Julia Kruse

Towards a universal barcode of oomycetes - a comparison of the cox1 and cox2 loci

Oomycetes are a diverse group of eukaryotes in terrestrial, limnic and marine habitats worldwide and include several devastating plant pathogens, for example Phytophthora infestans (potato late blight). The cytochrome c oxidase subunit 2 gene (cox2) has been widely used for identification, taxonomy and phylogeny of various oomycete groups. However, recently the cox1 gene was proposed as a DNA barcode marker instead, together with ITS rDNA. The cox1 locus has been used in some studies of Pythium and Phytophthora, but has rarely been used for other oomycetes, as amplification success of cox1 varies with different lineages and sample ages. To determine which out of cox1 or cox2 is best suited as a universal oomycete barcode, we compared these two genes in terms of (i) PCR efficiency for 31 representative genera, as well as for historic herbarium specimens, and (ii) sequence polymorphism, intra‐ and interspecific divergence. The primer sets for cox2 successfully amplified all oomycete genera tested, while cox1 failed to amplify three genera. In addition, cox2 exhibited higher PCR efficiency for historic herbarium specimens, providing easier access to barcoding‐type material. Sequence data for several historic type specimens exist for cox2, but there are none for cox1. In addition, cox2 yielded higher species identification success, with higher interspecific and lower intraspecific divergences than cox1. Therefore, cox2 is suggested as a partner DNA barcode along with ITS rDNA instead of cox1. The cox2‐1 spacer could be a useful marker below species level. Improved protocols and universal primers are presented for all genes to facilitate future barcoding efforts.

Asexual and sexual morphs of Moesziomyces revisited

Yeasts of the now unused asexually typified genus Pseudozyma belong to the smut fungi (Ustilaginales) and are mostly believed to be apathogenic asexual yeasts derived from smut fungi that have lost pathogenicity on plants. However, phylogenetic studies have shown that most Pseudozyma species are phylogenetically close to smut fungi parasitic to plants, suggesting that some of the species might represent adventitious isolations of the yeast morph of otherwise plant pathogenic smut fungi. However, there are some species, such as Moesziomyces aphidis (syn. Pseudozyma aphidis) that are isolated throughout the world and sometimes are also found in clinical samples and do not have a known plant pathogenic sexual morph. In this study, it is revealed by phylogenetic investigations that isolates of the biocontrol agent Moesziomyces aphidis are interspersed with M. bullatus sexual lineages, suggesting conspecificity. This raises doubts regarding the apathogenic nature of asexual morphs previously placed in Pseudozyma, but suggests that there might also be pathogenic sexual morph counterparts for those species known only from asexual morphs. The finding that several additional species currently only known from their yeast morphs are embedded within the genus Moesziomyces, suggests that the yeast morph might play a more dominant role in this genus as compared to other genera of Ustilaginaceae. In addition, phylogenetic reconstructions demonstrated that Moesziomyces bullatus has a narrow host range and that some previously described but not widely used species names should be applied for Moesziomyces on other host genera than Echinochloa.

New smut-specific primers for the ITS barcoding of Ustilaginomycotina

The smut fungi (Ustilaginomycotina) are a highly diverse group, containing about 115 genera and 1700 species, most of which are biotrophic plant pathogens. As for other fungal groups, the ITS rDNA region is widely used to determine smut fungi at species level due to its high discriminatory power and for phylogenetic reconstructions within genera. So far, two primer sets, ITS1/ITS4 and M-ITS1/ITS4, were generally used to amplify smut fungi, but these often co-amplify host plants or contaminant fungi and do not yield satisfactory amplification for a variety of smut fungi. In the present study, based on a selection of genera that include more than 90% of the species of smut fungi (more than half of the genera of smut fungi), three new primers, smITS-F, smITS-R1 and smITS-R2, situated in the SSU or LSU region, were designed to avoid the amplification of host plants and to extend the coverage of PCR amplification for as many smut genera as possible.

Revision of Plasmopara (Oomycota, Peronosporales) parasitic to Impatiens

The oomycete Plasmopara obducens was first described on wild Impatiens noli-tangere in Germany in 1877. About 125 years later the first occurrence of P. obducens on cultivated I. walleriana in the United Kingdom was reported, and a worldwide epidemic followed. Although this pathogen is a major threat for ornamental busy lizzy, the identity of the pathogen remained unconfirmed and the high host specificity observed for the genus Plasmopara cast doubts regarding its determination as P. obducens. In this study, using multigene phylogenies and morphological investigation, it is revealed that P. obducens on I. noli-tangere is not the conspecific with the pathogen affecting I. walleriana and another ornamental balsam, I. balsamina. As a consequence, the new names P. destructor and P. velutina are introduced for the pathogens of I. walleriana and I. balsamina, respectively.

Ustilago species causing leaf-stripe smut revisited

Leaf-stripe smuts on grasses are a highly polyphyletic group within Ustilaginomycotina, occurring in three genera, Tilletia, Urocystis, and Ustilago. Currently more than 12 Ustilago species inciting stripe smuts are recognised. The majority belong to the Ustilago striiformis-complex, with about 30 different taxa described from 165 different plant species. This study aims to assess whether host distinct-lineages can be observed amongst the Ustilago leaf-stripe smuts using nine different loci on a representative set. Phylogenetic reconstructions supported the monophyly of the Ustilago striiformis-complex that causes leaf-stripe and the polyphyly of other leaf-stripe smuts within Ustilago. Furthermore, smut specimens from the same host genus generally clustered together in well-supported clades that often had available species names for these lineages. In addition to already-named lineages, three new lineages were observed, and described as new species on the basis of host specificity and molecular differences: namely Ustilago jagei sp. nov. on Agrostis stolonifera, U. kummeri sp. nov. on Bromus inermis, and U. neocopinata sp. nov. on Dactylis glomerata.

Confirmation of Peronospora agrimoniae as a distinct species

Leaves with typical symptoms of downy mildew were found on common agrimony in the Czech Republic in 2014 and 2015 and at several locations in Germany from 2010 to 2014. The causal agent of downy mildew of agrimony was often reported as Peronospora agrimoniae, but sometimes also as P. sparsa. Morphological characteristics of the pathogens found in both countries are in the range of previous works for P. agrimoniae, but also other downy mildews parasitic on Rosaceae, rendering their discrimination based on published observations difficult. For molecular identification sequencing of several loci (nrITS rDNA, cox1 and cox2) was performed. Phylogenetic analyses based on nrITS rDNA clearly separated P. agrimoniae from other Peronospora species infecting Rosaceae. Thus, considering P. agrimoniae as separate species seems justified. Two German specimens were identical to two Czech samples in both nrITS rDNA and cox1 mtDNA sequences, but differed in a single nucleotide substitution in cox2 region. To our knowledge, this is the first verified record of P. agrimoniae on common agrimony in the Czech Republic.

The first smut fungus, Thecaphora anthemidis sp. nov. (Glomosporiaceae), described from Anthemis (Asteraceae)

Abstract There are 63 known species of Thecaphora (Glomosporiaceae, Ustilaginomycotina), a third of which occur on Asteraceae. These smut fungi produce yellowish-brown to reddish-brown masses of spore balls in specific, mostly regenerative, plant organs. A species of Thecaphora was collected in the flower heads of Anthemischia (Anthemideae, Asteraceae) on Rhodes Island, Greece, in 2015 and 2017, which represents the first smut record of a smut fungus on a host plant species in this tribe. Based on its distinctive morphology, host species and genetic divergence, this species is described as Thecaphoraanthemidissp. nov. Molecular barcodes of the ITS region are provided for this and several other species of Thecaphora. A phylogenetic and morphological comparison to closely related species showed that Th.anthemidis differed from other species of Thecaphora. Thecaphoraanthemidis produced loose spore balls in the flower heads and peduncles of Anthemischia unlike other flower-infecting species.

New smut-specific primers for multilocus genotyping and phylogenetics of Ustilaginaceae

The Ustilaginomycotina, often collectively referred to as smut fungi, represent one of the three subphyla of the Basidiomycota. Smut fungi predominantly parasitize Angiosperms, are globally distributed, and contain several economically important pathogens. The most species-rich family of the smut fungi is the Ustilaginaceae. To investigate the molecular phylogeny of smut fungi, most studies rely on nrDNA loci, such as ITS and LSU. Protein coding genes, like rpb1, rpb2, TeF1a, atp6, and ß-tubulin, have been used in some studies. However, because of the huge diversity of smut fungi and the lack of dedicated primers, amplification of these loci has proven difficult for several groups. Thus, it was the aim of the current study to develop primers for new loci for the smut fungi with the focus on the largest family, the Ustilaginaceae. Here, the development and testing of new primers for nine loci based on protein-coding genes is reported (myosin, map, rpl3, tif2, ssc1, ß-tubulin, sdh1, rpl4A and atp2). A list of various primer combinations for the amplification of the new loci is given, with the corresponding PCR conditions and the best combinations for several genera of the Ustilaginaceae and some other Ustilaginomycetes. We hope that the primers presented in this study will be useful in overcoming the limitations of currently-used loci in terms of phylogenetic resolution, especially with respect to resolving species complexes and providing a better resolution of the higher-level phylogenetic relationships of smut fungi.