Development of new immunoanalytical test systems for diagnostics of potato blackleg caused by Dickeya spp. bacteria

Cover Page

Abstract


Potato blackleg caused by Dickeya spp. bacteria is one of the most important bacterial diseases of potatoes. The rapid spread of this disease in the territory of Russia requires new effective diagnostic tools for the timely detection of infection. To solve this problem, antisera specific to Dickeya spp. were obtained. Polyclonal antibodies isolated from antisera have shown high affinity for the main species of Dickeya spp. ( D. solani, D. dianthicola, D. chrysanthemi, D. dadantii, D. paradisiaca ). Enzyme linked immunosorbent assay (ELISA) and lateral flow immunoassay (LFIA) test systems have been developed based on specific and high affinity antibodies that were obtained. For ELISA, the detection limit was 0.8 × 105 cells/mL for D. solani and 2 × 104 cells/mL for D. dianthicola . For LFIA, suitable for use in non-laboratory conditions, the detection limit of D. solani was 2 × 105 cells/mL and the analysis time was 15 minutes. When testing potato seed material, LFIA test system confirmed positive results of ELISA determination in 75 % of samples, and negative - in 100 % of samples.


Full Text

 

Fig. 1. Comparison of antisera specific to D. solani through ELISA, with absorption of D. solani bacteria (a) and P. atrosepticum (b) on a microplate: 1–4 — numbers of serum

 

Fig. 2. Concentration dependences of antibodies specific to D. solani cells through ELISA, absorbed in a microplate at a concentration of 1×108 cells/mL: 1, 2 — numbers of serum, from which antibodies were purified

Table 1. Results of testing pure cultures of bacteria (1 × 107 cells/mL) through ELISA and LFIA test systems

Collection number of Russian Plant Quarantine Center

Bacterium

Test system

ELISA

LFIA

OD450

Result

Result

1

0039

Ralstonia solanacearum, раса 3, bv.2

0

2

0040

Ralstonia solanacearum, раса 3, bv.2

0

3

0141

 Pectobacterium carotovorum subsp. carotovorum

0

4

0142

Pectobacterium atrosepticum

0

5

0143

Pectobacterium atrosepticum

0

6

0144

Diсkeya dianthicola

2.41

+

+

7

0235

Clavibacter michiganensis subsp. Sepedonicus

0

8

0239

Сlavibacter michiganensis subsp. michiganensis

0

9

0240

Сlavibacter michiganensis subsp. michiganensis

0

10

0222

Pseudomonas syringae pv. syringae

0

11

0223

Pseudomonas syringae pv. syringae

0

12

0327

Pectobacterium carotovorum subsp. odoriferum

0

13

0328

Pectobacterium wasabiae

0

14

0329

Pectobacterium betavasculorum

0

15

0330

Pectobacterium cacticida

0

16

0331

Dickeya chrysanthemi

2.46

+

+

17

0332

Dickeya dadantii subsp. Dadantii

1.89

+

+

18

0333

Dickeya paradisiaca

2.16

+

19

0334

Dickeya zeae

0.28

+

+

20

0335

Pseudomonas fuscovaginae

0

21

0336

Dickeya dadantii subsp. dieffenbachiae

2.19

+

+

22

0353

Dickeya solani

1.98

+

+

 

Fig 3. Concentration dependences obtained through ELISA from samples with different bacterial concentration of D. solani and D. dianthicola

Fig. 4. Test strips after analysis of samples with different concentrations of D. solani: 0 — negative control; 1–1.4×105; 2–4.1×105; 3–1.2×106; 4–3.7×106; 5–1.1×107; 6–3.3×107; 7–1×108 cells/mL and the corresponding dependence of the color intensity of the test zones on the concentration of D. solani

Table 2. Results of ELISA and LFIA testing of Dickeya spp. infection in potato tubers

Sample*

 

ELISA

LFIA

Cultivar, reproduction

OD450

Result

Result

1

Ledi Kler, 3 repr.

0.062

­-

­-

2

Opal, 3 repr.

0.088

­-

­-

3

Opal, 3 repr.

0.113

­-

­-

4

Korolek, 2 repr.

0.073

­-

­-

5

Ledi Kler, elite

1.289

+

+

6

Ledi Kler, 1 repr.

0.057

­-

­-

7

Ostin, 1 repr.

2.358

+

+

8

Korolek, 2 repr.

0.117

­-

­-

9

Nort, elite

0.073

­-

­-

10

Bars, elite

2.111

+

+

11

Opal, 3 repr.

2.635

+

+

12

Korolek, 2 repr.

0.155

­-

­-

13

Impala, SSE

2.308

+

+

14

Rivyera, SSE

0.095

­-

­-

15

Kollete, SSE

0.119

­-

­-

16

Udacha, FFR

0.120

­-

­-

17

Kollete, FFR

2.493

+

+

18

Dezire, FFR

0.427

+

­-

19

Fioletovy, FFR

0.656

+

­-

20

Fioletovy, SSE

0.303

+

­-

21

Arrou, SSE

0.070

­-

­-

22

Udacha, SSE

0.276

+

­-

23

Red Skarlet, FFR

1.477

+

+

24

Impala, FFR

2.476

+

+

25

Red Skarlet, SSE

0.092

­-

­-

26

Dezire, SSE

0.730

+

­-

27

Rivyera, FFR

0.116

­-

­-

28

Grand, FFR

0.439

+

+

29

Varyag, FFR

0.991

+

­-

30

Kumach, FFR

2.395

+

+

31

Gulliver, FFR

0.092

­-

­-

32

Meteor, FFR

1.800

+

+

33

Red Ledi, elite

1.977

+

+

34

Koroleva Anna, elite

0.170

­-

­-

35

Red Skarlet, A

0.110

­-

­-

36

Sante, elite

0.092

­-

­-

37

Gala, 3 repr.

2.818

+

+

38

VR­808, 2 repr.

2.281

+

+

39

Bruk, 1 repr.

2.120

+

+

40

Ledi Kler, 2 repr.

2.386

+

+

41

Ledi Kler, 2 repr

0.789

+

+

42

Gala, 2 repr.

1.177

+

+

43

Nakra, mini­tubers

0.064

­-

­-

*Samples received: 1–12 — from Kyrgyzstan; 13–32, 37–42 — from the Moscow region; 33–36 — from the Oryol region; 43 — from the Bryansk region.

About the authors

Shyatesa Razo

A.N. Bach Institute of Biochemistry, Research Centre of Biotechnology of the Russian Academy of Sciences; RUDN University

Author for correspondence.
Email: 1042175063@rudn.ru
ORCID iD: 0000-0002-4131-3797
33 Leninsky Prospect , Moscow, 119071, Russian Federation; 8/2 MiklukhoMaklaya st., Moscow, 117198, Russian Federation

PhD candidate, Agrarian and Technological Institute

Pavel A. Galushka

Russian Potato Research Center

Email: pavel_galushka@mail.ru
ORCID iD: 0000-0003-4680-9684
23 Lorkh st., Kraskovo, Moscow region, 140051, Russian Federation

Candidate of Biological Sciences, Senior Researcher

Yuri A. Varitsev

Russian Potato Research Center

Email: varyuriy@yandex.ru
ORCID iD: 0000-0002-2329-7965
23 Lorkh st., Kraskovo, Moscow region, 140051, Russian Federation

Candidate of Biological Sciences, Lead Senior Researcher

Anatoly V. Zherdev

A.N. Bach Institute of Biochemistry, Research Centre of Biotechnology of the Russian Academy of Sciences

Email: zherdev@inbi.ras.ru
ORCID iD: 0000-0003-3008-2839
33 Leninsky Prospect , Moscow, 119071, Russian Federation

Candidate of Biological Sciences, Lead Senior Researcher, A.N. Bach Institute of Biochemistry

Irina V. Safenkova

A.N. Bach Institute of Biochemistry, Research Centre of Biotechnology of the Russian Academy of Sciences

Email: saf-iri@yandex.ru
ORCID iD: 0000-0002-3621-4321
33 Leninsky Prospect , Moscow, 119071, Russian Federation

Candidate of Biological Sciences, Senior Researcher, A.N. Bach Institute of Biochemistry

Elena N. Pakina

RUDN University

Email: e-pakina@yandex.ru
ORCID iD: 0000-0002-1647-9138
8/2 MiklukhoMaklaya st., Moscow, 117198, Russian Federation

Candidate of Biological Sciences, Associate Professor, Director of Department of Agrobiotechnology, Agrarian and Technological Institute

Boris B. Dzantiev

A.N. Bach Institute of Biochemistry, Research Centre of Biotechnology of the Russian Academy of Sciences

Email: dzantiev@inbi.ras.ru
ORCID iD: 0000-0003-4008-4918
33 Leninsky Prospect , Moscow, 119071, Russian Federation

Doctor of Chemical Sciences, Professor, Head of Laboratory, A.N. Bach Institute of Biochemistry

References

  1. Toth I, Saddler G, Elphinstone J. Investigating the biology and appropriate control of Dickeya species affecting GB potatoes. Kenilworth: Potato Council; 2014.
  2. Stevenson WR, Loria R, Franc GD, Weingartner DP. (eds.) Compendium of potato diseases. 2nd edition. St Paul, Minnesota: APS Press; 2001.
  3. Karlov AN, Zotov VS, Pekhtereva ES, Matveeva EV. Dickeya dianthicola is a new bacterial pathogen of potatoes for Russia. Izvestiya of Timiryazev Agricultural Academy. 2010; (3):134—141. (In Russ.).
  4. Ignatov AN, Egorova MS, Khodykina MV. Spread of bacterial and phytoplasma diseases of plants in Russia. Plant protection and quarantine. 2015; (5):6—9. (In Russ.).
  5. Zaitsev IA, Varitsev YA, Lazarev AM, Galushka PA, Varitseva GP. Monitoring of hidden (latent) forms of the spread of pathogens of black leg and ring rot of potatoes in the Russian Federation. In: Agricultural sciences: scientific priorities of scientists. Permian: Evensis publ.; 2016. p.39–56. (In Russ.).
  6. Gill ED, Schaerer S, Dupuis B. Factors impacting blackleg development caused by Dickeya spp. in the field. European Journal of Plant Pathology. 2014; 140(2):317–327. doi: 10.1007/s10658–014–0465-y
  7. Ignatov AN, Panycheva JS, Voronina MV, Vasiliev DM, Dzhalilov FS. Dynamics of species composition of potato pathogens in the European part of the Russian Federation. Potato and vegetables. 2019; (9):28—32. (In Russ.). doi: 10.25630/PAV.2019.57.62.003
  8. Yerokhova MD, Kuznetsova MA. Blackleg of potato is a dangerous disease for national potato growing. Agrarian Science. 2019; 3:44—48. (In Russ.). doi: 10.32634/0869–8155–2019–326–3–44–48
  9. Toth IK, van der Wolf JM, Saddler G, Lojkowska E, Hélias V, Pirhonen M, Tsror L, Elphinstone JG. Dickeya species: an emerging problem for potato production in Europe. Plant Pathology. 2011; 60(3):385—399. doi: 10.1111/j.1365–3059.2011.02427.x
  10. Pritchard L, Humphris S, Saddler GS, Parkinson NM, Bertrand V, Elphinstone JG, Toth IK. Detection of phytopathogens of the genus Dickeya using a PCR primer prediction pipeline for draft bacterial genome sequences. Plant Pathology. 2013; 62(3):587—596. doi: 10.1111/j.13653059.2012.02678.x
  11. Varitsev YA, Belov GL, Uskov AI, Varitseva GP, Zavriev SK, Arshava NV et al. Metodicheskie ukazaniya po diagnostike vozbuditelei chernoi nozhki (Erwinia carotovora (Jones) Bergey et al.) i kol’tsevoi gnili kartofelya (Clavibacter michiganensis subsp. sepedonicus (Spieck. Et Kotth.) Skaptasson et Burk.) metodami immunofermentnogo analiza, immunofluores-tsentnoi mikroskopii i polimeraznoi tsepnoi reaktsii [Methodological guidelines for the diagnosis of causative agents of black leg (Erwinia carotovora (Jones) Bergey et al.) and ring rot of potatoes (Clavibacter michiganensis subsp. sepedonicus (Spieck. Et Kotth.) Skaptasson et Burk.) by enzyme-linked immunosorbent assay, immuno-microscopy and polymerase chain reaction]. Moscow: Russian Research Institute of Potato Economy publ.; 2003. (In Russ.).
  12. Frens G. Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions. Nature Physical Science. 1973; 241(105):20—22. doi: 10.1038/physci241020a0
  13. Byzova NA, Safenkova IV, Chirkov SN, Zherdev AV, Blintsov AN, Dzantiev BB, Atabekov IG. Development of immunochromatographic test systems for express detection of plant viruses. Applied Biochemisty and Microbiology. 2009; 45 (2): 204—209. doi: 10.1134/S000368380902015X
  14. Safenkova IV, Zaitsev IA, Varitsev YA, Byzova NA, Drenova NV, Zherdev AV, Dzantiev BB. Development of a lateral flow immunoassay for rapid diagnosis of potato blackleg caused by Dickeya species. Analytical and Bioanalytical Chemistry. 2017; 409(7):1915—1927. doi: 10.1007/s00216–016–0140–6
  15. Interstate Council for Standardization, Metrology and Certification. GOST 33996–2016. Seed potatoes. Specifications and methods of determining the quality. Moscow: Standartinform publ.; 2020. (In Russ.).

Supplementary files

Supplementary Files Action
1.
Fig. 1. Comparison of antisera specific to D. solani through ELISA, with absorption of D. solani bacteria (a) and P. atrosepticum (b) on a microplate: 1–4 — numbers of serum

View (72KB) Indexing metadata
2.
Fig. 2. Concentration dependences of antibodies specific to D. solani cells through ELISA, absorbed in a microplate at a concentration of 1×108 cells/mL: 1, 2 — numbers of serum, from which antibodies were purified

View (46KB) Indexing metadata
3.
Fig 3. Concentration dependences obtained through ELISA from samples with different bacterial concentration of D. solani and D. dianthicola

View (45KB) Indexing metadata
4.
Fig. 4. Test strips after analysis of samples with different concentrations of D. solani: 0 — negative control; 1–1.4×105; 2–4.1×105; 3–1.2×106; 4–3.7×106; 5–1.1×107; 6–3.3×107; 7–1×108 cells/mL and the corresponding dependence of the color intensity of the test zones on the concentration of D. solani

View (46KB) Indexing metadata

Statistics

Views

Abstract - 354

PDF (Russian) - 88

Cited-By


PlumX

Dimensions


Copyright (c) 2021 Razo S.C., Galushka P.A., Varitsev Y.A., Zherdev A.V., Safenkova I.V., Pakina E.N., Dzantiev B.B.

Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.

This website uses cookies

You consent to our cookies if you continue to use our website.

About Cookies