Information to authors
To the question of genotyping strains of bacteria of the genera Pectobacterium and Pseudomonas Ц the pathogens of bacterioses of potatoes
SUMMARY. Molecular genetic research methods make it possible to identify phytopathogens with comprehensive characteristics of their hereditary material. Our method of genotyping phytopathogenic bacteria is based on the use of two restriction endonucleases for digesting the genomic DNA (DDSL). The Taq DNA polymerase included in the reaction mixture provides labeling of DNA fragments with biotinylated deoxycytosinetriphosphate (Bio-dCTP). The label is incorporated only into DNA having 3-prime recessed ends formed by the first enzyme. The second restriction endonuclease produces only blunt ends of fragments that are unable to incorporate a label. As a result of the DDSL reaction, 20Ц50 distinct DNA fragments are visualized on the filter, the amount and distribution of which is characteristic for each bacterial strain. The work was carried out using two pairs of restriction enzy-mes Ц XbaI/DraI and XbaI/Eco24I; the results indicate the same discriminatory ability of these combinations of enzymes. Some advantage is noted for the combination XbaI/DraI, which allows detecting differences in genetic profiles in the region of longer DNA fragments. The genetic profile generated by enzyme pair BcuI-Eco32I in Ps. fluorescens 894, was significantly different from that of Ps. xanthochlora, which could be anticipated considering that these are two different bacterial species. In general, genotyping data correlate with data concerning the origin of bacterial strains. In particular, strains with the same genetic profile, D822 and D828, G399 and G400, C960 and C966 were obtained from affected tubers of the same experimental fields, which allow the possibility of contact spread of the pathogen. In some cases, the identity of genetic profiles is demonstrated, despite their different geographic origins. This fact confirms the contamination that occurred in the past during the exchange of seed material.
Key words: potato pathogens, strains, restriction endonucleases, genotyping
E-mail: valeriter mail.ru, allazar54 mail.ru
1. Nabhan, S., De Boer, S.H., Maiss, E., and Wydra, K., Taxonomic relatedness between Pectobacterium carotovorum subsp. carotovorum, Pectobacterium carotovorum subsp. odoriferum and Pectobacterium carotovorum subsp. brasiliense subsp. nov., J. Appl. Microbiol., 2012, vol. 113, no. 4, pp. 904Ц913. https://doi.org/10.1111/j.1365-2672.2012.05383.x
2. Vernon-Shirley, M. and Burns, R., The development and use of monoclonal antibodies for detection of Erwinia, J. Appl. Microbiolю, 1992, vol. 72, no. 2, pp. 97Ц102.
3. Peltzer, S. and Sivasithamparam, K., Sero-groups of Ec associated with water, soil, tuber and stems of potato plants in Western Australia, N. Z. J. Agric., 1988, vol. 16, no. 3, pp. 265Ц270.
4. Faure, D. and Dessaux, Y., Quorum sensing as a target for developing control strategies for the plant pathogen Pectobacterium, Eur. J. Plant Pathol., 2007, vol. 119, pp. 353Ц365.
5. Essarts, Y.R., Cigna, J., Quetu-Laurent, A., Caron, A., Munier, E., Beury-Cirou, A., Helias, V., and Faurea, D., Biocontrol of the potato blackleg and soft rot diseases caused by Dickeya dianthicola, Appl. Environ. Microbiol., 2016, vol. 82, no. 1, pp. 268Ц278. https://doi.org/10.1128/AEM.02525-15
6. Pishchik, V.N., Chernyaeva, I.I., Vorobjev, N.I., and Lazarev, A.M., Characteristic features of virulent and avirulent strains of Erwinia carotovora, Microbiology (Moscow), 1996, vol. 65, no. 2, pp. 232Ц237.
7. Pishchik, V.N., Provorov, N.A., Vorobjov, N.I., Chizevskaya, E.P., Safronova, V.I., Kozhemyakov, A.P., and Tuev, A.N., Interactions between plants and associated bacteria in soils contaminated with heavy metals, Microbiology (Moscow), 2009, vol. 78, no. 6, pp. 785Ц793. https://doi.org/10.1134/S0026261709060162
8. Xiu, J.H., Ji, G.H., Wang, M., Yang, Y.L., and Li, C.Y., Molecular identification and genetic diversity in KonnyakuТs soft rot bacteria, Acta Microbiol. Sin., 2006, vol. 46, no. 4, pp. 522Ц525.
9. Nabhan, S., Wydra, K., Linde, M., and Debener, T., The use of two complementary DNA assays, AFLP and MLSA, for epidemic and phylogenetic studies of pectolytic enterobacterial strains with focus on the heterogeneous species Pectobacterium carotovorum, Plant Pathol., 2012, vol. 61, pp. 498Ц508. https://doi.org/10.1111/j.1365-3059.2011.02546.x
10. Terletskiy, V.P., Tyshchenko, V.I., Novikova, O.B., Borisenkova, A.N., Belash, D.E., and Yakovlev, A.F., The efficient molecular genetic technique for identification of Salmonella and Proteus strains, Russ. Agricul. Sci., 2013, no. 5, pp. 60Ц63. https://doi.org/10.3103/S1068367413060220
11. Czajkowski, R., Perombelon, M.C.M., Jafra, S., Lojkowska, E., Potrykus, M., van der Wolf, J.M., and Sledz, W., Detection, identification and differentiation of Pectobacterium and Dickeya species causing potato blackleg and tuber soft rot: a review, Ann. Appl. Biol., 2015, vol. 166, pp. 18Ц38. https://doi.org/10.1111/aab.12166
12. Terletskiy, V.P., Tyshchenko, V.I., Novikova, I.I., Boikova, I.V., Tyulebaev, S.D., and Shakhtamirov, I.Y., An efficient method for genetic certification of bacillus subtilis strains, prospective producers of biopreparations, Microbiology (Moscow), 2016, vol. 85, no. 1, pp. 71Ц76. https://doi.org/10.1134/S0026261716010136
13. Terletskiy, V., Kuhn, G., Francioli, P., and Blanc, D., Application and evaluation of double digest selective label (DDSL) typing technique for Pseudomonas aeruginosa hospital isolates, J. Microbiol. Methods, 2008, vol. 72, no. 3, pp. 283Ц287. https://doi.org/10.1016/j.mimet.2007.12.006
14. Vinogradova, S.V., Kyrova, E.I., and Ignatov, A.N., Complete genome sequencing of phytopathogenic bacteria, Potato Protection, 2014, no. 2, pp. 15Ц17.
15. Khayi, S., Blin, P., Chong, T.M., Chan, K.G., and Faure, D., Complete genome anatomy of the emerging potato pathogen Dickeya solani type strain IPO 2222(T), Stand. Genomic Sci., 2016, vol. 11, pp. 87. https://doi.org/10.1186/s40793-016-0208-0
16. Ignatov, A.N., Lazarev, A.M., Panycheva, J.S., Provorov, N.A., and Chebotar, V.K., Potato phytopathogens of genus DickeyaЧa mini review of systematic and etiology of diseases, Agricult. Biol., 2018, vol. 53, no. 1, pp. 123Ц131. https://doi.org/10.15389/agrobiology.2018.1.123eng
17. Toth, I.K., van der Wolf, J.M., Saddler, G., Lojkowska, E., Helias, V., Pirhonen, M., and Elphinstone, J.G., Dickeya species: an emerging problem for potato production in Europe, Plant Pathol., 2011, vol. 60, no. 3, pp. 385Ц399.
|Coded & Designed by Volodymyr Duplij||Modified 10.04.21|