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.

About the authors

Yulia Vladislavovna Tsvetkova

All-Russian Plant Quarantine Center; Lomonosov Moscow State University

Author for correspondence.
Email: yutska@mail.ru
32, Pogranichnaya st., Bykovo, Ramensky district, Moscow region, 140150, Russian Federation; 1/12 Leninskie gory, Moscow, 119991, Russian Federation

Junior Researcher, Mycology Laboratory, All-Russian Plant Quarantine Center; PhD student, Lomonosov Moscow State University, Faculty of Biology

Vera Alekseevna Yakovleva

All-Russian Plant Quarantine Center

Email: yakovleva_va@mail.ru
32, Pogranichnaya st., Bykovo, Ramensky district, Moscow region, 140150, Russian Federation

PhD in Biological Sciences, Leading Researcher, Head of Phytosanitary Biology Department

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