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

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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.

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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 Federation

Vera 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 Federation

References

  1. Braun H. Biologische Spezialisierung bei Synchytrium endobioticum (Schilb.) Perc. Zeitschrift Für Pflanzenkrankheiten (Pflanzenpathologie) Und Pflanzenschutz. 1942; 52(11):481-486.
  2. Hey A. Die Biotypenforschung beim Erreger des Kartoffelkrebses Synchytrium endobioticum (Schilb.) Perc. in Deutschland. Nachrichtenbl. Dtsch. Pflanzenschutzdienst. 1948; 2:1-3.
  3. Hey A. Zur Biotypenfrage des Kartoffelkrebses. Mitt. Biol. Zentralanst. f. Land u. Forstwirtschaft (Berlin-Dahlem). 1953; 75:173-175.
  4. Hey A. Stand und Aussichten der Pflanzenquarantäne im Kartoffelbau. Leipzig: S. Hirzel; 1954.
  5. Baayen RP, Bonthuis H, Withagen JCM, Wander JGN, Lamers JL, Meffert JP, et al. Resistance of potato cultivars to Synchytrium endobioticum in field and laboratory tests, risk of secondary infection, and implications for phytosanitary regulations. EPPO Bulletin. 2005; 35(1):9-23. doi: 10.1111/j.1365-2338.2005.00775.x
  6. Baayen RP, Cochius G, Hendriks H, Meffert JP, Bakker J, Bekker M, et al. History of potato wart disease in Europe - a proposal for harmonisation in defining pathotypes. European Journal of Plant Pathology. 2006; 116:21-31. doi: 10.1007/s10658-006-9039-y
  7. Çakir E, Van Leeuwen GCM, Flath K, Meffert JP, Janssen WAP, Maden S. Identification of pathotypes of Synchytrium endobioticum found in infested fi lds in Turkey. EPPO Bulletin. 2009; 39(2):175-178. doi: 10.1111/j.1365-2338.2009.02285.x
  8. Przetakiewicz J. First report of new pathotype 39(P1) of Synchytrium endobioticum causing potato wart disease in Poland. Plant Disease. 2015; 99(2):285-286. doi: 10.1094/PDIS-06-14-0636-PDN
  9. EPPO Standards. PM 9/5 (2). National regulatory control systems for Synchytrium endobioticum. EPPO Bulletin; 2017.
  10. van de Vossenberg BTLH., van Gent-Pelzer MPE, Boerma M, van der Gouw LP, van der Lee TAJ, Vossen JH. An alternative bioassay for Synchytrium endobioticum demonstrates the expression of potato wart resistance in aboveground plant parts. Phytopathology. 2019; 109(6):1043-1052. doi: 10.1094/PHYTO-01-19-0024-R
  11. Dimitrova L, Laginova M, Becheva A, van Leeuwen GCM. Occurrence of potato wart disease (Synchytrium endobioticum) in Bulgaria: identifi of pathotype(s) present. EPPO Bulletin. 2011; 41(2):195-202. doi: 10.1111/j.1365-2338.2011.02453.x
  12. Vloutoglou I, van Leeuwen GCM, Eleftheriadis E, Sarigkoli I, Simoglou KB, Tsirogiannis D, et al. First report of potato wart disease caused by Synchytrium endobioticum (Schilb.) Perc. in Greece: detection, impacts and pathotype identification. Hellenic Plant Protection Journal. 2015; 8 (S1):9Special issue.
  13. Dorozhkin NA, Sharikov KE. Instruktsiya po opredeleniyu zarazhennosti pochvy rakom kartofelya (Synchytri-um endobioticum (Schilb.) Perc. [Instructions on determining soil infection with potato wart (Synchytrium endobioticum (Schilb.) Perc.)]. Minsk; 1950. (In Russ).
  14. EasyWay product line kits. Available from: https://www.syntol.ru/catalog/nabory-reagentov-dlyavydeleniya-dnk-i-rnk/nabory-reagentov-easyway.html [Accessed 11.03.2020]. (In Russ.)

Copyright (c) 2021 Tsvetkova Y.V., Yakovleva V.A.

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