Sebastian Ploch

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.

The local environment determines the assembly of root endophytic fungi at a continental scale.

Root endophytic fungi are found in a great variety of plants and ecosystems, but the ecological drivers of their biogeographic distribution are poorly understood. Here, we investigate the occurrence of root endophytes in the non-mycorrhizal plant genus Microthlaspi, and the effect of environmental factors and geographic distance in structuring their communities at a continental scale. We sampled 52 plant populations across the northern Mediterranean and central Europe and used a cultivation approach to study their endophytic communities. Cultivation of roots yielded 2601 isolates, which were grouped into 296 operational taxonomic units (OTUs) by internal transcribed spacer sequencing of 1998 representative colonies. Climatic and spatial factors were the best descriptors of the structure of endophytic communities, outweighing soil characteristics, host genotype and geographical distance. OTU richness was negatively affected by precipitation, and the composition of communities followed latitudinal gradients of precipitation and temperature. Only six widespread OTUs belonging to the orders Pleosporales, Hypocreales and Helotiales represented about 50% of all isolates. Assessments of their individual distribution revealed particular ecological preferences or a cosmopolitan occurrence. Our findings support a strong influence of the local environment in determining root endophytic communities, and show a different niche occupancy by individual endophytes.

Evolution of diversity in Albugo is driven by high host specificity and multiple speciation events on closely related Brassicaceae.

The Albuginaceae, responsible for white blister rust disease on various angiosperms, are obligate biotrophic oomycetes that are only distantly related to downy mildews (Peronosporaceae). Their diversity has been much underestimated during the past decades, mainly because of the paucity of morphological characters for species delimitation, which led to the application of a broad species concept. Recent phylogenetic analyses have revealed three new species within Albugo parasitic to Brassicaceae, but the overall evolution of these plant pathogens remains poorly understood. Especially the diversity of Albugo in various plant genera is almost completely unknown. Based on ITS and cox2 sequence data of 72 Albugo specimens, predominantly from herbarium archives, and focusing on the widespread genus Cardamine, a high degree of phylogenetic diversity was revealed in Albugo. In particular, the hypothesis that one host genus can be colonised by more than one white blister rust species is confirmed. In addition, it is revealed that there are hitherto overlooked lineages with close relationships to the generalist species Albugo candida. Evidence for at least three different species of Albugo infecting Cardamine is presented in this study. Based on molecular phylogenetic and morphological data three new white blister rust species are described, Albugo hohenheimia, Albugo hesleri, and Albugo leimonios infecting Cardamine hirsuta, Cardamine diphylla and Cardamine pratensis, respectively. The fact that these species each have different ecological niches, suggests that environmental factors may have played a role in the speciation process in Albugo. Our findings suggest that other larger genera of the Brassicaceae may harbour unrecognized white blister rust species and that only a small fraction of the true biodiversity of white blister rusts is known at present.

Obligate biotrophic pathogens of the genus Albugo are widespread as asymptomatic endophytes in natural populations of Brassicaceae

Mutualistic interactions of plants with true fungi are a well‐known and widespread phenomenon, which includes mycorrhiza and non‐mycorrhizal endophytes like species of Epichloë. Despite the fact that these organisms intrude into plants, neither strong defence reactions nor the onset of symptoms of disease can be observed in most or even all infested plants, in contrast to endophytic pathogens. Oomycetes are fungal‐like organisms belonging to the kingdom Straminipila, which includes diatoms and seaweeds. Although having evolved many convergent traits with true fungi and occupying similar evolutionary niches, widespread oomycete endophytes are not known to date, although more than 500 endophytic pathogens, including species of the obligate biotrophic genus Albugo, have been described. Here, we report that oomycetes of the genus Albugo are widespread in siliques of natural host populations. A total of 759 plants, encompassing four genera with rare reports of white blister incidents and one with common incidents, were collected from 25 sites in Germany. Nested PCR with species‐specific primers revealed that 5–27% of the hosts with rare disease incidence carried asymptomatic Albugo in their siliques, although only on a single plant of 583 individuals, an isolated pustule on a single leaf could be observed. Control experiments confirmed that these results were not because of attached spores, but because of endophytic mycelium. Vertical inheritance of oomycete infections has been reported for several plant pathogens, and it seems likely that in nature this way of transmission plays an important role in the persistence of asymptomatic endophytic Albugo species.

The inclusion of downy mildews in a multi-locus-dataset and its reanalysis reveals a high degree of paraphyly in Phytophthora.

Pathogens belonging to the Oomycota, a group of heterokont, fungal-like organisms, are amongst the most notorious pathogens in agriculture. In particular, the obligate biotrophic downy mildews and the hemibiotrophic members of the genus Phytophthora are responsible for a huge variety of destructive diseases, including sudden oak death caused by P. ramorum, potato late blight caused by P. infestans, cucurbit downy mildew caused by Pseudoperonospora cubensis, and grape downy mildew caused by Plasmopara viticola. About 800 species of downy mildews and roughly 100 species of Phytophthora are currently accepted, and recent studies have revealed that these groups are closely related. However, the degree to which Phytophthora is paraphyletic and where exactly the downy mildews insert into this genus in relation to other clades could not be inferred with certainty to date. Here we present a molecular phylogeny encompassing all clades of Phytophthora as represented in a multi-locus dataset and two representatives of the monophyletic downy mildews from divergent genera. Our results demonstrate that Phytophthora is at least six times paraphyletic with respect to the downy mildews. The downy mildew representatives are consistently nested within clade 4 (contains Phytophthora palmivora), which is placed sister to clade 1 (contains Phytophthora infestans). This finding would either necessitate placing all downy mildews and Phytopthora species in a single genus, either under the oldest generic name Peronospora or by conservation the later name Phytophthora, or the description of at least six new genera within Phytophthora. The complications of both options are discussed, and it is concluded that the latter is preferable, as it warrants fewer name changes and is more practical.

Root-associated fungi of Arabidopsis thaliana and Microthlaspi perfoliatum

Root-associated fungi, with the focus on endophytic species, were isolated from healthy Arabidopsis thaliana and Microthlaspi perfoliatum plants collected at different locations in Germany. A large number of fungal taxa were discovered with a small-scale approach. This provides additional evidence that root-associated and endophytic fungi are common in Brassicaceae. The most prevalent genera associated with A. thaliana roots were Trichoderma and Fusarium, while the roots of M. perfoliatum were dominated by different species of Fusarium and Penicillium. Differences in species composition and richness might be due to preferences and life-cycle of the two plant species. Strains of endophyte species that did not have closely related species in GenBank searches and those already known as root endophytes were chosen for preliminary co-cultivation experiments using germinating host plants on agar medium to observe effects on plant growth and health. Under these conditions several fungal isolates had an adverse effect on plant growth and health, especially on Arabidopsis thaliana. Some isolates did not adversely affect biomass during initial plant growth, while they altered the shoot-root ratio in favour of the shoot, especially in Microthlaspi perfoliatum. These strains are promising candidates for future research on endophytes as they might have some effects in Brassicaceae that are similar to mycorrhizal fungi. They are also promising candidates for investigating interactions with their host plants.

Three new phylogenetic lineages are the closest relatives of the widespread species Albugo candida

White blister rust caused by the obligate biotroph Albugo candida (Albuginaceae; Oomycota) is one of the most notorious and common diseases of Brassicaceae. During the past 5 y, A. candida specimens collected from about 30 host genera were phylogenetically and morphologically investigated in several studies. These not only revealed that A. candida s.str. has a broad host range, encompassing a large number of host plants belonging to Brassicales, but also the presence of previously overlooked species of Albugo with hosts in this order. In this study, we examined specimens from Alyssum, Barbarea, and Rorippa, of which many species were commonly recorded as host plants of A. candida but could not be included in previous works due to the paucity of specimens available. It was revealed that Albugo specimens from Alyssum montanum, Barbarea vulgaris, and various Rorippa species, were placed in three phylogenetically distinct clades, but closer to A. candida s.str. than any previously reported species. Oospores were observed from Albugo specimens parasitic to Rorippa and could be distinguished morphologically from A. candida. Therefore, Albugo rorippae sp. nov. is described and illustrated here. In addition, a key of Albugo species described previously from Brassicales is given. The present study reveals that a large number of Albugo species remain still undiscovered, and that species close to A. candida exist. This could help elucidating the basis of the broad host range of A. candida as opposed to the narrow specialisation that is seemingly present in other species of Albugo on the Brassicaceae.

Seed transmission of Pseudoperonospora cubensis.

Pseudoperonospora cubensis, an obligate biotrophic oomycete causing devastating foliar disease in species of the Cucurbitaceae family, was never reported in seeds or transmitted by seeds. We now show that P. cubensis occurs in fruits and seeds of downy mildew-infected plants but not in fruits or seeds of healthy plants. About 6.7% of the fruits collected during 2012–2014 have developed downy mildew when homogenized and inoculated onto detached leaves and 0.9% of the seeds collected developed downy mildew when grown to the seedling stage. This is the first report showing that P. cubensis has become seed-transmitted in cucurbits. Species-specific PCR assays showed that P. cubensis occurs in ovaries, fruit seed cavity and seed embryos of cucurbits. We propose that international trade of fruits or seeds of cucurbits might be associated with the recent global change in the population structure of P. cubensis.

The molecular phylogeny of the white blister rust genus Pustula reveals a case of underestimated biodiversity with several undescribed species on ornamentals and crop plants

Despite their economic importance, the knowledge of the biodiversity of many plant pathogens is still fragmentary. In this study we show that this is true also for the white blister rust genus Pustula that is parasitic on several genera in the asterids, including sunflower and the gentian, Eustoma. It is revealed that several distinct species exist in Pustula, suggesting that species are mostly host genus specific. No geographic patterns were observed in the occurrence of Pustula, the host range of which includes the Araliaceae, Asteraceae, Gentianaceae, and Goodeniaceae. Evidence points to these becoming hosts as a result of jumps from the Asteraceae, with subsequent host-specific adaptation and speciation. Among the undescribed species are pathogens of economic importance, e.g. the white blister rusts of sunflower, or with still restricted geographical ranges, e.g. Pustula centaurii, which could potentially spread with international seed trade, if no quarantine restrictions are implemented.

High Diversity Revealed in Leaf‐Associated Protists (Rhizaria: Cercozoa) of Brassicaceae

The largest biological surface on earth is formed by plant leaves. These leaf surfaces are colonized by a specialized suite of leaf‐inhabiting microorganisms, recently termed “phyllosphere microbiome”. Microbial prey, however, attract microbial predators. Protists in particular have been shown to structure bacterial communities on plant surfaces, but virtually nothing is known about the community composition of protists on leaves. Using newly designed specific primers targeting the 18S rDNA gene of Cercozoa, we investigated the species richness of this common protist group on leaves of four Brassicaceae species from two different locations in a cloning‐based approach. The generated sequences revealed a broad diversity of leaf‐associated Cercozoa, mostly bacterial feeders, but also including known plant pathogens and a taxon of potential endophytes that were recently described as algal predators in freshwater systems. This initial study shows that protists must be regarded as an integral part of the microbial diversity in the phyllosphere of plants.