventory is available at the website of the International Subcommission on Taxonomy of Trichoderma www.trichoderma.info ). Among the 375 species with valid names as of July 2020, 361 (96%) have been cultivated in vitro and DNA barcoded. Thus, we have developed a protocol for molecular identification of Trichoderma that requires analysis of the three DNA barcodes (ITS, tef1 , and rpb2 ), and it is supported by online tools that are available on www.trichokey.info . We then used all the whole-genome sequenced (WGS) Trichoderma strains that are available in public databases to provide versatile practical examples of molecular identification, reveal shortcomings, and discuss possible ambiguities. Based on the Trichoderma example, this study shows why the identification of a fungal species is an intricate and laborious task that requires a background in mycology, molecular biological skills, training in molecular evolutionary analysis, and knowledge of taxonomic literature. We provide an in-depth discussion of species concepts that are applied in Trichoderma taxonomy, and conclude that these fungi are particularly suitable for the implementation of a polyphasic approach that was first introduced in Trichoderma taxonomy by John Bissett (1948–2020), whose work inspired the current study. We also propose a regulatory and unifying role of international commissions on the taxonomy of particular fungal groups. An important outcome of this work is the demonstration of an urgent need for cooperation between Trichoderma researchers to get prepared to the efficient use of the upcoming wave of Trichoderma genomic data.

The 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. We 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. Our 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.

Background: Trichoderma is a genus of mycotrophic filamentous fungi (teleomorph Hypocrea) which possess a bright variety of biotrophic and saprotrophic lifestyles. The ability to parasitize and/or kill other fungi (mycoparasitism) is used in plant protection against soil-borne fungal diseases (biological control, or biocontrol). To investigate mechanisms of mycoparasitism, we compared the transcriptional responses of cosmopolitan opportunistic species and powerful biocontrol agents Trichoderma atroviride and T. virens with tropical ecologically restricted species T. reesei during confrontations with a plant pathogenic fungus Rhizoctonia solani. Results: The three Trichoderma spp. exhibited a strikingly different transcriptomic response already before physical contact with alien hyphae. T. atroviride expressed an array of genes involved in production of secondary metabolites, GH16 ß-glucanases, various proteases and small secreted cysteine rich proteins. T. virens, on the other hand, expressed mainly the genes for biosynthesis of gliotoxin, respective precursors and also glutathione, which is necessary for gliotoxin biosynthesis. In contrast, T. reesei increased the expression of genes encoding cellulases and hemicellulases, and of the genes involved in solute transport. The majority of differentially regulated genes were orthologues present in all three species or both in T. atroviride and T. virens, indicating that the regulation of expression of these genes is different in the three Trichoderma spp. The genes expressed in all three fungi exhibited a nonrandom genomic distribution, indicating a possibility for their regulation via chromatin modification. Conclusion: This genome-wide expression study demonstrates that the initial Trichoderma mycotrophy has differentiated into several alternative ecological strategies ranging from parasitism to predation and saprotrophy. It provides first insights into the mechanisms of interactions between Trichoderma and other fungi that may be exploited for further development of biofungicides.