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.

Efficiency of detecting resting spores of Synchytrium endobioticum (potato wart disease) in infested sites of Ukraine and Georgia

Aim. To determine the occurrence and persistence of Synchytrium endobioticum, resting spore contamination in a small survey of (known infested) potato plots in Ukraine and Georgia; to compare the detection efficiency for resting spores (winter sporangia) of S. endobioticum using an extraction method, routinely applied in Ukraine, based on the use of sodium iodide (NaI) and an extraction method largely based on EPPO Standard PM 7/28(2) (2017), using kaolin and calcium chloride (CaCl2) for extraction. Methods. The examination of fields, aimed at detecting of S. endobioticum in 22 infested plots in Georgia, was conducted following the standard European Plant Protection Organisation (EPPO) phytosanitary procedure PM 3/59(3) (2017), and in Ukraine in 11 plots according to the Ukrainian Standard ‘Methodological recommendations on sampling during quarantine inspection and evaluation’ (Omeluta V P et al, 1996). Resting spores were extracted using kaolin and CaCl2 (following largely EPPO Standard PM 7/28(2) 2017), and floatation in a solution of NaI (Zelya et al, 2005), respectively. The content of soil organic matter (SOM), collected in potato wart infested plots in Ukraine and Georgia, was determined according to the ‘Method of laboratory determination of the content of organic matter’, largely following the method of Tyurin to determine total soil organic carbon (Jankauskas B et al, 2006). The persistence or decline of potato wart in infested plots was evaluated, based on our own observations and the data of the national phytosanitary services (https://dpss.gov.ua; http://agr.georgia.gov). The results of the study were subjected to statistical analysis, using Statistica 5 software. Results. It was found that under a low up to high level of organic matter in soil samples (2.0–2.7 and 3.1–3.9 %, respectively) and a moderate to high level of inoculum in soil (3–15 up to 41–65 resting spores/g soil, respectively) there were no significant differences in the efficiency of two extraction methods under consideration: the floatation in a solution of NaI and the application of kaolin and CaCl2. However, at a low number of resting spores present in soil (1–2 resting spores/g soil), the efficiency of the method using NaI decreased statistically significant by 20–30 % as opposed to the method using kaolin/CaCl2. A relatively high level of soil contamination with resting spores was found in 11 investigated plots of 4 Ukrainian regions: 41–46 resting spores/g soil in the Ivano-Frankivsk Region, 49 in the Lviv Region, 40–65 in the Zakarpattia and 52–65 Chernivtsi Regions. The majority of the 22 investigated Georgian plots showed a low inoculum level (1–7 resting spores/g soil) and only in one village their level amounted to 15 resting spores/g soil (Uchguli village, Mestia municipality). Conclusions. The method applying sodium iodide was found to be comparable to the method applying kaolin and CaCl2 under conditions of moderate to high inoculum levels (15–65 resting spores/g soil) but the latter method was more efficient under conditions of high content of organic matter and very low inoculum level (

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 phytosanitary term of old sources potato wart Synchytrium endobioticum (Schilb.) Perc. in Ukraine

Goal. To do analyze phitosanitary state of wart potato sources in Carpathian region of Ukraine with determining infectious load of soil. Identify existing anti-cancer patatoes in Ukraine. Methods. The selection of soil’s samples for zoosporangia of wart pathogen was provided by standard method as per DSTU 3355-96. The sample’s selection points fixed by GPS-navigator Garmin eTrex Legend. The potato wart zoosporangia extraction was conducted by floatation in solution of sodium iodide. The solution Coomassi blue G-250 used for zoosporangia viability determining. Results. Of potato wart pathogens infectious load in Carpathian region of Ukraine 2016—2018 showed the different level of zoosporangia soil’s infection in researched areas. In the hearths of the Chernivtsi region the infectious load was reached 56—68 zoosporangia/1g in sources of Carpathian region, 42—48 zoosporangioa was in the sources of Zakarpattia region, 40—45/1g zoosporangia was determined in Ivano-Frankivsk region, 49 zoosporangia /1g was determined in town Turka Lviv region. The common (D1) pathotype and four aggressive pathotypes of wart potato: 11 (M1) - Mizhgirrya, 13 (R2) - Rachiv, 18 (Ya) - Yasynya and 22 (B1) - Bystrytsya were determined by pathotypes identification in old sources of Zakarpattia, Ivano-Frankivsk, Lviv and Chernivtsi region Conclusions. Analysis of phitosanitary state of wart potato sources in Carpathian region of Ukraine showed the different level of soil’s infectious load in researched areas. There is a common pathotype (D1) and 4 aggressive pathogen disease in Ukraine for present time. The identification of Ukrainian wart pothotypes is planned to identify by European potato cultivar test-assortment usage in perspective.

An Alternative Bioassay for Synchytrium endobioticum Demonstrates the Expression of Potato Wart Resistance in Aboveground Plant Parts

The obligate biotrophic chytrid species Synchytrium endobioticum is the causal agent of potato wart disease. Currently, 39 pathotypes have been described based on their interaction with a differential set of potato varieties. Wart resistance and pathotyping is performed using bioassays in which etiolated tuber sprouts are inoculated. Here, we describe an alternative method in which aboveground plant parts are inoculated. Susceptible plants produced typical wart symptoms in developing but not in fully expanded aboveground organs. Colonization of the host by S. endobioticum was verified by screening for resting spores by microscopy and by molecular techniques using TaqMan polymerase chain reaction and RNAseq analysis. When applied to resistant plants, none of these symptoms were detectable. Recognition of S. endobioticum pathotypes by differentially resistant potato varieties was identical in axillary buds and the tuber-based bioassays. This suggests that S. endobioticum resistance genes are expressed in both etiolated “belowground” sprouts and green aboveground organs. RNAseq analysis demonstrated that the symptomatic aboveground materials contain less contaminants compared with resting spores extracted from tuber-based assays. This reduced microbial contamination in the aboveground bioassay could be an important advantage to study this obligate biotrophic plant–pathogen interaction. Because wart resistance is active in both below- and aboveground organs, the aboveground bioassay can potentially speed up screening for S. endobioticum resistance in potato breeding programs because it omits the requirement for tuber formation. In addition, possibilities arise to express S. endobioticum effectors in potato leaves through agroinfiltration, thereby providing additional phenotyping tools for research and breeding. [Formula: see text] Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .