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Improvement of soil testing techniques for detecting spores of potato wart disease Synchytrium endobioticum using molecular methods
- Authors: Tsvetkova Y.V.1,2, Yakovleva V.A.1
-
Affiliations:
- All-Russian Plant Quarantine Center
- Lomonosov Moscow State University
- Issue: Vol 16, No 1 (2021)
- Pages: 66-76
- Section: Plant protection
- URL: https://agrojournal.rudn.ru/agronomy/article/view/19641
- DOI: https://doi.org/10.22363/2312-797X-2021-16-1-66-76
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Abstract
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.
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Table 1. Soil samples used for isolation of S. endobioticum spores
No. | Soil samples | Number of fungal spores per 100 g of soil |
1* | Artificial inoculation (loamy sand) | 5 |
2* | Artificial inoculation (loam) | 5 |
3* | Artificial inoculation (loamy sand) | 50 |
4* | Artificial inoculation (loam) | 50 |
5 | Artificial inoculation (loamy sand) | 500 |
6 | Artificial inoculation (loamy sand) | 5000 |
7 | Artificial inoculation (loamy sand) | 20000 |
8 | Moscow Region, old outbreak (peaty soil) | Unknown |
9 | Moscow Region, outbreak (loam) | Unknown |
10 | Voronezh Region, old outbreak (loamy sand) | Unknown |
11 | Negative control | 0 |
* The soil samples were used only for the direct DNA extraction method.
Table 2. Results of direct testing of soil samples for presence of S. endobioticum spores using carbon tetrachloride
No. | Number of fungal spores per 100 g of soil | Number of fungal spores isolated from 100 g of soil | Effectiveness, % |
5 | 500 | 180.87 ± 44.9 | 36.17 |
6 | 5000 | 492.64± 53.66 | 9.85 |
7 | 20000 | 849.45 ± 54.52 | 4.24 |
8 | Unknown | Not conducted | — |
9 | Unknown | 230.2 ± 44.7 | — |
10 | Unknown | 0 | — |
Negative control K | 0 | 0 | — |
Table 3. Analytical sensitivity of «Synchytrium endobioticum-RT»
DNA dilution | Concentration of fungal cells, cells/ml | Synchytrium endobioticumRT | ||
Cycle threshold Ct | ||||
1 | 2 | 3 | ||
0 | 105 | 17.86 | 17.76 | 18.03 |
1 | 104 | 22.02 | 21.53 | 21.99 |
2 | 103 | 32.06 | 36.06 | 30.92 |
3 | 102 | 39.49 | — | — |
4 | 101 | — | — | — |
5 | 100 | — | — | — |
| K+ | 31.98 | 31.15 | 30.92 |
| Kv | — | — | — |
| Kch | — | — | — |
Table 4. DNA extraction from soil samples with Realtime PCR identification
Sample | Number of fungal spores per 100 g of soil | Cycle threshold Ct | ||
1 | 2 | 3 | ||
1 | 5 | — | — | — |
2 | 5 | — | — | — |
3 | 50 | 39.61 | — | — |
4 | 50 | 39.24 | — | — |
5 | 500 | 35.42 | 35.10 | 39.47 |
6 | 5000 | 34.16 | 32.21 | 33.49 |
7 | 20000 | 34.41 | 32.80 | 34.65 |
8 | Unknown | 38.15 | 28.74 | 33.88 |
9 | Unknown | 37.41 | 35.55 | 36.97 |
10 | Unknown | 41.61 | 33.20 | 33.56 |
11 | 0 | — | — | — |
K+* | — | 33.48 | 31.98 | 32.17 |
K+z** | — | 17.86 | 17.76 | 18.03 |
K-v | — | — | — | — |
K-ch | -— | — | — | — |
* Positive control of «Synchytrium endobioticum-RT» reagent kit.
** Positive control, DNA, extracted from a potato wart.
About the authors
Yulia Vladislavovna Tsvetkova
All-Russian Plant Quarantine Center; Lomonosov Moscow State University
Author for correspondence.
Email: yutska@mail.ru
Junior Researcher, Mycology Laboratory, All-Russian Plant Quarantine Center; PhD student, Lomonosov Moscow State University, Faculty of Biology
32, Pogranichnaya st., Bykovo, Ramensky district, Moscow region, 140150, Russian Federation; 1/12 Leninskie gory, Moscow, 119991, Russian FederationVera Alekseevna Yakovleva
All-Russian Plant Quarantine Center
Email: yakovleva_va@mail.ru
PhD in Biological Sciences, Leading Researcher, Head of Phytosanitary Biology Department
32, Pogranichnaya st., Bykovo, Ramensky district, Moscow region, 140150, Russian FederationReferences
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