Improvement of soil testing techniques for detecting spores of potato wart disease Synchytrium endobioticum using molecular methods

Synchytrium endobioticum (Schilb.) Percival. is a pathogen of potato wart disease and has a restricted distribution on the territory of the Russian Federation. Its main pathways are infected potato tubers and different planting material containing soil particles infected with spores of the fungus. One of the main problems is the use of toxic chemicals during detecting the disease in laboratory methods of direct soil testing to identify resting spores. This paper presents the assessment of molecular methods of soil diagnosis for detection of S. endobioticum by direct extraction of fungal DNA from soil samples using the MetaGen reagent kit. Identification was performed using the Fitoskrin. Synchytrium endobioticum-RT kit. The kit was pre-tested using DNA isolated from potato warts by various commercial kits. It was found that the optimal method of DNA isolation from the warts was using the FitoSorb-Avtomat 48 kit at the Tecan robotic station. Studies have shown that the sensitivity of the direct DNA extraction method from soil samples with various infection levels is the same as that of flotation method using carbon tetrachloride. Moreover, this method makes it possible to work with soil samples of different types, including peaty soils.

Transcriptome analysis of resistance mechanism to potato wart disease

To understand the molecular mechanism of the resistance to potato wart disease, we used the potato cultivar Qingshu 9 as the experimental material and prepared potato samples with different levels of disease through inoculation. The RNAs of the samples were extracted, and transcriptome analysis was performed on the samples not infected by the disease (control group) and also on the samples with different levels of disease, with the aid of high-throughput sequencing. Next, the differentially expressed genes (DEGs) associated with the resistance to potato wart disease were identified based on the analysis results. Using bioinformatic tools, the DEGs were functionally annotated, classified, and enriched in related metabolic pathways. The main results are as follows: Compared with the control group, 4 DEGs were identified in the samples with light disease, 52 were found in the samples with medium disease, and 214 were discovered in the samples with heavy disease. Potato mainly resists the wart disease by suppressing its gene expression, and the degree of suppression depends on the level of the disease; the disease resistance might be dominated by cellular nucleic acid-binding protein, AP2-like transcription factor, and E3 ubiquitin-protein ligase. This research provides new insights into the molecular mechanism of potato resistance against wart disease.

A Hitchhiker’s guide to the potato wart disease resistance galaxy

Key message Two novel major effect loci (Sen4 and Sen5) and several minor effect QTLs for potato wart disease resistance have been mapped. The importance of minor effect loci to bring full resistance to wart disease was investigated. Using the newly identified and known wart disease resistances, a panel of potato breeding germplasm and Solanum wild species was screened. This provided a state-of-the-art “hitch-hikers-guide” of complementary wart disease resistance sources. Abstract Potato wart disease, caused by the obligate biotrophic soil-born fungus Synchytrium endobioticum, is the most important quarantine disease of potato. Because of its huge impact on yield, the lack of chemical control and the formation of resting spores with long viability, breeding for resistant varieties combined with strict quarantine measures are the only way to efficiently and durably manage the disease. In this study, we set out to make an inventory of the different resistance sources. Using a Genome-Wide Association Study (GWAS) in the potato breeding genepool, we identified Sen4, associated with pathotypes 2, 6 and 18 resistance. Associated SNPs mapped to the south arm of chromosome 12 and were validated to be linked to resistance in one full-sib population. Also, a bulked segregant analysis combined with a Comparative Subsequence Sets Analysis (CoSSA) resulted in the identification of Sen5, associated with pathotypes 2, 6 and 18 resistance, on the south arm of chromosome 5. In addition to these two major effect loci, the GWAS and CoSSA allowed the identification of several quantitative trait loci necessary to bring full resistance to certain pathotypes. Panels of varieties and Solanum accessions were screened for the presence of Sen1, Sen2, Sen3, Sen4 and Sen5. Combined with pedigree analysis, we could trace back some of these genes to the ancestral resistance donors. This analysis revealed complementary resistance sources and allows elimination of redundancy in wart resistance breeding programs.

The Synchytrium endobioticum AvrSen1 triggers a Hypersensitive Response in Sen1 potatoes while natural variants evade detection

Synchytrium endobioticum is an obligate biotrophic fungus of the phylum Chytridiomycota. It causes potato wart disease, has a world-wide quarantine status and is included on the HHS and USDA 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. So far, thirty-nine pathotypes are reported. A single dominant gene (Sen1) governs pathotype 1 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 fourteen 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, which co-segregated 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.Author SummaryPlant pathogens can have a great social and economic impact, and are a continuous threat to food security. A clear example is Synchytrium endobioticum, the fungus causing potato wart disease. The impact of the pathogen, lack of effective chemical control agents and the longevity of resting spores produced by the pathogen led to a world-wide quarantine status for S. endobioticum. Strict phytosanitary measures and the use of resistance potato varieties are currently the only way to prevent the spread of the disease. The emergence of new pathotypes that overcome resistance urged to study the underlying molecular mechanisms of S. endobioticum recognition by the plant. Here we describe the identification of the first effector (AvrSen1) of S. endobioticum that is recognized by the Sen1 resistance gene product. Also, we report the loss of AvrSen1 in other pathotypes thus avoiding recognition by the plant and triggering immune responses. AvrSen1 represents the first effector to be identified in the basal fungal lineage Chytridiomycota. The discovery of AvrSen1 provides an important tool to manage potato wart disease. Moreover, knowledge about Chytridiomycota effectors will shed light on other (pathogenic) interactions and the co-evolution of Chytridiomycota species with their hosts.

First Report of New Pathotype 39(P1) of Synchytrium endobioticum Causing Potato Wart Disease in Poland

Potato wart disease, caused by Synchytrium endobioticum (Schilb.) Perc., is one of the most dangerous diseases of cultivated potato. S. endobioticum is an obligate soil-borne fungus. The pathogen originated in the Andean zones of South America, from which it spread to North America and Europe at the end of the nineteenth century. The typical symptoms of cauliflower-like galls can develop on all meristematic tissues of potato except roots. The pathogen is on the European and Mediterranean Plant Protection Organization (EPPO) A2 list of quarantine pests. Since the discovery of pathotype 2(G1) in Germany, more than 40 pathotypes have been reported in Europe (1). In 2009, warted tubers were collected from Lesser Poland. Direct microscopic examination confirmed the presence of summer and winter sporangia in galls. The galls were multiplied on cv. Eersteling using the Glynne-Lemmerzahl method (GL), according to EPPO standard PM 7/28 (3). To identify the pathotype of S. endobioticum, 10 differential potato cultivars were inoculated with fresh galls following the GL method. The reaction types were evaluated 4 weeks after incubation in sand in a growth chamber. Cultivars Combi, Delcora, Deodara, Eersteling, Miriam, Producent, and Tomensa were extremely susceptible (predominant tumor formation); cv. Karolin was slightly susceptible (small galls with numerous winter sporangia); and cvs. Saphir and Ulme were resistant (early and late defense necrosis). This virulence profile was different from patterns of known European (1), Turkish (2), and Polish local pathotypes (4,5). The virulence of the pathogen to cv. Karolin was unique. Winter sporangia were isolated from galls formed on cv. Karolin and used in a modified Potoček's tube test (5) to obtain fresh galls with summer sporangia that were necessary to reconfirm the virulence profile of the pathotype. The reaction of all cultivars to the new pathotype was the same after the GL was performed. In accordance with the summation of known pathotypes, it is proposed to encode this new pathotype as pathotype 39(P1), using an Arabic number to designate the subsequent pathotype (the last one, 38 [Nevşehir], was identified in Turkey) (2) and the first letter of the locality where it was found (Piekielnik). The presence of pathotype 39(P1) was confirmed in four out of six districts where the Polish local pathotype 2(Ch1) was prevalent. The virulence profiles of both pathotypes were compared using differential set and cv. Asche Sämling, which is differential only for pathotype 2(Ch1). In contrast to all other pathotypes (big galls), no galls were observed on A. Sämling inoculated with pathotype 2(Ch1) and 39(P1). From this observation it appears that the new pathotype probably has been selected from the Polish local pathotype 2(Ch1). Pathotype 39(P1) was detected in small garden potato plots in the rainy mountainous area, a non-economically important potato-growing region where the old traditional cultivars of potato are cultivated without crop rotations. It seems, therefore, that where climatic conditions are suitable for S. endobioticum and the growing of slightly susceptible cultivars is possible, development of the new pathotype is favored. References: (1) R. P. Baayen et al. Eur. J. Plant Pathol. 116:21, 2006. (5) E. Çakir et al. OEPP/EPPO Bull. 39:175, 2009. (2) EPPO. EPPO Bull. 34:213, 2004. (3) J. Przetakiewicz. Biull. IHAR 257/258:207, 2010. (4) J. Przetakiewicz. Plant Dis. 98:688, 2014.

First Report of Synchytrium endobioticum (Potato Wart Disease) Pathotype 18(T1) in Poland

Synchytrium endobioticum (Schilb.) Perc. is a soil-borne biotrophic fungus causing potato wart disease (PWD) of cultivated potato, one of the most important crops in Poland. S. endobioticum infects epidermal cells of young potato organs, such as eyes, sprouts, young tubers, stolons, stems, leaves, and even flowers, but never roots. S. endobioticum survives in the soil as winter (resting) spores, which germinate, infect the plant, and produce secondary sporangia (summer spores); infection results in galls on the stolons and tubers, in which the pathogen multiplies. Its long persistence in soil and the severe losses it inflicts to potato crops have prompted its inclusion into the A2 quarantine list. The fungus originates from the Andean zones of South America, from where it spread to North America and Europe at the end of the 19th century. S. endobioticum was first reported in the United Kingdom in 1876. This pathogen is quite variable. The first discovery of a new pathotype in Europe occurred in former East Germany in 1941, and there have been 38 S. endobioticum pathotypes identified so far. Pathotypes 1(D1), 2(G1), 6(O1), 8(F1), and 18(T1) are of greatest relevance in Europe. In 2008, the Polish Inspectorate of Plant Health and Seed Inspection (PIORiN) collected soil samples from Mazowieckie Region in Central Poland. Microscopic examinations revealed the presence of viable resting spores of S. endobioticum in a soil sample collected from a crop plantation of ornamentals intended for export. One kilogram of soil contained an average of 300 viable spores. A bioassay (pot tests), recommended by the EPPO standard PM 3/59 (1), showed no wart symptoms because of the very low sporangium density of S. endobioticum. However, concentrating S. endobioticum inoculum by centrifugation and using for a bioassay modified Potocek's tube test (1) allowed us to obtain fresh galls with summer sporangia. The first symptoms of PWD were visible on sprouts of extremely susceptible potato genotypes 7 weeks after inoculation. To identify the pathotype of S. endobioticum, 10 differential potato cultivars (Deodara, Tomensa, Eersteling, Producent, Combi, Saphir, Delcora, Miriam, Karolin, and Ulme) were inoculated with fresh galls of S. endobioticum using the Glynne-Lemmerzahl method (2), according to EPPO standard PM 7/28. Galls were formed on all cultivars except Saphir, Karolin, and Ulme (resistant cultivars). This virulence profile was identical to that of European pathotype 18(T1) of S. endobioticum. This is the first detection in Poland of pathotype 18(T1), which is one of the most virulent pathotypes of this fungus. It should be noted that in 2004, prior to planting, the field was investigated by PIORiN and found to be free of S. endobioticum. The winter sporangia were found on the field with ornamental plants originating from Western Europe, where pathotype 18(T1) is still occurring. S. endobioticum is a classic example of the distribution of plant pathogens by man. Although an infection source has not been determined, the field was probably infested by soil connected with roots of the plantlets. This is an example of alternative ways for S. endobioticum spreading without potato as a main host. References: (1) EPPO. Bull. OEPP/EPPO Bull. 29:225, 1999. (2) EPPO. Bull. OEPP/EPPO Bull. 34:213, 2004.