Hypocrea/Trichoderma bioresources from Southern Europe

Hypocrea is a genus of the fungal phylum Ascomycota. It is particularly well known in its asexual state, Trichoderma, which is one of the principal fungal components in soil playing a key role for soil health. Many species of Hypocrea/Trichoderma have economic and ecological importance, as sources of pharmaceuticals and mycotoxins, as sources of industrial enzymes, and as biological control agents of plant pathogenic fungi, but they also pose a serious threat to cultivated mushrooms and to immune-suppressed patients. Thus Hypocrea species are valuable biological resources. Utilization of these and new bioresources require their detection in nature and preservation in collections of living cultures. It is essential to clearly define and characterize the taxonomy and systematic position of the species, to be able to distinguish them from other species and to recognize them. While many studies have been carried out on species isolated from soil, little has been known about the biodiversity and ecology of the genus above soil level. Hypocrea teleomorphs occur commonly on wood, other plant material and various fungi. In a previous project Hypocrea was collected in 14 European countries. Due to limited morphological variation, identification has to be based on gene sequences. To do this, DNA is extracted from cultures prepared from ascospores. This work resulted in several unexpected findings. In temperate Europe the biodiversity of the genus Hypocrea exceeds the biodiversity of Trichoderma in soil and it exceeds by far expectations regarding the number of species. 75 species instead of ca. 20 expected species were identified, and 30 are new to science. All species known to form a teleomorph in Europe will be described in a monograph. Despite previous efforts we do not know anything about the genus from warmer regions of Europe, particularly the Mediterranean and Macaronesia, regions that contain many biodiversity hotspots, but are endangered by increasing drought periods and forest fires. Only two studies on soil inhabiting Trichoderma anamorphs from Sardinia and Tenerife have been conducted, which do not appear to be representative for the mycoflora above the rhizosphere level in light of the previous projects. The new project is therefore proposed to (1) analyze the biodiversity of Hypocrea / Trichoderma on and above soil in these regions, which are characterized by climates and plant diversity deviating substantially from temperate Europe, (2) search for new and endemic species evolved on islands due to geographic separation, to describe and conserve them as living cultures, (3) to improve molecular species delimitation currently based on a single gene (tef1) within complex clades by development of additional phylogenetic markers, and (4) to analyze and document the ecology of the species collected.

In this third part of a project series southern Europe including the Canary Islands was investigated regarding biodiversity, ecology and DNA-based phylogeny of the economically important fungal genus Trichoderma (formerly Hypocrea) and some other representatives of the order Hypocreales. The primary question to answer was how many and which species of Trichoderma exist in Europe. Of more than 650 specimens, freshly collected in nature, living cultures were prepared and DNA sequences determined. As a main result 74 Trichoderma species were detected and 30 new species characterized by morphology and growth properties. DNA sequences were used to elucidate species limits and phylogenetic relationships. Taken all 3 projects together, 120 species were detected in Europe. Cultures of the new species represent potential bioresources and were therefore preserved in an official strain collection (CBS, Utrecht), where they are accessible for future research. Fungi are not well known, nor very popular in everyday life. Scientific studies on fungi leap far behind those on other organisms like plants and animals, and still many new species and genera are being discovered, even more so after introduction of gene sequences in species identification and definition ca. 20 years ago. Accurate observations of organisms on one hand and chemical analysis of secondary metabolites in the natural setting and in the laboratory on the other provide knowledge about co-existence and interaction of organisms. Such knowledge helps to infer usefulness of certain organisms. One example: Trichoderma species attack other fungi and this property is utilized in biocontrol of harmful fungi, e.g. those which cause plant or other diseases. This is not the only reason why the genus Trichoderma is important, but also because several species are known to produce a large number of substances, such as antibiotics (called peptaibols or peptaibiotics in Trichoderma) and enzymes like cellulases (to degrade plant material) or chitinases (to degrade fungi). Thus, they are applied in several spheres of life such as detergents, textile and paper industry, but also in the production of biofuels. In the present project we did not only considerably increase the number of Trichoderma species, but also constructed the hitherto most complete phylogenetic tree containing 228 named species of Trichoderma. For the first time we present in addition a genus-wide phylogenetic tree based on a newly sequenced gene (acl1). The numerous cultures prepared in this project enabled us to resolve difficult species groups, to corroborate that Trichoderma is a species-rich genus, that the species diversity on plant and fungal materials exceeds that in soil and to show that the occurrence of various species depends strongly on ecological conditions such as climate. Accordingly, southern Europe, particularly regions with marked Mediterranean climate types, differs considerably in species occurrence from other European regions, and only 37 species were found in southern, central and northern Europe. Within this project also several new groups of peptaibol antibiotics were detected, and the defence of Trichoderma species against competing organisms like bacteria and other fungi in the environment was confirmed experimentally. In conclusion, owing to the current work, Europe is the best-investigated continent with regard to species diversity in Trichoderma. These projects may stimulate and guide future researchers in accomplishing similar tasks in other regions. The recognition, characterisation and naming of species within difficult complexes will help to better understand conflicting results of trials in applied research such as biocontrol or antibiotics and toxins research, but also regarding infections of immunocompromised patients.