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Development of new immunoanalytical test systems for diagnostics of potato blackleg caused by Dickeya spp. bacteria
- Authors: Razo S.1,2, Galushka P.A.3, Varitsev Y.A.3, Zherdev A.V.1, Safenkova I.V.1, Pakina E.N.2, Dzantiev B.B.1
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Affiliations:
- A.N. Bach Institute of Biochemistry, Research Centre of Biotechnology of the Russian Academy of Sciences
- RUDN University
- Russian Potato Research Center
- Issue: Vol 16, No 3 (2021)
- Pages: 198-214
- Section: Plant protection
- URL: https://agrojournal.rudn.ru/agronomy/article/view/19672
- DOI: https://doi.org/10.22363/2312-797X-2021-16-3-198-214
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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.
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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
PhD candidate, Agrarian and Technological Institute
33 Leninsky Prospect , Moscow, 119071, Russian Federation; 8/2 MiklukhoMaklaya st., Moscow, 117198, Russian FederationPavel A. Galushka
Russian Potato Research Center
Email: pavel_galushka@mail.ru
ORCID iD: 0000-0003-4680-9684
Candidate of Biological Sciences, Senior Researcher
23 Lorkh st., Kraskovo, Moscow region, 140051, Russian FederationYuri A. Varitsev
Russian Potato Research Center
Email: varyuriy@yandex.ru
ORCID iD: 0000-0002-2329-7965
Candidate of Biological Sciences, Lead Senior Researcher
23 Lorkh st., Kraskovo, Moscow region, 140051, Russian FederationAnatoly 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
Candidate of Biological Sciences, Lead Senior Researcher, A.N. Bach Institute of Biochemistry
33 Leninsky Prospect , Moscow, 119071, Russian FederationIrina 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
Candidate of Biological Sciences, Senior Researcher, A.N. Bach Institute of Biochemistry
33 Leninsky Prospect , Moscow, 119071, Russian FederationElena N. Pakina
RUDN University
Email: e-pakina@yandex.ru
ORCID iD: 0000-0002-1647-9138
Candidate of Biological Sciences, Associate Professor, Director of Department of Agrobiotechnology, Agrarian and Technological Institute
8/2 MiklukhoMaklaya st., Moscow, 117198, Russian FederationBoris 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
Doctor of Chemical Sciences, Professor, Head of Laboratory, A.N. Bach Institute of Biochemistry
33 Leninsky Prospect , Moscow, 119071, Russian FederationReferences
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