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Identification of Pseudomonas fuscovaginae, Pseudomonas syringae and Xanthomonas translucens in wheat seeds using PCR
- Authors: Muvingi M.1, Slovareva O.Y.2, Zargar M.1
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Affiliations:
- Peoples’ Friendship University of Russia
- All-Russian Plant Quarantine Center
- Issue: Vol 17, No 4 (2022)
- Pages: 473-483
- Section: Plant protection
- URL: https://agrojournal.rudn.ru/agronomy/article/view/19830
- DOI: https://doi.org/10.22363/2312-797X-2022-17-4-473-483
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Abstract
The causative agents of grain crops bacteriosis viz. Pseudomonas fuscovaginae , Pseudomonas syringae and Xanthomonas translucens are regulated by phytosanitary requirements of the largest importers of Russian grain - Egypt, Turkey, Bangladesh, Nigeria and Pakistan. Therefore, it requires the development of rapid methods for their diagnosis. The PCR method, which is the fastest and most reliable in testing laboratories, needs optimal preparation of the test material. The aim of the study was to optimize the process of preparing seed samples for subsequent detection and identification of P. fuscovaginae, P. syringae and X. translucens by PCR. Wheat grain samples were soaked in phosphate-buffered saline (PBS) for 2 hours and infected with suspensions of P. fuscovaginae, P. syringae pv. coronafaciens and X. translucens at various concentrations. Then, the infected grain samples were crushed and subjected to two-stage centrifugation. DNA was isolated from the obtained analytical samples and species-specific PCR was performed for each bacterial species. It was found that a two-hour soaking of the seeds and their treatment with a homogenizer is sufficient to effectively destroy each grain in the sample and ensure the release of bacteria into the liquid part of the sample. The first low-speed centrifugation allowed the crushed grain to settle efficiently and remove excess starch from the supernatant. High-speed centrifugation of the supernatant made it possible to obtain a concentrated microbiota contained in the grain sample. To obtain DNA of sufficient quality for PCR test, the kit ‘Proba-GS’ (AgroDiagnostika, Russia) was used for DNA extraction. Using ‘Pseudomonas fuscovaginae-RT’ kit (Syntol, Russia) and PsyF/PsyR and 4F1/4R 1 primers, DNA of P. fuscovaginae P. syringae and X. translucens , respectively, was successfully detected in each of the samples infected with these bacteria at concentrations of 103 CFU/ml. The absence of PCR inhibition was noted. The method of removing starch from samples for molecular diagnostics of phytopathogens was used for the first time. Application of these methods will allow diagnosing pathogens of bacterioses within one day.
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Table 1. The number of colony-forming units (CFU/ml) in the tested bacterial suspensions 7 days after plating
Strain | Dilution of the initial suspension | ||||
2 | 3 | 4 | 5 | 6 | |
0335 | 4.2×107 | 4.2×106 | 4.2×105 | 4.2×104 | 4.2×103 |
0440 | 3.7×107 | 3.7×106 | 3.7×105 | 3.7×104 | 3.7×103 |
0337 | 1.5×107 | 1.5×106 | 1.5×105 | 1.5×104 | 1.5×103 |
Table 2. The result of DNA-testing by ‘Pseudomonas fuscovaginae-RT’ kit (Sintol, Russia)
Test-tube identification | Ct, FAM | Ct, HEX | Result |
0335—106-PBS | 27.0 | 34.0 | + |
0335—105-PBS | 29.7 | 33.8 | + |
0335—104-PBS | 30.8 | 34.1 | + |
0335—103-PBS | 31.6 | 34.2 | + |
0335—106-Extract-1 | 25.4 | 34.2 | + |
0335—106-Extract-2 | 25.2 | 34.6 | + |
0335—105-Extract-1 | 29.2 | 34.3 | + |
0335—105-Extract-2 | 29.4 | 34.3 | + |
0335—104-Extract-1 | 32.3 | 34.3 | + |
0335—104-Extract 2 | 31.6 | 34.2 | + |
0335—103-Extract-1 | 34.3 | 34.5 | + |
0335—103-Extract-2 | 35.3 | 34.5 | + |
0335—107-PBS-PCS | 22.9 | 34.1 | + |
Extract1-NCS | 34.4 | – | |
Extract2-NCS | 34.6 | – | |
PBS-NCS | 34.6 | – |
Note: Ct — PCR threshold cycle; FAM — PCR specific detection channel; HEX — PCR internal positive control detection channel; «+» — positive; «–» — negative; PBS — phosphate-buffered saline; PCS — positive control sample; NCS — negative control sample.
Fig. 1. Electropherogram of PCR testing of samples infected with strain 0440 for the presence of Pseudomonas syringae with PsyF/PsyR primers: 1, 10, 11 and 20—100 bp DNA length marker; 2, 3 — Negative control sample (Extract); 4 — pure Phosphate buffered saline (PBS); 5, 8 — PBS infected with strain 0440 at concentrations of 3.7×106—3.7×103 respectively; 9, 12 — strain 0440 in extract (3.7×106); 13, 14 — strain 0440 in extract (3.7×105); 15, 16 — strain 0440 in extract (3.7×104); 17,18 — strain 0440 in extract (3.7×105); 19 — Positive control sample
Fig. 2. Electropherogram of PCR testing of samples infected with strain 0337 for the presence of Xanthomonas translucens with primers 4F1/4R 1: 1, 10, 11 and 20—100 bp DNA length marker; 2, 3 — Negative control sample (Extract); 4 — Pure Phosphate buffered saline (PBS); 5, 8 — PBS infected with strain 0337 at concentrations of 1.5×106—1.5×103 respectively; 9, 12 — strain 0337 in extract (1.5×106); 13, 14 — strain 0337 in extract (1.5×105); 15, 16 — strain 0337 in extract (1.5×104); 17, 18 — strain 0337 in extract (1.5×105); 19 — Positive control sample
About the authors
Mufaro Muvingi
Peoples’ Friendship University of Russia
Author for correspondence.
Email: mufaromuvingi@gmail.com
ORCID iD: 0000-0001-7700-1296
PhD student, Agrobiotechnology Department, Agrarian and Technological Institute
8 Miklukho-Maklaya st., Moscow, 117198, Russian FederationOlga Y. Slovareva
All-Russian Plant Quarantine Center
Email: slovareva.olga@gmail.com
ORCID iD: 0000-0001-6022-5955
Candidate of Science (Biology), Junior Researcher, Department of Organization of Interlaboratory Comparison Tests
32 Pogranichnaya st., Bykovo vill., Ramenskoye, Moscow Region, 140150, Russian FederationMeisam Zargar
Peoples’ Friendship University of Russia
Email: zargar-m@rudn.ru
ORCID iD: 0000-0002-5208-0861
Candidate of Science (Biology), Junior Researcher, Department of Organization of Interlaboratory Comparison Tests
8 Miklukho-Maklaya st., Moscow, 117198, Russian FederationReferences
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