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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.

In Poland the Plant Breeding and Acclimatization Institute - National Research Institute (IHAR-PIB) is responsible for officially assessing the resistance of potato breeding lines and cultivars to Synchytrium endobioticum. In the official assessment of wart resistance the modified Glynne-Lemmerzahl method is used. A full cycle of assessment of the wart disease resistance requires 42 - 45 tubers per cultivar/breeding line. Forty two tubers are used in laboratory tests. To complete the laboratory tests, another 10 tubers are inoculated with winter sporangia of the fungus, using ring test. The final results are available after 3 years of investigation. If necessary, the full cycle of resistance tests to S. endobioticum can be performed during one year on 15 tubers in each of the 3 replications (45 tubers in total). Molecular verification of potato genotypes resistance to pathotype 1(D1) is possible with the use of SCAR marker Nl25-1400. Nevertheless, an official phenotypical assessment of advanced breeding lines, as the final verification of their resistance, is required.

This study investigated changes in the amount of sporangium of Synchytrium endobioticum causing Potato Wart Disease by years and cultivated crop design in the districts of Derinkuyu in Niğde province and Kaymakli in Nevsehir for three-years. The experiment was carried out in total of 28 fields selected 16 from the district of Derinkuyu and 12 from Kaymaklı between 2006-2008. In the study it was tried to sample from different production areas and the concentration of the inoculum in the soil was tried to be determined according to the years and the crop designs. There was no correlation between the rates of increase or decrease in the level of sporangium and crop design in the survey studies. The contaminated soil samples which were kept in six different temperatures ranging from 45-70 °C and varying durations depending on temperatures were examined in order to determine the mortality rates of the winter sporangium of Synchytrium endobioticum at different temperatures and durations. 100% sporangium death was observed in the duration of 204 hours at 60ºC, 34 hours at 65ºC and 8 hours at 70ºC in the experiments conducted in order to determine the temperature of thermal death and mortality rates.

Background Standard strategies to identify genomic regions involved in a specific trait variation are often limited by time and resource consuming genotyping methods. Other limiting pre-requisites are the phenotyping of large segregating populations or of diversity panels and the availability and quality of a closely related reference genome. To overcome these limitations, we designed efficient Comparative Subsequence Sets Analysis (CoSSA) workflows to identify haplotype specific SNPs linked to a trait of interest from Whole Genome Sequencing data. Results As a model, we used the resistance to Synchytrium endobioticum pathotypes 2, 6 and 18 that co-segregated in a tetraploid full sib population. Genomic DNA from both parents, pedigree genotypes, unrelated potato varieties lacking the wart resistance traits and pools of resistant and susceptible siblings were sequenced. Set algebra and depth filtering of subsequences (k-mers) were used to delete unlinked and common SNPs and to enrich for SNPs from the haplotype(s) harboring the resistance gene(s). Using CoSSA, we identified a major and a minor effect locus. Upon comparison to the reference genome, it was inferred that the major resistance locus, referred to as Sen3, was located on the north arm of chromosome 11 between 1,259,552 and 1,519,485 bp. Furthermore, we could anchor the unanchored superscaffold DMB734 from the potato reference genome to a synthenous interval. CoSSA was also successful in identifying Sen3 in a reference genome independent way thanks to the de novo assembly of paired end reads matching haplotype specific k-mers. The de novo assembly provided more R haplotype specific polymorphisms than the reference genome corresponding region. CoSSA also offers possibilities for pedigree analysis. The origin of Sen3 was traced back until Ora. Finally, the diagnostic power of the haplotype specific markers was shown using a panel of 56 tetraploid varieties. Conclusions CoSSA is an efficient, robust and versatile set of workflows for the genetic analysis of a trait of interest using WGS data. Because the WGS data are used without intermediate reads mapping, CoSSA does not require the use of a reference genome. This approach allowed the identification of Sen3 and the design of haplotype specific, diagnostic markers. Electronic supplementary material The online version of this article (10.1186/s13007-019-0445-5) contains supplementary material, which is available to authorized users.

Background Standard strategies to identify genomic regions involved in a specific trait variation are often limited by time and resource consuming genotyping methods. Other limiting pre-requisites are the phenotyping of large segregating populations or of diversity panels and the availability and quality of a closely related reference genome. To overcome these limitations, we designed efficient Comparative Subsequence Sets Analysis (CoSSA) workflows to identify haplotype specific SNPs linked to a trait of interest from Whole Genome Sequencing data. Results As a model, we used the resistance to Synchytrium endobioticum pathotypes 2, 6 and 18 that co-segregated in a tetraploid full sib population. Genomic DNA from both parents, pedigree genotypes, unrelated potato varieties lacking the wart resistance traits and pools of resistant and susceptible siblings were sequenced. Set algebra and depth filtering of subsequences (k-mers) were used to delete unlinked and common SNPs and to enrich for SNPs from the haplotype(s) harboring the resistance gene(s). Using CoSSA, we identified a major and a minor effect locus. Upon comparison to the reference genome, it was inferred that the major resistance locus, referred to as Sen3, was located on the north arm of chromosome 11 between 1,259,552 and 1,519,485 bp. Furthermore, we could anchor the unanchored superscaffold DMB734 from the potato reference genome to a synthenous interval. CoSSA was also successful in identifying Sen3 in a reference genome independent way thanks to the de novo assembly of paired end reads matching haplotype specific k-mers. The de novo assembly provided more R haplotype specific polymorphisms than the reference genome corresponding region. CoSSA also offers possibilities for pedigree analysis. The origin of Sen3 was traced back until Ora. Finally, the diagnostic power of the haplotype specific markers was shown using a panel of 56 tetraploid varieties. Conclusions CoSSA is an efficient, robust and versatile set of workflows for the genetic analysis of a trait of interest using WGS data. Because the WGS data are used without intermediate reads mapping, CoSSA does not require the use of a reference genome. This approach allowed the identification of Sen3 and the design of haplotype specific, diagnostic markers.

Die Kartoffel ist ein Wirt für eine Vielzahl von Erregern. Ein bedeutender Vertreter stellt Synchytrium endobioticum dar, der Erreger des Kartoffelkrebses. Bei diesem Pathogen handelt es sich aufgrund seiner schweren Bekämpfbarkeit um einen Quarantäneschaderreger. Eine chemische Bekämpfung des Erregers ist nicht möglich, hauptsächlich der Anbau resistenter Sorten kann zu einer Eindämmung der Krankheit führen. S. endobioticum ist ein obligat biotropher, bodenbürtiger Pilz der Ordnung Chytridiales. Er zeichnet sich durch die Bildung von Dauersporen aus, welche unter optimalen Bedingungen bis zu 40 Jahre im Boden überdauern und eine erneute Infektion hervorrufen können. S. endobioticum infiziert die unterirdischen Pflanzenteile der Kartoffel (außer Wurzeln) durch Zoosporen, die durch Bewegungen im Bodenwasser meristematische Gewebe erreichen und befallen. Es kommt zur Ausbildung von tumorähnlichen Wucherungen. Es sind verschiedene Pathotypen des Erregers beschrieben, von welchen die Pathotypen 1, 2, 6 und 18 die bedeutendsten darstellen. Ein kleiner Teil zugelassener deutscher Kartoffelsorten ist gegen alle vier Pathotypen resistent. Das resistenzvermittelnde Gen ist bisher nicht bekannt, ebenso liegen kaum Informationen zum Befallsmechanismus auf molekularer Ebene vor. Ziel der vorliegenden Arbeit war es, die Interaktion von S. endobioticum mit resistenten und anfälligen Kartoffelsorten auf Basis von shotgun Proteomdaten zu charakterisieren. Die Proteomanalyse lieferte einen Einblick in die Proteine der Pathogenabwehr in der inkompatiblen Interaktion. Hierbei handelte es sich um PR-2, sowie andere Glucanasen. In der kompatiblen Interaktion waren Proteine des Zellzyklus erhöht vertreten. Ebenso sollten erste Sequenzen des Erregers anhand von Transkriptom- und Genomdaten identifiziert und bioinformatisch analysiert werden. Es war möglich, eine Sequenzsammlung von insgesamt 8.866 ESTs und 423 genomischen Contigs zu generieren. Aus dieser Sequenzsammlung wurden 66 Effektorkandidaten identifiziert. Ebenso wurden 45 Effektorkandidaten aus einer Sequenzsammlung ohne Homologien zu bekannten Taxa bestimmt. Sequenzen wurden als Effektorkandidat gewertet, wenn sie unter 300 Aminosäuren aufwiesen und ein Signalpeptid enthielten, sowie am sekretorischen Weg der Zelle beteiligt waren. Eine Gruppe von 14 Effektorkandidaten beider Gruppen wurde in transienten Expressionsanalysen mittels Agroinfiltration in Tabak mit Hilfe einer nachfolgenden TRV-Infektion funktional untersucht. Für vier Kandidaten zeigte sich eine reduzierte Pathogenabwehr der infiltrierten Blätter. Im Rahmen der Arbeit war es erstmals möglich, Sequenzen des Erregers zu charakterisieren, sowie Effektorkandidaten zu identifizieren. Erstmals wurde eine Proteomanalyse zur Analyse des Kartoffelkrebses durchgeführt.

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. Electronic supplementary material The online version of this article (10.1007/s10658-017-1411-6) contains supplementary material, which is available to authorized users.

Bu doktora çalışmasında, patates siğil hastalığının kalıtımı ve siğil hastalığına dayanıklı genotiplerin seleksiyonunda bazı moleküler markörlerin kullanım olanakları araştırılmıştır. Denemeler siğil hastalığı ile bulaşık tarlalarda 34 patates çeşidi ve 10 farklı melez ailesine ait 1.000 genotiple yürütülmüştür. Dayanıklılığın belirlenmesinde bazı Basit Dizi Tekrarları (SSR) markörlerinin de etkinlikleri araştırılmıştır. İki yıl yürütülen çeşitlerin bulaşık alan tarla denemesinde en düşük bulaşıklık oranları Lindita, Andante ve Van Gogh çeşitlerinde, en yüksek bulaşıklık oranları ise Lady Olympia, Lady Claire ve Folva çeşitlerinde gözlemlenmiştir. Üç yıl yürütülen genotiplerin bulaşık alan tarla denemesinde ise en düşük bulaşık genotip sayısı Van Gogh x Megusta ve Megusta x Lindita melez ailelerinde, en yüksek bulaşık genotip sayısı ise Agria x Granola ve Megusta x Granola melez ailelerinde tespit edilmiştir. STM2030 ve STM3023b SSR markörleri ile çeşit ve genotiplerin hastalık belirtileri arasında ilişki saptanamamıştır. Megusta x Granola melez ailesinden PAİ-12-11-76 ve PAİ-12-11-143 genotipleri patates siğil hastalığına dayanıklı hatlar olarak seçilerek tescil ettirilmek üzere tohumluk çoğaltımına alınmışlardır., The objectives of this dissertation project were to investigate inheritance of potato wart disease and to determine reliability of some molecular markers for resistance genotype selection against the wart disease. The experiments were conducted at wart infected fields with 34 potato varieties and 1.000 genotypes from 10 different crossing family. Two SSR markers were used to determine efficacy of molecular marker for resistance determination. According to two years infected field trial results Lindita, Andante and Van Gogh varieties were found the most resistance varieties in spite of that Lady Olympia, Lady Claire and Folva varieties were found the most susceptible varieties. The lowest infection ratios were determined at Van Gogh x Megusta and Megusta x Lindita families whereas the highest infection ratios were determined at Agria x Granola and Megusta x Granola families. There was not found any significant relationship between STM2030 and STM3023b SSR markers with disease infection symptom. At the end of three years infected field trials, PAĐ-12-11-76 and PAĐ-12-11- 143 genotypes from Megusta x Granola family were selected as wart resistant breeding lines, and their seed multiplication have been started.

Bu doktora çalışmasında, patates siğil hastalığının kalıtımı ve siğil hastalığına dayanıklı genotiplerin seleksiyonunda bazı moleküler markörlerin kullanım olanakları araştırılmıştır. Denemeler siğil hastalığı ile bulaşık tarlalarda 34 patates çeşidi ve 10 farklı melez ailesine ait 1.000 genotiple yürütülmüştür. Dayanıklılığın belirlenmesinde bazı Basit Dizi Tekrarları (SSR) markörlerinin de etkinlikleri araştırılmıştır. İki yıl yürütülen çeşitlerin bulaşık alan tarla denemesinde en düşük bulaşıklık oranları Lindita, Andante ve Van Gogh çeşitlerinde, en yüksek bulaşıklık oranları ise Lady Olympia, Lady Claire ve Folva çeşitlerinde gözlemlenmiştir. Üç yıl yürütülen genotiplerin bulaşık alan tarla denemesinde ise en düşük bulaşık genotip sayısı Van Gogh x Megusta ve Megusta x Lindita melez ailelerinde, en yüksek bulaşık genotip sayısı ise Agria x Granola ve Megusta x Granola melez ailelerinde tespit edilmiştir. STM2030 ve STM3023b SSR markörleri ile çeşit ve genotiplerin hastalık belirtileri arasında ilişki saptanamamıştır. Megusta x Granola melez ailesinden PAİ-12-11-76 ve PAİ-12-11-143 genotipleri patates siğil hastalığına dayanıklı hatlar olarak seçilerek tescil ettirilmek üzere tohumluk çoğaltımına alınmışlardır., The objectives of this dissertation project were to investigate inheritance of potato wart disease and to determine reliability of some molecular markers for resistance genotype selection against the wart disease. The experiments were conducted at wart infected fields with 34 potato varieties and 1.000 genotypes from 10 different crossing family. Two SSR markers were used to determine efficacy of molecular marker for resistance determination. According to two years infected field trial results Lindita, Andante and Van Gogh varieties were found the most resistance varieties in spite of that Lady Olympia, Lady Claire and Folva varieties were found the most susceptible varieties. The lowest infection ratios were determined at Van Gogh x Megusta and Megusta x Lindita families whereas the highest infection ratios were determined at Agria x Granola and Megusta x Granola families. There was not found any significant relationship between STM2030 and STM3023b SSR markers with disease infection symptom. At the end of three years infected field trials, PAİ-12-11-76 and PAİ-12-11-143 genotypes from Megusta x Granola family were selected as wart resistant breeding lines, and their seed multiplication have been started.

Bu doktora çalışmasında, patates siğil hastalığının kalıtımı ve siğil hastalığına dayanıklı genotiplerin seleksiyonunda bazı moleküler markörlerin kullanım olanakları araştırılmıştır. Denemeler siğil hastalığı ile bulaşık tarlalarda 34 patates çeşidi ve 10 farklı melez ailesine ait 1.000 genotiple yürütülmüştür. Dayanıklılığın belirlenmesinde bazı Basit Dizi Tekrarları (SSR) markörlerinin de etkinlikleri araştırılmıştır. İki yıl yürütülen çeşitlerin bulaşık alan tarla denemesinde en düşük bulaşıklık oranları Lindita, Andante ve Van Gogh çeşitlerinde, en yüksek bulaşıklık oranları ise Lady Olympia, Lady Claire ve Folva çeşitlerinde gözlemlenmiştir. Üç yıl yürütülen genotiplerin bulaşık alan tarla denemesinde ise en düşük bulaşık genotip sayısı Van Gogh x Megusta ve Megusta x Lindita melez ailelerinde, en yüksek bulaşık genotip sayısı ise Agria x Granola ve Megusta x Granola melez ailelerinde tespit edilmiştir. STM2030 ve STM3023b SSR markörleri ile çeşit ve genotiplerin hastalık belirtileri arasında ilişki saptanamamıştır. Megusta x Granola melez ailesinden PAİ-12-11-76 ve PAİ-12-11-143 genotipleri patates siğil hastalığına dayanıklı hatlar olarak seçilerek tescil ettirilmek üzere tohumluk çoğaltımına alınmışlardır., The objectives of this dissertation project were to investigate inheritance of potato wart disease and to determine reliability of some molecular markers for resistance genotype selection against the wart disease. The experiments were conducted at wart infected fields with 34 potato varieties and 1.000 genotypes from 10 different crossing family. Two SSR markers were used to determine efficacy of molecular marker for resistance determination. According to two years infected field trial results Lindita, Andante and Van Gogh varieties were found the most resistance varieties in spite of that Lady Olympia, Lady Claire and Folva varieties were found the most susceptible varieties. The lowest infection ratios were determined at Van Gogh x Megusta and Megusta x Lindita families whereas the highest infection ratios were determined at Agria x Granola and Megusta x Granola families. There was not found any significant relationship between STM2030 and STM3023b SSR markers with disease infection symptom. At the end of three years infected field trials, PAĐ-12-11-76 and PAĐ-12-11- 143 genotypes from Megusta x Granola family were selected as wart resistant breeding lines, and their seed multiplication have been started.

A zonal centrifugation method, known as the Hendrickx centrifuge technique, was tested for routine detection of winter sporangia of Synchytrium endobioticum in soil. In four experiments the ability of the Hendrickx centrifuge to extract the sporangia from soil was compared with a method used by the Dutch Plant Protection Service, which is a modification of the recommended EPPO method. Naturally and artificially contaminated soil samples were used to study the recovery percentage of and variation in numbers of winter sporangia. The effects of soil type and inoculum density were studied. The Hendrickx centrifuge method, developed originally for extraction of free living nematodes from soil, performed better than the method used by the Dutch Plant Protection Service. This was due to a better extraction recovery (60% higher), a lower measurement error (50% lower) and a lower detection level (down to 0.02 sporangia g(-16) soil). The Hendrickx centrifuge method is much less labour-intensive than the method used by the Dutch Plant Protection Service. It can be used to extract many different organisms from soil, and DNA can be subsequently extracted from the supernatant for further PCR analysis. Inclusion of the Hendrickx centrifuge method in the official EPPO diagnostic protocol for regulated pests is recommended as an alternative method for detection of sporangia in soil

Synchytrium endobioticum is a severe pathogen of potato causing wart disease. For this obligate fungus many pathotypes exist, which are pathogenic or non-pathogenic to different cultivars of potato. To determine to which pathotype an isolate belongs, two biological assays are used on a set of different potato cultivars: the Spieckermann and the Glynne-Lemmerzahl test. A differential set of cultivars has been recommended by EPPO (European Plant Protection Organization) for these tests. Drawbacks of these tests are that it can take up to several months to score the interaction and results are difficult to score and also often not conclusive. Therefore, possibilities were investigated to look for molecular markers for pathotype specificity. An extremely low level of diversity was observed in a set of eight isolates using CRoPS™ (Complexity Reduction of Polymorphic Sequences) technology. Only 191 sequence polymorphisms were found in 14,660 contigs representing approximately 195 Kb of sequence for eight isolates. Nine sequence polymorphisms could be related to pathotype specificity. Two of these polymorphisms were transferred into a real-time TaqMan PCR assay to discriminate between pathotype specific groups. Validation of the assays was performed using multiple isolates from different countries for which the bioassay had been performed.