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 (

Применение биологических препаратов в защите картофеля против возбудителя рака Synchytrium endobioticum (Schilb.) Perc. и золотистой картофельной цистообразующей нематоды Globodera rosthochiensis (Woll.)

The researches results of the following biological preparations Planrise, Paurin, Micosan, Actofit, Phitodoctor usage were presented. The biological preparations Planrise and Paurin (32,6%) against causa-tive agent of wart potato Synchytrium endobioticum (Schilb.) Perc. were shown the highest efficiency among the biological preparations by the research results. The efficiency of Phitodoctor consisted 25,8 %, Micosan was 23,1, respectively. The preparations efficiency were Micosan (42,8%), Phitodoctor and Paurin (30,2%); Actofit (34,3%); Bitobacsicillin (28,5%) against potato yellow cyst nematodes Globod-era rostochiensis (Woll.).

Відбір сортів картоплі з комплексною стійкістю до збудника раку Synchytrium endobioticum Schilbersky Perc.

Мета. Оцінити та відібрати сорти картоплі україн- ської та іноземної селекції з комплексною стійкістю до всіх патотипів раку, що існують в Україні, та виділити стійкі сорти для занесення до Державного реєстру сортів рослин, придатних для поширення в Україні, які можна впровадити у вогнищах хвороби. Методи. У дослі- дження залучено 15 сортів картоплі: 5 сортів української селекції та 10 – іноземної. Оцінку селекційного матері- алу на стійкість до звичайного 1(D1) і агресивних пато- типів збудника раку картоплі проводили в лабораторних умовах Української науково-дослідної станції карантину рослин ІЗР НААН на штучному інфекційному фоні згідно з «Методикою оцінки та відбору селекційного матері- алу картоплі, стійкого до раку Synchytrium endobioticum (Schilb.) Perc., гармонізованою з вимогами ЄС» у лабо- раторних та польових умовах. Результати. У результаті проведених лабораторних та польових досліджень з оцінки та відбору сортів картоплі з комплексною стій- кістю до всіх патотипів раку відібрано три сорти кар- топлі української селекції: Родинна та Містерія селек- ції Інституту картоплярства НААН і Княжа селекції Львівського національного аграрного університету; три сорти картоплі Алюетт, Лаундин та Фонтане фірми «Агріко Б.В.», Нідерланди. Усі досліджувані сорти (100%) виявилися стійкими до звичайного патотипу; до 11 (Міжгірського) патотипу відібрано 66,6% стійких сортів; стійких до 13 (Рахівського) агресивного пато- типу виявлено 73,3%; до 18 (Ясінівського) – 80,0%; до 22 (Бистрецького) – 93,3%. Ураження позитивного кон- трольного сорту картоплі Поліська рожева у лаборатор- них та польових умовах становило 100%; негативного контрольного сорту Божедар – 0%. Висновки. Сорти картоплі, які не уразилися патотипами збудника раку, рекомендовано для занесення до Державного реєстру сортів рослин, придатних для поширення в Україні та для впровадження у вогнищах хвороби.

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.