Grit Walther

One stop shop: backbones trees for important phytopathogenic genera: I (2014)

Many fungi are pathogenic on plants and cause significant damage in agriculture and forestry. They are also part of the natural ecosystem and may play a role in regulating plant numbers/density. Morphological identification and analysis of plant pathogenic fungi, while important, is often hampered by the scarcity of discriminatory taxonomic characters and the endophytic or inconspicuous nature of these fungi. Molecular (DNA sequence) data for plant pathogenic fungi have emerged as key information for diagnostic and classification studies, although hampered in part by non-standard laboratory practices and analytical methods. To facilitate current and future research, this study provides phylogenetic synopses for 25 groups of plant pathogenic fungi in the Ascomycota, Basidiomycota, Mucormycotina (Fungi), and Oomycota, using recent molecular data, up-to-date names, and the latest taxonomic insights. Lineage-specific laboratory protocols together with advice on their application, as well as general observations, are also provided. We hope to maintain updated backbone trees of these fungal lineages over time and to publish them jointly as new data emerge. Researchers of plant pathogenic fungi not covered by the present study are invited to join this future effort. Bipolaris, Botryosphaeriaceae, Botryosphaeria, Botrytis, Choanephora, Colletotrichum, Curvularia, Diaporthe, Diplodia, Dothiorella, Fusarium, Gilbertella, Lasiodiplodia, Mucor, Neofusicoccum, Pestalotiopsis, Phyllosticta, Phytophthora, Puccinia, Pyrenophora, Pythium, Rhizopus, Stagonosporopsis, Ustilago and Verticillium are dealt with in this paper.

DNA barcoding in Mucorales: an inventory of biodiversity.

The order Mucorales comprises predominantly fast-growing saprotrophic fungi, some of which are used for the fermentation of foodstuffs but it also includes species known to cause infections in patients with severe immune or metabolic impairments. To inventory biodiversity in Mucorales ITS barcodes of 668 strains in 203 taxa were generated covering more than two thirds of the recognised species. Using the ITS sequences, Molecular Operational Taxonomic Units were defined by a similarity threshold of 99 %. An LSU sequence was generated for each unit as well. Analysis of the LSU sequences revealed that conventional phenotypic classifications of the Mucoraceae are highly artificial. The LSU- and ITS-based trees suggest that characters, such as rhizoids and sporangiola, traditionally used in mucoralean taxonomy are plesiomorphic traits. The ITS region turned out to be an appropriate barcoding marker in Mucorales. It could be sequenced directly in 82 % of the strains and its variability was sufficient to resolve most of the morphospecies. Molecular identification turned out to be problematic only for the species complexes of Mucor circinelloides, M. flavus, M. piriformis and Zygorhynchus moelleri. As many as 12 possibly undescribed species were detected. Intraspecific variability differed widely among mucorealean species ranging from 0 % in Backusella circina to 13.3 % in Cunninghamella echinulata. A high proportion of clinical strains was included for molecular identification. Clinical isolates of Cunninghamella elegans were identified molecularly for the first time. As a result of the phylogenetic analyses several taxonomic and nomenclatural changes became necessary. The genus Backusella was emended to include all species with transitorily recurved sporangiophores. Since this matched molecular data all Mucor species possessing this character were transferred to Backusella. The genus Zygorhynchus was shown to be polyphyletic based on ITS and LSU data. Consequently, Zygorhynchus was abandoned and all species were reclassified in Mucor. Our phylogenetic analyses showed, furthermore, that all non-thermophilic Rhizomucor species belong to Mucor. Accordingly, Rhizomucor endophyticus was transferred to Mucor and Rhizomucor chlamydosporus was synonymised with Mucor indicus. Lecto-, epi- or neotypes were designated for several taxa.

Species Recognition and Clinical Relevance of the Zygomycetous Genus Lichtheimia (syn. Absidia Pro Parte, Mycocladus)

ABSTRACT The zygomycete genus Lichtheimia (syn. Absidia pro parte, Mycocladus) consists of saprotrophic fungi inhabiting soil or dead plant material. Lichtheimia corymbifera (syn. Absidia corymbifera, Mycocladus corymbifer) and Lichtheimia ramosa (syn. Absidia ramosa, Mycocladus ramosus) may cause fulminant infections in patients with impaired immunity. The present study investigated the species boundaries in Lichtheimia using genealogical concordance phylogenetic species recognition (by comparison of the genealogies of the internal transcribed spacer [ITS] sequence, the D1/D2 region of the large subunit [LSU], and actin), biological species recognition by mating tests, as well as morphological and physiological characteristics. The three molecular markers used were selected by evaluating the polymorphisms and paralogies of several loci, including those for β-tubulin, translation elongation factor 1α, the two largest subunits of the RNA polymerase II (RPB1 and RPB2), the mitochondrial cytochrome c oxidase subunit I (COI), and the mitochondrial small-subunit (mtSSU) rDNA, among four strains belonging to different putative species. Comparing the genealogies of the ITS, LSU, and actin genes, we recognized seven phylogenetic species. However, mating tests did not show intrinsic reproductive barriers for two pairs of the phylogenetic species. Therefore, we regard five species in Lichtheima to be confirmed: Lichtheimia corymbifera, L. ornata comb. nov., L. ramosa, L. hyalospora, and L. sphaerocystis sp. nov. Only the first three species seem to have clinical relevance. Lichtheimia blakesleeana is reduced to a synonym of Lichtheimia hyalospora. We provide a detailed description of Lichtheimia sphaerocystis sp. nov. and a key for the identification of all accepted species identified in the present study on the basis of their morphological traits and growth at different temperatures.

Antifungal Susceptibility and Phylogeny of Opportunistic Members of the Order Mucorales

ABSTRACT The in vitro susceptibilities of 66 molecularly identified strains of the Mucorales to eight antifungals (amphotericin B, terbinafine, itraconazole, posaconazole, voriconazole, caspofungin, micafungin, and 5-fluorocytosine) were tested. Molecular phylogeny was reconstructed based on the nuclear ribosomal large subunit to reveal taxon-specific susceptibility profiles. The impressive phylogenetic diversity of the Mucorales was reflected in susceptibilities differing at family, genus, and species levels. Amphotericin B was the most active drug, though somewhat less against Rhizopus and Cunninghamella species. Posaconazole was the second most effective antifungal agent but showed reduced activity in Mucor and Cunninghamella strains, while voriconazole lacked in vitro activity for most strains. Genera attributed to the Mucoraceae exhibited a wide range of MICs for posaconazole, itraconazole, and terbinafine and included resistant strains. Cunninghamella also comprised strains resistant to all azoles tested but was fully susceptible to terbinafine. In contrast, the Lichtheimiaceae completely lacked strains with reduced susceptibility for these antifungals. Syncephalastrum species exhibited susceptibility profiles similar to those of the Lichtheimiaceae. Mucor species were more resistant to azoles than Rhizopus species. Species-specific responses were obtained for terbinafine where only Rhizopus arrhizus and Mucor circinelloides were resistant. Complete or vast resistance was observed for 5-fluorocytosine, caspofungin, and micafungin. Intraspecific variability of in vitro susceptibility was found in all genera tested but was especially high in Mucor and Rhizopus for azoles and terbinafine. Accurate molecular identification of etiologic agents is compulsory to predict therapy outcome. For species of critical genera such as Mucor and Rhizopus, exhibiting high intraspecific variation, susceptibility testing before the onset of therapy is recommended.

The systematic relevance of conidiogenesis modes in the gilled Agaricales

Dikaryotic and haploid mycelia of more than 150 gilled species of euagarics were studied morphologically and by molecular phylogenetic methods. The morphological investigations revealed anamorphs in more than 90 species that were often specific at the genus or family level. Thallic conidiogenesis dominated and varied from fragmentation of normally branched hyphae to the formation of differentiated sympodially branched conidiophores. Secession modes, coiling of the conidiogenous hyphae or the swelling of the conidia were additional distinguishing features. Phylogenetic analysis of the D1-D3 domains of the nuclear gene for the ribosomal large subunit using a Bayesian Markov chain Monte Carlo approach resulted in several well-supported groups that are consistent with anamorph morphology. These results indicate that the anamorphs provide valuable characters for a natural classification of the Agaricales.

The family structure of the Mucorales: a synoptic revision based on comprehensive multigene-genealogies

The Mucorales (Mucoromycotina) are one of the most ancient groups of fungi comprising ubiquitous, mostly saprotrophic organisms. The first comprehensive molecular studies 11 yr ago revealed the traditional classification scheme, mainly based on morphology, as highly artificial. Since then only single clades have been investigated in detail but a robust classification of the higher levels based on DNA data has not been published yet. Therefore we provide a classification based on a phylogenetic analysis of four molecular markers including the large and the small subunit of the ribosomal DNA, the partial actin gene and the partial gene for the translation elongation factor 1-alpha. The dataset comprises 201 isolates in 103 species and represents about one half of the currently accepted species in this order. Previous family concepts are reviewed and the family structure inferred from the multilocus phylogeny is introduced and discussed. Main differences between the current classification and preceding concepts affects the existing families Lichtheimiaceae and Cunninghamellaceae, as well as the genera Backusella and Lentamyces which recently obtained the status of families along with the Rhizopodaceae comprising Rhizopus, Sporodiniella and Syzygites. Compensatory base change analyses in the Lichtheimiaceae confirmed the lower level classification of Lichtheimia and Rhizomucor while genera such as Circinella or Syncephalastrum completely lacked compensatory base changes.

Direct Analysis and Identification of Pathogenic Lichtheimia Species by Matrix-Assisted Laser Desorption Ionization–Time of Flight Analyzer-Mediated Mass Spectrometry

ABSTRACT Zygomycetes of the order Mucorales can cause life-threatening infections in humans. These mucormycoses are emerging and associated with a rapid tissue destruction and high mortality. The resistance of Mucorales to antimycotic substances varies between and within clinically important genera such as Mucor, Rhizopus, and Lichtheimia. Thus, an accurate diagnosis before onset of antimycotic therapy is recommended. Matrix-assisted laser desorption ionization (MALDI)–time of flight (TOF) mass spectrometry (MS) is a potentially powerful tool to rapidly identify infectious agents on the species level. We investigated the potential of MALDI-TOF MS to differentiate Lichtheimia species, one of the most important agents of mucormycoses. Using the Bruker Daltonics FlexAnalysis (version 3.0) software package, a spectral database library with m/z ratios of 2,000 to 20,000 Da was created for 19 type and reference strains of clinically relevant Zygomycetes of the order Mucorales (12 species in 7 genera). The database was tested for accuracy by use of 34 clinical and environmental isolates of Lichtheimia comprising a total of five species. Our data demonstrate that MALDI-TOF MS can be used to clearly discriminate Lichtheimia species from other pathogenic species of the Mucorales. Furthermore, the method is suitable to discriminate species within the genus. The reliability and robustness of the MALDI-TOF-based identification are evidenced by high score values (above 2.3) for the designation to a certain species and by moderate score values (below 2.0) for the discrimination between clinically relevant (Lichtheimia corymbifera, L. ramosa, and L. ornata) and irrelevant (L. hyalospora and L. sphaerocystis) species. In total, all 34 strains were unequivocally identified by MALDI-TOF MS with score values of >1.8 down to the generic level, 32 out of 34 of the Lichtheimia isolates (except CNM-CM 5399 and FSU 10566) were identified accurately with score values of >2 (probable species identification), and 25 of 34 isolates were identified to the species level with score values of >2.3 (highly probable species identification). The MALDI-TOF MS-based method reported here was found to be reproducible and accurate, with low consumable costs and minimal preparation time.

Molecular phylogeny of the Entomophthoromycota.

The Entomophthoromycota is a ubiquitous group of fungi best known as pathogens of a wide variety of economically important insect pests, and other soil invertebrates. This group of fungi also includes a small number of parasites of reptiles, vertebrates (including humans), macromycetes, fern gametophytes, and desmid algae, as well as some saprobic species. Here we report on recent studies to resolve the phylogenetic relationships within the Entomophthoromycota and to reliably place this group among other basal fungal lineages. Bayesian Interference (BI) and Maximum Likelihood (ML) analyses of three genes (nuclear 18S and 28S rDNA, mitochondrial 16S, and the protein-coding RPB2) as well as non-molecular data consistently and unambiguously identify 31 taxa of Entomophthoromycota as a monophyletic group distinct from other Zygomycota and flagellated fungi. Using the constraints of our multi-gene dataset we constructed the most comprehensive rDNA phylogeny yet available for Entomophthoromycota. The taxa studied here belong to five distinct, well-supported lineages. The Basidiobolus clade is the earliest diverging lineage, comprised of saprobe species of Basidiobolus and the undescribed snake parasite Schizangiella serpentis nom. prov. The Conidiobolus lineage is represented by a paraphyletic grade of trophically diverse species that include saprobes, insect pathogens, and facultative human pathogens. Three well supported and exclusively entomopathogenic lineages in the Entomophthoraceae center around the genera Batkoa, Entomophthora and Zoophthora, although several genera within this crown clade are resolved as non-monophyletic. Ancestral state reconstruction suggests that the ancestor of all Entomophthoromycota was morphologically similar to species of Conidiobolus. Analyses using strict, relaxed, and local molecular clock models documented highly variable DNA substitution rates among lineages of Entomophthoromycota. Despite the complications caused by different rates of molecular evolution among lineages, our dating analysis indicates that the Entomophthoromycota originated 405±90 million years ago. We suggest that entomopathogenic lineages in Entomophthoraceae probably evolved from saprobic or facultatively pathogenic ancestors during or shortly after the evolutionary radiation of the arthropods.

Understanding the Evolutionary Processes of Fungal Fruiting Bodies: Correlated Evolution and Divergence Times in the Psathyrellaceae

Fruiting body evolution is one of the central topics in fungal evolutionary biology. A number of hypotheses have been developed to explain the contemporary diversity of fruiting body forms, but their evaluation has been hampered by the lack of well-sampled data sets and suitable statistical methods. Phylogenetic evidence of the physiological changes that accompany switches in fruiting body type is lacking, and very little is known about the age of major events of fruiting body evolution. Based on a new multigene phylogeny, by using Bayesian methods, we demonstrate the existence of correlation between a number of morphological features and switches from nondeliquescent to deliquescent (autodigesting) fruiting bodies in the mushroom family Psathyrellaceae. Our results show that switches in the anatomy of two types of spacer cells (cystidia and pseudoparaphyses) and basidia (bimorphic or monomorphic) as well as the structure of the mushroom cap follow the evolution of deliquescent fruiting bodies, which suggests strong functional linkage between these traits. We performed Bayes factor-based tests, referred hereafter to as evolutionary pathway test (EPT), to decide which of the correlated characters were gained first during evolution. The EPTs strongly suggest that deliquescence was gained first, followed after short waiting times by the other morphological features. Bayesian relaxed molecular clock analyses suggest that the various events of switching between fruiting body types occurred independently at various ages during the history of the family. The utility of two mushroom fossils (Archaemarasmius and Protomycena), the only ones with unambiguous taxonomic positions, for the calibration of agaric trees were also examined. Based on our results, we suggest that the evolutionary benefit of deliquescence may be prevention against desiccation via accelerated ontogeny of the fruiting body. Hypotheses regarding the functional significance of the correlated evolution are presented and discussed. Further, we argue that the changes in fruiting body types in mushrooms in general can be attributed to independent events (e.g., dispersal and adaptation) and not to particular geologic ages.

Diversity and delimitation of Rhizopus microsporus

Rhizopus microsporus has been used for centuries in the production of oriental fermented foods, but the species is also known as a toxin producer and from severe human infections. To study the diversity and species delimitation of Rhizopus microsporus, 48 isolates from the reference collection of the CBS-KNAW Fungal Biodiversity Centre, comprising nine environmental, ten clinical, and 23 foodborne strains, in addition to six strains from unknown sources and representing all existing varieties of the species, were examined. Sequence diversity was based on the internal transcribed spacer (ITS), and on a part of the actin (ACT) and translation elongation factor 1-α (TEF) genes. Differences in physiological properties were assessed including temperature relationships. Spore morphology was studied, mating type tests were performed, and MALDI-ToF profiles were generated. Clinical and food-associated strains as well as members of different varieties mated successfully and consequently they belong to a single biological species. Molecular differences did not match with any other parameter investigated. Based on these results the varieties of Rhizopus microsporus are reduced to synonyms.