Valentin Queloz

Hymenoscyphus pseudoalbidus, the causal agent of European ash dieback

UNLABELLED The ascomycete Hymenoscyphus pseudoalbidus (anamorph Chalara fraxinea) causes a lethal disease known as ash dieback on Fraxinus excelsior and Fraxinus angustifolia in Europe. The pathogen was probably introduced from East Asia and the disease emerged in Poland in the early 1990s; the subsequent epidemic is spreading to the entire native distribution range of the host trees. This pathogen profile represents a comprehensive review of the state of research from the discovery of the pathogen and points out knowledge gaps and research needs. TAXONOMY Members of the genus Hymenoscyphus (Helotiales, Leotiomycetidae, Leotiomycetes, Ascomycota) are small discomycetes which form their ascomata on dead plant material. A phylogeny based on the internal transcribed spacers (ITSs) of the rDNA indicated the avirulent Hymenoscyphus albidus, a species native to Europe, as the closest relative of H. pseudoalbidus. SYMPTOMS Hymenoscyphus pseudoalbidus causes necrotic lesions on leaves, twigs and stems, eventually leading to wilting and dieback of girdled shoots. Bark lesions are characterized by a typical dark- to cinnamon-brown discoloration. LIFE CYCLE Hymenoscyphus pseudoalbidus is heterothallic and reproduces sexually on ash petioles in the litter once a year. Ascospores are wind dispersed and infect ash leaves during the summer. The asexual spores only serve as spermatia. TOOLS AND TECHNIQUES The most important techniques for fungal handling, such as detection, isolation, culturing, storage, crossing and ascocarp production, are briefly described. MANAGEMENT Once the disease is established, management is hardly possible. The occurrence of a small fraction of partially tolerant trees constitutes hope for resistance breeding in the future. Healthy-looking trees should be preserved.

Hymenoscyphus fraxineus, the correct scientific name for the fungus causing ash dieback in Europe.

Under the rules for the naming of fungi with pleomorphic life-cycles adopted in July 2011, the nomenclaturally correct name for the fungus causing the current ash dieback in Europe is determined to be Hymenoscyphus fraxineus, with the basionym Chalara fraxinea, and Hymenoscyphus pseudoalbidus as a taxonomic synonym of H. fraxineus.

Improving ITS sequence data for identification of plant pathogenic fungi

SummaryPlant pathogenic fungi are a large and diverse assemblage of eukaryotes with substantial impacts on natural ecosystems and human endeavours. These taxa often have complex and poorly understood life cycles, lack observable, discriminatory morphological characters, and may not be amenable to in vitro culturing. As a result, species identification is frequently difficult. Molecular (DNA sequence) data have emerged as crucial information for the taxonomic identification of plant pathogenic fungi, with the nuclear ribosomal internal transcribed spacer (ITS) region being the most popular marker. However, international nucleotide sequence databases are accumulating numerous sequences of compromised or low-resolution taxonomic annotations and substandard technical quality, making their use in the molecular identification of plant pathogenic fungi problematic. Here we report on a concerted effort to identify high-quality reference sequences for various plant pathogenic fungi and to re-annotate incorrectly or insufficiently annotated public ITS sequences from these fungal lineages. A third objective was to enrich the sequences with geographical and ecological metadata. The results – a total of 31,954 changes – are incorporated in and made available through the UNITE database for molecular identification of fungi (http://unite.ut.ee), including standalone FASTA files of sequence data for local BLAST searches, use in the next-generation sequencing analysis platforms QIIME and mothur, and related applications. The present initiative is just a beginning to cover the wide spectrum of plant pathogenic fungi, and we invite all researchers with pertinent expertise to join the annotation effort.

Population structure of the invasive forest pathogen Hymenoscyphus pseudoalbidus

Understanding the genetic diversity and structure of invasive pathogens in source and in introduced areas is crucial to the revelation of hidden biological features of an organism, to the reconstruction of the course of invasions and to the establishment of effective control measures. Hymenoscyphus pseudoalbidus (anamorph: Chalara fraxinea) is an invasive and highly destructive fungal pathogen found on common ash Fraxinus excelsior in Europe and is native to East Asia. To gain insights into its dispersal mechanisms and history of invasion, we used microsatellite markers and characterized the genetic structure and diversity of H. pseudoalbidus populations at three spatial levels: (i) between Europe and Japan, (ii) in Europe and (iii) at the epidemics front in Switzerland. Phylogenetic and network analysis demonstrated that individuals from both regions are conspecific. However, populations from Japan harboured a higher genetic diversity and were genetically differentiated from European ones. No evident population structure was found among the 1208 European strains using Bayesian and multivariate clustering analysis. Only the distribution of genetic diversity in space, pairwise population differentiation (GST) and the spatial analysis of principal components revealed a faint geographical pattern around Europe. A significant allele deficiency in most European populations pointed to a recent genetic bottleneck, whereas no pattern of isolation by distance was found. Our data suggest that H. pseudoalbidus was introduced just once by at least two individuals. The potential source region of H. pseudoalbidus is vast, and further investigations are required for a more accurate localization of the source population.

Phylogeny of Phaeomollisia piceae gen. sp. nov.: a dark, septate, conifer-needle endophyte and its relationships to Phialocephala and Acephala.

Dark, septate endophytes (DSE) were isolated from roots and needles of dwarf Picea abies and from roots of Vaccinium spp. growing on a permafrost site in the Jura Mountains in Switzerland. Two of the isolates sporulated after incubation for more than one year at 4 degrees C. One of them was a hitherto undescribed helotialean ascomycete Phaeomollisia piceae gen. sp. nov., the other was a new species of Phialocephala, P. glacialis sp. nov. Both species are closely related to DSE of the Phialocephala fortinii s. lat.-Acephala applanata species complex (PAC) as revealed by phylogenetic analyses of the ITS and 18S rDNA regions. Morphologically dissimilar fungi, such as Vibrissea and Loramyces species, are phylogenetically also closely linked to the new species and the PAC. Cadophora lagerbergii and C. (Phialophora) botulispora are moved to Phialocephala because Phialocephala dimorphospora and P. repens are the closest relatives. Several Mollisia species were closely related to the new species and the PAC according to ITS sequence comparisons. One DSE from needles of Abies alba and one from shoots of Castanea sativa formed Cystodendron anamorphs in culture. Their identical 18S sequences and almost identical ITS sequences indicated Mollisia species as closest relatives, suggesting that Mollisia species are highly euryoecious.

Microsatellite size homoplasies and null alleles do not affect species diagnosis and population genetic analysis in a fungal species complex

The suitability of 13 microsatellite loci for species diagnosis and population genetics in 11 species of the Phialocephala fortinii s.l.–Acephala applanata species complex (PAC) was assessed. Two data sets were compared to test possible biases in species typing and clone detection resulting from null alleles and size homoplasies. The first data set was based on fragment lengths derived from a multiplex polymerase chain reaction (PCR) assay and the second data set was received from singleplex PCR at lower stringency and sequencing. Most null alleles observed in the multiplex PCR assay could be amplified during singleplex PCR under less stringent conditions. Size homoplasies resulting from mutations in flanking regions and differences in microsatellite structures were observed. For example, Phialocephala uotolensis possessed a (CT)13 in addition to the (GT)x motif at locus mPF_0644. Despite the occurrence of null alleles and size homoplasies, species diagnosis and population genetic analysis studies were not affected. These markers will facilitate studies on population biology, ecology and biogeography of PAC species.

Structure of Diversity in Dark Septate Endophytes: From Species to Genes

Dark septate endophytes (DSE) are among the most abundant colonizers of plant roots. The form taxon DSE includes a broad range of fungal species that are only distantly related and the taxonomy of DSE has puzzled mycologists for years. In the following chapter we discuss the structure of diversity in dark septate endophytes. In the first part, we give an overview of the taxonomic placement of DSE and present a reference dataset of ITS sequences of well-characterized DSE taxa that can be used to classify DSE in future studies. The second part is dedicated to the diversity in the Phialocephala fortinii s.l. – Acephala applanata species complex (PAC), which includes some of the most frequent and widespread DSE. Diversity of PAC is discussed on various levels ranging from single root segments to continents and from communities to genes.

Isolation and characterization of microsatellite markers for the tree-root endophytes Phialocephala subalpina and Phialocephala fortinii s.s.

Species of the Phialocephala fortinii s.l.–Acephala applanata complex are the dominant dark septate endophytes (DSE) in roots of species belonging to the Pinaceae. The two species Phialocephala subalpina and P. fortinii s.s. belong to the most widely distributed species within this complex. In the present study, 15 polymorphic microsatellite loci were developed for these two closely related species. Strains of a community which were analysed previously using single‐copy restriction fragment length polymorphism were screened with the new markers. Microsatellites were suitable to classify the two species and to recognize individuals within species.

Sex in the PAC: a hidden affair in dark septate endophytes?

BackgroundFungi are asexually and sexually reproducing organisms that can combine the evolutionary advantages of the two reproductive modes. However, for many fungi the sexual cycle has never been observed in the field or in vitro and it remains unclear whether sexual reproduction is absent or cryptic. Nevertheless, there are indirect approaches to assess the occurrence of sex in a species, such as population studies, expression analysis of genes involved in mating processes and analysis of their selective constraints. The members of the Phialocephala fortinii s. l. - Acephala applanata species complex (PAC) are ascomycetes and the predominant dark septate endophytes that colonize woody plant roots. Despite their abundance in many ecosystems of the northern hemisphere, no sexual state has been identified to date and little is known about their reproductive biology, and how it shaped their evolutionary history and contributes to their ecological role in forest ecosystems. We therefore aimed at assessing the importance of sexual reproduction by indirect approaches that included molecular analyses of the mating type (MAT) genes involved in reproductive processes.ResultsThe study included 19 PAC species and > 3, 000 strains that represented populations from different hosts, continents and ecosystems. Whereas A. applanata had a homothallic (self-fertile) MAT locus structure, all other species were structurally heterothallic (self-sterile). Compatible mating types were observed to co-occur more frequently than expected by chance. Moreover, in > 80% of the populations a 1:1 mating type ratio and gametic equilibrium were found. MAT genes were shown to evolve under strong purifying selection.ConclusionsThe signature of sex was found in worldwide populations of PAC species and functionality of MAT genes is likely preserved by purifying selection. We hypothesize that cryptic sex regularely occurs in the PAC and that further field studies and in vitro crosses will lead to the discovery of the sexual state. Although structurally heterothallic species prevail, it cannot be excluded that homothallism represents the ancestral breeding system in the PAC.

The Endophytic Mycobiome of European Ash and Sycamore Maple Leaves – Geographic Patterns, Host Specificity and Influence of Ash Dieback

The European ash (Fraxinus excelsior) is threatened by the introduced ascomycete Hymenoscyphus fraxineus, the causal agent of ash dieback. Endophytic fungi are known to modulate their host’s resistance against pathogens. To understand possible consequences of ash dieback on the endophytic mycobiome, F. excelsior leaves were collected in naturally regenerated forests and the fungal communities analyzed by classic culture and Illumina amplicon sequencing using a newly developed and validated fungal-specific primer. Collections were done in the area infested by ash dieback north of the Alps, and in the disease free area on the south side. Sycamore maple (Acer pseudoplatanus) was additionally collected, as well as the flowering ash (F. ornus), which occurs naturally in the south and shows tolerance to ash dieback. Both cultivation and amplicon sequencing revealed characteristic endophytic fungal communities dominated by several strictly host specific Venturia species. On A. pseudoplatanus, a hitherto undescribed Venturia species was identified. Due to its dominance on F. excelsior, V. fraxini is unlikely to go extinct in case of reduced host densities. A majority of species was not strictly host specific and is therefore likely less affected by ash dieback in the future. Still, shifts in community structure and loss of genetic diversity cannot be excluded. The potentially endangered endophyte Hymenoscyphus albidus was rarely found. In addition to host specificity, species with preferences for leaf laminae or petioles were found. We also detected considerable geographical variation between sampling sites and clear differences between the two sides of the Alps for endophytes of F. excelsior, but not A. pseudoplatanus. Since sycamore maple is not affected by an epidemic, this could point toward an influence of ash dieback on ash communities, although firm conclusions are not possible because of host preferences and climatic differences. Furthermore, the mycobiota of F. excelsior trees with or without dieback symptoms were compared, but no clear differences were detected. Besides methodical refinement, our study provides comprehensive data on the ash mycobiome that we expect to be subject to changes caused by an emerging disease of the host tree.