Resolving the conundrum of trichoderma taxonomy: how ecological genetics coupled with evolutionary analysis enhances the polyphasic species concept in Fungi

Abstract

Pilze der Gattung Trichoderma (Hypocreales, Ascomycota) sind für den Menschen von besonderer Bedeutung aufgrund der Verwendung einzelner Arten in der Enzym produzierenden Biotechnologie, als biologische Pflanzenschutzmittel und Stimulatoren des Pflanzenwachstums in der Landwirtschaft, oder aber als Besiedler immunschwacher Menschen und Tiere. Gegenwärtig sind fast 400 Spezies bekannt, aber deren sichere Identifizierung ist schwierig. Ich habe mich daher in meiner Arbeit mit der Identifizierung der einzelnen Spezies sowie den möglichen Mechanismen der Artenbildung bei Trichoderma beschäftigt.Als ersten Schritt hierzu habe ich eine auf DNA-Sequenzanalyse beruhende Methode („DNA Barcoding“) entwickelt: zu diesem Zweck habe ich die DNA-Sequenzen dreier Barcode-Marker (ITS, tef1 und rpb2) aller Spezies verglichen, und eine bioinformatische Analysenmethode erarbeitet die eine sichere Speziesidentifizierung ermöglicht.Die Methode wurde in der Folge anhand nicht identifizierter Trichoderma Isolate aus einem Salzmarsch-Ökosystem an der Küste des Gelben Meers getestet und verifiziert, was auch zur Identifizierung und Beschreibung einer neuen Trichoderma Art – T. arenarium sp. nov. -führte. Im Zuge dieser Untersuchung konnten – als sekundärer „Benefit“ - auch zahlreiche Isolate gefunden werden welche als Bioeffektoren für die Pflanzenzucht auf salzreichen Böden geeignet sind.Die oben beschriebenen Ergebnisse zeigten aber auch dass die bei der Speziesbildung wirksamen Mechanismen in Trichoderma nur ungenügend bekannt sind. Ich habe daher in Weiterführung meiner Arbeit die Theorie aufgestellt dass die Speziesbildung in der Evolution von Genen für die Fitness im Ökosystem reflektiert sein müsste. Ich habe dies am Beispiel zweier Typen sogenannter “surface active, small secreted and cysteine rich” (saSSCPs) Proteine – der Hydrophobine (HFB) und der Ceratoplatanine (CPs) – analysiert. Meine Untersuchungen an T. harzianum und T. guizhouense zeigen dass die Funktion und Evolution der HFBs tatsächlich mit der Anpassung sympatrischer Spezies an ökologische Nischen korreliert. Im Falle der CPs konnte ich eine auf stabilisierender Selektion beruhende Evolution zeigen, was deren Rolle bei der Anpassung an das jeweilige Ökosystem verdeutlicht. Meine Arbeit hat daher einen neuen Weg zur Identifizierung von Trichoderma Arten aufgezeigt und darüber hinaus Mechanismen der Artenbildung entdeckt. Diese Befunde sind nicht nur für Trichoderma bedeutsam sondern können auch als Vorlage für ähnliche Untersuchungen bei anderen Pilzgattungen dienen.

Fungi comprise one of the most diverse groups of eukaryotes on Earth. Nearly two thousand new species are described each year, while the number of known fungal species - approximately 150,000, is estimated to hardly excess only a few percent of the total diversity in this kingdom. The rapid growth of fungal taxonomy is escalated by the outstanding role that fungi play for humankind because new species may possess new properties. Fungi produce a rich spectrum of hydrolytic enzymes, surfactant proteins, and secondary metabolites that found their application in industry, pharmaceutics, and agriculture. Many fungi are valuable for food production; others are exploited as industrial cell factories to synthesize bioactive compounds or serve as models for cell biology research. Some biotrophic fungi can cause diseases in humans, and cattle or parasitize crops. Being ubiquitous in most ecosystems, fungi perform essential ecological processes as symbionts and decomposers. Fungi have a simple structure of their body that is either tubular (mycelium, or hyphae) or single-celled (spores, or yeasts). This morpho-anatomical organization frequently undergoes convergent evolution making the precise and accurate species identification required to predict their application or pathology-relevant properties challenging. In the first part of this thesis, we focused on the diversity, species delimitation, and molecular identification of nearly four hundred species of the plant-beneficial and industrially-relevant filamentous fungi from the genus Trichoderma (Hypocreales, Ascomycota). We compiled a complete inventory of all Trichoderma species and DNA barcoding materials deposited in public databases. We have then developed an authoritative guideline for molecular identification of Trichoderma that requires analysis of the three DNA barcodes (ITS, tef1, and rpb2) and supported it by several online tools. We then used all the whole-genome sequenced (WGS) Trichoderma strains to provide versatile, practical examples of Trichoderma DNA Barcoding, reveal methodological and theoretical shortcomings, and discuss possible ambiguities. The work provides an in-depth discussion of species concepts applied in Trichoderma taxonomy. We conclude that these fungi are particularly suitable for implementing integrative taxonomy that fuses DNA Barcoding and polyphasic phenotyping. Subsequently, we tested the applicability of the developed DNA Barcoding protocol on the collection of Trichoderma spp. strains isolated from the emerging salt marches on the Yellow Sea coastal tidal flat zones. This study resulted in the discovery of T. arenarium sp. nov. and other fungal bioeffectors suitable for biosaline agriculture. The above taxonomic studies and the review of the ecological genomics of the most industrially-relevant species of Trichoderma - T. reesei revealed the critical shortage of species recognition criteria in the genus and highlighted the theoretical shortcomings of our understanding of the speciation process in fungi. To overcome it, we have proposed that speciation can be reflected in the evolution of genes relevant to fungal fitness (ecological genetics). For this purpose, in the second part of the thesis, we optimized the toolbox for the genetic recombination of Trichoderma and reviewed the strain improvement techniques available for these fungi. Our previous study discovered that the highly surface-active small secreted cysteine-rich proteins (saSSCPs) – hydrophobins (HFBs), strongly influence the fitness of the two sister species from the Harzianum Clade of Trichoderma. It showed that HFB evolution and function analysis could reveal distinct adaptations of sympatric species to microecological niches. Therefore, in the third part of the thesis, we studied the other family of saSSCPs that are massively secreted by Trichoderma – cerato-platanins (CPs). The in silico analysis of 283 CPs from 157 fungal genomes revealed the long evolutionary history of CPs in Dikarya fungi that have undergone several lateral gene transfer events and multiple gene duplications. Three genes were maintained in the core genome of Trichoderma, while some species have up to four CP-encoding genes. However, the functional analysis of CPs revealed that only EPL1 is active at all development stages but plays a minor role in interactions with other fungi and bacteria. The deletion of this gene resulted in increased colonization of rhizosphere by Trichoderma spp. Similarly, the biochemical tests of the heterologously produced EPL1 by Pichia pastoris support the above claims. Overall, this study pointed to the evolutionary and functional paradox of CPs in fungi. The high diversity and stabilizing selection suggest their importance for the speciation process. In summary, the studies presented in this thesis provide the conceptual and methodological framework for establishing the integrative taxonomy of Trichoderma spp. that should rely on DNA Barcoding and consider the genetic background of ecological adaptations driving the evolution and speciation. We conclude that applying the polyphasic approach to species recognition in Trichoderma and other highly diverse genera of environmentally opportunistic fungi will finally resolve the conundrum of fungal taxonomy.