Potato Wart Isolates from Europe and North America Form Distinct Clusters of Genetic Variation.

We have extended previously published sets of simple sequence repeat markers for Synchytrium endobioticum, selected to be polymorphic for the German-standard isolates of pathotypes P1, P2, P6, P8, and P18. These markers also complement the extensive published information on DNA polymorphisms for the mitogenomes of Synchytrium endobioticum. This extended set of 35 markers representing 73 alleles differentiated 51 isolates from Europe and North America into three large, well-separated clusters and subclusters using dendrogram analysis, principal coordinates analysis (PCoA), and population substructure analysis using STRUCTURE 2.3.4 software. This suggests a limited number of introgressions of the wart disease pathogen into current potato growing areas, followed by recombination and admixture of populations through human activities. The new markers extend the published marker sets and are useful tools for future analyses of population structure and dynamics in Synchytrium endobioticum, which are necessary to understand the biology of the interaction between the pathogen and its potato host and to develop future control strategies.

Quantification of DNA of Fusarium culmorum and Trichothecene Genotypes 3ADON and NIV in the Grain of Winter Wheat.

Fusarium head blight (FHB) is a wheat disease caused by fungi of the genus Fusarium. The aim of the study was to find relationships between the weather conditions in the experimental years and the locations and the amount of F. culmorum DNA and trichothecene genotypes, as well as the proportions between them. A three-year field experiment (2017, 2018 and 2019) was established in two locations (Poznan, Radzików). The DNA of F. culmorum was detected in all grain samples in an average amount of 20,124 pg per 1 µg of wheat DNA. The average amount of DNA from the 3ADON genotype was 4879 pg/µg and the amount of DNA from the NIV genotype was 3330 pg/µg. Weather conditions strongly affected the amount of DNA of F. culmorum and trichothecene genotypes detected in the grain. In the three experimental years, a high variability was observed in the coefficients of correlation between DNA concentrations and the FHB index, FDK, ergosterol and the corresponding toxins. There were significant correlations between disease incidence, fungal biomass (quantified as the total amount of fungal DNA or DNA trichothecene genotypes) and toxins (DON, 3AcDON and NIV) concentrations. The 3ADON trichothecene genotype dominated over the NIV genotype (ratio 1.5); however, this varied greatly depending on environmental conditions.

Comparative proteomic analysis of resistant and susceptible potato cultivars during Synchytrium endobioticum infestation.

MAIN CONCLUSION: We report the first comparative study of protein expression profiles in tuber sprouts between Katahdin-derived potato cultivars resistant and susceptible to Synchytrium endobioticum. Synchytrium endobioticum causes wart disease in potato (Solanum tuberosum L.) and is considered as the most important quarantine pathogen in almost all countries where potatoes are grown. We performed a comparative analysis of differentially expressed proteins in the tuber sprouts of potato cultivars differing in resistance to pathotype 1(D1) of S. endobioticum using two-dimensional electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) approaches. Bulks prepared from two resistant (Calrose and Humalda) and three susceptible (Sebago, Seneca and Wauseon) potato cultivars were studied. When protein profiles were compared between mock- and S. endobioticum-inoculated sprouts, 35 and 63 protein spots, indicating qualitative or quantitative differences, were detected in the resistant and susceptible cultivars, respectively. In turn, 24 proteins associated with resistance to S. endobioticum were revealed by comparison of the resistant and susceptible bulks. These proteins were changed in a constitutive or induced manner and were grouped into four categories: stress and defence, cell structure, protein turnover, and metabolism. Among the 13 proteins classified into the stress and defence group, seven proteins were related to heat-shock proteins (HSPs)/chaperone factors. In addition, four proteins, S-adenosyl-L-homocysteine hydrolase-like, superoxide dismutase [Mn], inactive patatin-3-Kuras 1 and patatin-15, were induced in the resistant bulk; whereas two proteins, patatin-01 and nucleoredoxin 1, showed significant differences in expression between the S. endobioticum-inoculated resistant and susceptible bulks. The detection of such a large number of S. endobioticum-mediated proteins representing the HSP70, HSP60 and HSP20 families suggests their significant role in restricting wart disease in potato tubers.

The Synchytrium endobioticum AvrSen1 Triggers a Hypersensitive Response in Sen1 Potatoes While Natural Variants Evade Detection.

Synchytrium endobioticum is an obligate biotrophic fungus of division Chytridiomycota. It causes potato wart disease, has a worldwide quarantine status and is included on the Health and Human Services and United States Department of Agriculture Select Agent list. S. endobioticum isolates are grouped in pathotypes based on their ability to evade host resistance in a set of differential potato varieties. Thus far, 39 pathotypes are reported. A single dominant gene (Sen1) governs pathotype 1 (D1) resistance and we anticipated that the underlying molecular model would involve a pathogen effector (AvrSen1) that is recognized by the host. The S. endobioticum-specific secretome of 14 isolates representing six different pathotypes was screened for effectors specifically present in pathotype 1 (D1) isolates but absent in others. We identified a single AvrSen1 candidate. Expression of this candidate in potato Sen1 plants showed a specific hypersensitive response (HR), which cosegregated with the Sen1 resistance in potato populations. No HR was obtained with truncated genes found in pathotypes that evaded recognition by Sen1. These findings established that our candidate gene was indeed Avrsen1. The S. endobioticum AvrSen1 is a single-copy gene and encodes a 376-amino-acid protein without predicted function or functional domains, and is the first effector gene identified in Chytridiomycota, an extremely diverse yet underrepresented basal lineage of fungi.

The linear mitochondrial genome of the quarantine chytrid Synchytrium endobioticum; insights into the evolution and recent history of an obligate biotrophic plant pathogen.

BACKGROUND: Chytridiomycota species (chytrids) belong to a basal lineage in the fungal kingdom. Inhabiting terrestrial and aquatic environments, most are free-living saprophytes but several species cause important diseases: e.g. Batrachochytrium dendrobatidis, responsible for worldwide amphibian decline; and Synchytrium endobioticum, causing potato wart disease. S. endobioticum has an obligate biotrophic lifestyle and isolates can be further characterized as pathotypes based on their virulence on a differential set of potato cultivars. Quarantine measures have been implemented globally to control the disease and prevent its spread. We used a comparative approach using chytrid mitogenomes to determine taxonomical relationships and to gain insights into the evolution and recent history of introductions of this plant pathogen. RESULTS: We assembled and annotated the complete mitochondrial genome of 30 S. endobioticum isolates and generated mitochondrial genomes for five additional chytrid species. The mitochondrial genome of S. endobioticum is linear with terminal inverted repeats which was validated by tailing and PCR amplifying the telomeric ends. Surprisingly, no conservation in organisation and orientation of mitochondrial genes was observed among the Chytridiomycota except for S. endobioticum and its sister species Synchytrium microbalum. However, the mitochondrial genome of S. microbalum is circular and comprises only a third of the 72.9 Kbp found for S. endobioticum suggesting recent linearization and expansion. Four mitochondrial lineages were identified in the S. endobioticum mitochondrial genomes. Several pathotypes occur in different lineages, suggesting that these have emerged independently. In addition, variations for polymorphic sites in the mitochondrial genome of individual isolates were observed demonstrating that S. endobioticum isolates represent a community of different genotypes. Such communities were shown to be complex and stable over time, but we also demonstrate that the use of semi-resistant potato cultivars triggers a rapid shift in the mitochondrial haplotype associated with increased virulence. CONCLUSIONS: Mitochondrial genomic variation shows that S. endobioticum has been introduced into Europe multiple times, that several pathotypes emerged multiple times, and that isolates represent communities of different genotypes. Our study represents the most comprehensive dataset of chytrid mitogenomes, which provides new insights into the extraordinary dynamics and evolution of mitochondrial genomes involving linearization, expansion and reshuffling.

Novel gene Sen2 conferring broad-spectrum resistance to Synchytrium endobioticum mapped to potato chromosome XI.

Key message Sen2 gene for potato wart resistance, located on chromosome XI in a locus distinct from Sen1 , provides resistance against eight wart pathotypes, including the virulent ones important in Europe. Synchytrium endobioticum causes potato wart disease imposing severe losses in potato production, and as a quarantine pathogen in many countries, it results in lost trade markets and land for potato cultivation. The resistance to S. endobioticum pathotype 1(D1) is widespread in potato cultivars but new virulent pathotypes appear and the problem re-emerges. To characterize and map a new gene for resistance to potato wart, we used diploid F1 potato population from a cross of potato clone resistant to S. endobioticum pathotype 1(D1) and virulent pathotypes: 2(G1), 6(O1), 8(F1), 18(T1), 2(Ch1), 3(M1) and 39(P1) with a potato clone resistant to pathotype 1(D1) only. The 176 progeny clones were tested for resistance to eight wart pathotypes with a modified Glynne-Lemmerzahl method. Bimodal distributions and co-segregation of resistance in the population show that a single resistance gene, Sen2, underlies the resistance to eight pathotypes. Resistance to pathotype 1(D1) was additionally conferred by the locus Sen1 inherited from both parents. Sen2 was mapped to chromosome XI using DArTseq markers. The genetic and physical distances between Sen1 and Sen2 loci were indirectly estimated at 63 cM and 32 Mbp, respectively. We developed PCR markers co-segregating with the Sen2 locus that can be applied in marker-assisted selection of potatoes resistant to eight important pathotypes of S. endobioticum. Wide spectrum of the Sen2 resistance may be an indication of durability which can be enhanced by the pyramiding of the Sen2 and Sen1 loci as in 61 clones selected within this study.

Euphresco Sendo: An international laboratory comparison study of molecular tests for Synchytrium endobioticum detection and identification.

An international test performance study (TPS) was organised to generate validation data for three molecular Synchytrium endobioticum tests: van den Boogert et al. (European Journal of Plant Pathology 113, 47-57, 2005), and van Gent-Pelzer et al. (European Journal of Plant Pathology, 126, 129-133, 2010) for the detection of S. endobioticum, and the pathotype 1(D1) identification test described by Bonants et al. (European Journal of Plant Pathology, 143, 495-506, 2015). Two TPS rounds were organised focussing on different test matrices, i.e. round 1: warted potato tissue, and round 2: resting spore suspensions. When using the tests for detection and identification of S. endobioticum in warted potato tissue, no significant differences were observed for diagnostic sensitivity, diagnostic specificity, overall accuracy, analytical sensitivity and robustness. When using the tests for detection and identification of S. endobioticum in resting spore suspensions, the van den Boogert and van Gent-Pelzer tests significantly outperform the Bonants test for diagnostic sensitivity and diagnostic specificity. For overall accuracy and analytical sensitivity, the van Gent-Pelzer significantly outperforms the van den Boogert and Bonants tests and is regarded as the test of choice when identifying S. endobioticum from resting spores. Tests regarded fit for purpose for routine testing of wart material and resting spore suspensions are proposed for the update of EPPO standard PM7/28(1) Synchytrium endobioticum.

Genomic and Transcriptomic Resources for Marker Development in Synchytrium endobioticum, an Elusive but Severe Potato Pathogen.

Synchytrium endobioticum is an obligate biotrophic fungus that causes wart diseases in potato. Like other species of the class Chytridiomycetes, it does not form mycelia and its zoospores are small, approximately 3 µm in diameter, which complicates the detection of early stages of infection. Furthermore, potato wart disease is difficult to control because belowground organs are infected and resting spores of the fungus are extremely durable. Thus, S. endobioticum is classified as a quarantine organism. More than 40 S. endobioticum pathotypes have been reported, of which pathotypes 1(D1), 2(G1), 6(O1), 8(F1), and 18(T1) are the most important in Germany. No molecular methods for the differentiation of pathotypes are available to date. In this work, we sequenced both genomic DNA and cDNA of the German pathotype 18(T1) from infected potato tissue and generated 5,422 expressed sequence tags (EST) and 423 genomic contigs. Comparative sequencing of 33 genes, single-stranded confirmation polymorphism (SSCP) analysis with polymerase chain reaction fragments of 27 additional genes, as well as the analysis of 41 simple sequence repeat (SSR) loci revealed extremely low levels of variation among five German pathotypes. From these markers, one sequence-characterized amplified region marker and five SSR markers revealed polymorphisms among the German pathotypes and an extended set of 11 additional European isolates. Pathotypes 8(F1) and 18(T1) displayed discrete polymorphisms which allow their differentiation from other pathotypes. Overall, using the information of the six markers, the 16 isolates could be differentiated into three distinct genotype groups. In addition to the presented markers, the new collection of EST from genus Synchytrium might serve in the future for molecular taxonomic studies as well as for analyses of the host-pathogen interactions in this difficult pathosystem. [Formula: see text] Copyright © 2017 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Tetraploid somatic hybrids of potato (Solanum tuberosum L.) obtained from diploid breeding lines.

Intraspecific somatic hybrids between 16 different diploid breeding lines of Solanum tuberosum L. were produced by PEG-induced fusion. Manually selected heterokaryons were cultured in a Millicells-CM using a post-fusion protoplast mixture. Plants were regenerated from calli derived from heterokaryons obtained from 10 out of 38 combinations of diploid lines. Of the tested putative somatic hybrids, 14.2% were diploid, 72.8% were tetraploid and 13% pentaploid. The DNA amplification pattern obtained with RAPD or semi-random primers confirmed that 6 fusion combinations were hybrids. In most cases, the morphological traits were intermediate to those of the diploid fusion partners. About 23.0% of the tested somatic hybrids showed variation in their morphology. Of the tested somatic hybrids, 78.0% flowered and 86.0% tuberized. The cytoplasm of 9 diploid lines and 6 somatic hybrid combinations was analysed. Two of the diploid lines had W/S chloroplasts and alpha or epsilon mitochondria; the remainder contained T chloroplasts and beta mitochondria. All the analysed somatic hybrids carried T chloroplasts and beta mitochondria.

The use of RAPD and semi-random markers to verify somatic hybrids between diploid lines of Solanum tuberosum L.

Markers specific to diploid lines of cultivated potato were identified using the polymerase chain reaction (PCR), primed with arbitrary 10-mers generating RAPDs, or with semi-random primers targeting intron-exon semi-conservative sequences of plant genes. One hundred and fifty RAPD primers and twelve semi-random primers were tested. Selected primers were subsequently used for verification of putative somatic hybrids.