The mycobiome of symptomatic wood of Prunus trees in Germany Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) Naturwissenschaftliche Fakultät I – Biowissenschaften – der Martin-Luther-Universität Halle-Wittenberg vorgelegt von Herrn Steffen Bien Geb. am 29.07.1985 in Berlin Copyright notice Chapters 2 to 4 have been published in international journals. Only the publishers and the authors have the right for publishing and using the presented data. Any re-use of the presented data requires permissions from the publishers and the authors. Content III Content Summary .................................................................................................................. IV Zusammenfassung .................................................................................................. VI Abbreviations ......................................................................................................... VIII 1 General introduction ............................................................................................. 1 1.1 Importance of fungal diseases of wood and the knowledge about the associated fungal diversity ...................................................................................... 1 1.2 Host-fungus interactions in wood and wood diseases ....................................... 2 1.3 The genus Prunus ............................................................................................. 4 1.4 Diseases and fungal communities of Prunus wood ........................................... 5 1.5 The German Barcoding of Life project ............................................................. 15 1.6 Objectives of the study .................................................................................... 15 1.7 References ...................................................................................................... 16 2 Manuscript I ......................................................................................................... 23 Bien, S., Kraus, C., Damm, U. (2020). Novel collophorina-like genera and species from Prunus trees and vineyards in Germany. Persoonia, 45, 46–67. 3 Manuscript II ........................................................................................................ 46 Bien, S., Damm, U. (2020). Arboricolonus simplex gen. et sp. nov. and novelties in Cadophora, Minutiella and Proliferodiscus from Prunus wood in Germany. MycoKeys, 63, 119–161. 4 Manuscript III ....................................................................................................... 90 Bien, S., Damm, U. (2020). Prunus trees in Germany – a hideout of unknown fungi? Mycological Progress, 19, 667–690. 5 General discussion............................................................................................ 115 5.1 Species delimitation and descriptions ........................................................... 115 5.2 Comparison of the fungal diversity in Prunus wood ....................................... 117 5.3 General remarks on the diversity assessment and taxonomic studies .......... 120 5.4 Outlook .......................................................................................................... 122 5.5 References .................................................................................................... 122 Acknowledgements .............................................................................................. 125 Statement on the personal contribution to the publications ............................ 126 Curriculum Vitae ................................................................................................... 127 Eidesstattliche Erklärung ..................................................................................... 130 IV Summary Summary Diseases on woody plant hosts caused by fungal pathogens have far-reaching ecological and economic consequences. In spite of their importance, the knowledge about the fungal diversity associated with symptomatic wood, even in economically important crops like stone fruits (Prunus spp.) is meagre. Furthermore, the taxonomic basis for the species identification of many pathogenic fungal taxa is insufficient, which impedes quick and targeted control measures in cases of sudden disease outbreaks. In order to fill this knowledge gap, an extensive assessment was conducted in orchards of Prunus avium, P. cerasus and P. domestica in three important fruit production areas in Germany. The fungi were isolated from symptomatic woody tissue and examined by means of morphological and molecular data. Selected genera were analysed in more depth; some additional strains from other origins were included as well. Fungi with reduced conidiogenous cells and a yeast-like phase were frequently isolated from all three Prunus species and in all sampling areas in Germany. Initial molecular identifications placed these fungi and ten similar strains from spore traps in a vineyard in Rhineland-Palatinate in the genus Collophorina (Leotiomycetes, Tympanidaceae). A family-wide phylogenetic analysis of the Tympanidaceae revealed the genus Collophorina to be polyphyletic. Consequently, C. paarla and C. euphorbiae were transferred to the newly described genera Pallidophorina and Ramoconidiophora, respectively. The fungi from wood of Prunus belonged to five species, namely Pa. paarla, C. africana and three new Collophorina species, described as C. badensis, C. germanica and C. neorubra. The strains from spore traps belonged to seven new species, including C. badensis, in five genera, of which three were newly described as well, namely Capturomyces, Variabilispora and Vexillomyces. The results of this study provide far-reaching insights into this group of understudied fungi that revealed an unknown diversity and high abundance in Prunus trees in Germany. One strain isolated from P. domestica resembled Collophorina as well. However, a preliminary molecular analysis indicated a relationship to the Hyaloscyphaceae (Leotiomycetes). A family-wide molecular and morphological analysis did not reveal a close relationship or resemblance, respectively, to any known species or genus, and consequently the new genus Arboricolonus and the new species A. simplex were described. The morphology reminiscent of Collophorina was attributed to convergent evolution, since both genera occupy the same habitat. Species of the genus Cadophora (Leotiomycetes) are known from different habitats like plants, fruits and deadwood, and were frequently isolated from Prunus wood in this study. An analysis of the genus comprised 29 strains from Prunus in Germany and three additional strains from Prunus in South Africa. In total, five species of Cadophora were differentiated, including two known species, Ca. luteo-olivacea and Ca. novi-eboraci from Germany and three new species, namely Ca. prunicola from Summary V Germany and South Africa, Ca. africana from South Africa, and Ca. ramosa from Germany. Moreover, Margarinomyces bubakii was combined in Cadophora, while Ca. obscura was resurrected. Based on DNA sequence data, eight strains from Prunus forming asexual morphs were assigned to the genus Proliferodiscus (Leotiomycetes). In the past, species of this genus were almost exclusively described based on the morphology of their sexual morphs, while few sequence data and data on asexual morphs were available. Our study resulted in the first comprehensive phylogenetic analysis of this genus. The new species Pr. ingens was described, and detailed descriptions of the asexual morphs of this species and a second species referred to as Proliferodiscus sp. were provided. Two species of the genus Minutiella (Eurotiomycetes) were isolated, one of which was newly described as M. pruni-avium. Within the general assessment of the fungal diversity, a total of 1,018 isolates from wood of the three Prunus species in Germany were obtained, morphologically observed, and their ITS and LSU sequences generated. A combination of blastn searches with strong focus on reliable reference sequences and thorough phylogenetic analyses revealed a diversity of 172 species of Ascomycota, Basidiomycota and Mucoromycota with the majority of strains belonging to the Ascomycota classes Sordariomycetes, Leotiomcetes and Dothideomycetes. Based on the number of nucleotide differences to reference sequences, species identifications were divided into categories. According to this scheme, 82 species were identified with high and 20 species with low certainty. By far the most dominant species detected were the coelomycetous species Aposphaeria corallinolutea (Dothideomycetes) and the collophorina-like species Pallidophorina paarla, both isolated from all three host species and in all sampling regions. The remaining 70 taxa could not be assigned to any species; most of them were therefore regarded as potentially new species. On this basis, Prunus wood, even in a well-explored region like Germany, was recognised as a habitat harbouring high numbers of potentially new species. Culturing and preservation of all strains isolated from Prunus wood in this study allow further follow-up studies of their taxonomy, physiology, ecology, pathogenicity and bio-chemistry. VI Zusammenfassung Zusammenfassung Durch pilzliche Erreger verursachte Krankheiten an Bäumen haben weitreichende ökologische und ökonomische Folgen. Trotz ihrer Bedeutung bestehen große Wissenslücken bezüglich der Pilzgemeinschaften in symptomatischem Holz. Dies betrifft auch ökonomisch bedeutende Pflanzengruppen