Genotyping pathogenic strains of genus <em>Xanthomonas</em> causing bacterioses in a number of plants by DDSL technique

Alexander Lazarev; Valery Terletskiy; Vladimir Chebotar

doi:10.21638/spbu03.2019.302

Authors

Alexander Lazarev Federal State Budget Scientific Institution “All-Russian Institute of Plant Protection”, Shosse Podbelskogo, 3, Saint Petersburg, 196608, Russian Federation https://orcid.org/0000-0002-4282-0141
Valery Terletskiy Federal State Budget Scientific Institution “All-Russian Institute of Plant Protection”, Shosse Podbelskogo, 3, Saint Petersburg, 196608, Russian Federation; Puskin Leningrad State University, Peterburgskoye Shosse, 10, Saint Petersburg, 196605, Russian Federation https://orcid.org/0000-0003-4043-3823
Vladimir Chebotar All-Russia Research Institute for Agricultural Microbiology, Shosse Podbel'skogo, 3, Saint Petersburg, 190608, Russian Federation https://orcid.org/0000-0001-9762-989X

DOI:

https://doi.org/10.21638/spbu03.2019.302

Abstract

In the genus Xanthomonas, specialists consider a significant number of species and varieties (pathovars) of phytopathogenic bacteria that infect many agricultural and ornamental plants (about 400 species), which leads to serious economic losses. For the timely detection of these pathogens, accurate diagnosis is necessary, allowing correct and prompt identification. Molecular genetic methods are able to identify populations of Xanthomonas strains with a fairly complete characterization of their hereditary material. The proposed method of genotyping — double digest and selective label (DDSL) — is based on the use of two restriction endonucleases for the separation of bacterial genomic DNA. The DNA polymerase (Taq) present in the reaction mixture along with biotinylated deoxycytosine triphosphate (Bio–dCTP) allows for the visualization of DNA fragments. The tag only labels DNA fragments that have 3'-recessed ends formed by the first enzyme (BcuI). The second restriction endonuclease (Eco147I) produces blunt ends that are unable to incorporate the label. As a result, in the DDSL reaction, 20–50 clearly distinguishable DNA fragments are visualized on the filter. The number and distribution of fragments are characteristic for each bacterial strain of the genus Xanthomonas. Genotyping these microorganisms makes it possible to identify the specific profile of each strain, i.e., assign it a sort of “bar code” for individual specification. The strains of bacteria of the genus Xanthomonas, obtained from different species (tomato, radish, sorghum) are genetically separated from each other, showing a specific pattern in terms of the distribution of DNA fragments, despite the common geographical origin. A comparatively rare case of the identity of strains, despite their geographical and temporal unrelatedness and different cultures, has been recorded.

Keywords:

Xanthomonas, strain, genotyping, double digest and selective label (DDSL), restriction endonucleases

Downloads

Download data is not yet available.

References

Barak, J. D. and Glibertson, R. L. 2003. Genetic diversity of Xanthomonas capestris pv. vitians, the causal agent of bacterial leafspot of lettuce. Phytopathology 93(5):596–603. https://doi.org/10.1094/PHYTO.2003.93.5.596

Bui, T. N., Veniera, C., Jarne, P., Brisse, S., Guerin, F., Boutry,S., Gagnevin, L., and Puvost, O. 2009. From local surveys to global surveillance: three high-throughput genotyping methods for epidemiological monitoring of Xanthomonas citri pv. citri pathotypes. Applied and Environmental Microbiology 75(4):1173–1184. https://doi.org/10.1128/AEM.02245-08

Ignatov, A., Sechler, A., Schuenzel, E. L., Agarkova, I., Oliver, B., Vidaver, A. K., and Schaad, N. W. 2007. Genetic diversity in populations of Xanthomonas campestris pv. campestris in cruciferous weeds in central coastal California. Phytopathology 97(7):803–812. https://doi.org/10.1094/PHYTO-97-7-0803

Jacques, M.-A., Arlat, M., Boulanger, A., Boureau, T., Carrère, S., Cesbron, S., Chen, N. W. G., Cociancich, S., Darrasse, A., Denancé, N., Fischer-Le Saux, M., Gagnevin, L., Koebnik, R., Lauber, E., Noël, L. D., Pieretti, I., Portier, P., Pruvost, O., Rieux, A., Robène, I., Royer, M., Szurek, B., Verdier, V., and Vernière, C. 2016. Using ecology, physiology, and genomics to understand host specificity in Xanthomonas. Annual Review of Phytopathology 54:163–187. https://doi.org/10.1146/annurev-phyto-080615-100147

Kizheva, Y, Urshev, Z., Rasheva, I., Vancheva, T., Hristova, P., Bogatzevska, N., and Moncheva, P. 2018. PFGE: a tool for examination of heterogeneity between the bacterial spot-causing xanthomonads of tomato plants in Bulgaria. Zeitschrift für Naturforschung C 73(7–8):257–264. https://doi.org/10.1515/znc-2016-0205

Lopez, R., Asensio, C., and Gilbertson, R. L. 2006. Phenotypic and genetic diversity in strains of common blight bacteria (Xanthomonas campestris pv. phaseoli and X. campestris pv. phaseoli var. fuscans) in a secondary center of diversity of the common bean host suggests multiple introduction events. Phytopathology 96(11):1204–1213. https://doi.org/10.1094/PHYTO-96-1204

Mansfield, J., Genin, S., Magori, S., Citovsky, V., Sriariyanum, M., Ronald, P., Dow, M., Verdier, V., Beer, S. V., Machado, M. A., Toth, I., Salmond G., and Foster, G. D. 2012. Top 10 plant pathogenic bacteria in molecular plant pathology. Molecular Plant Pathology 13:614–629. https://doi.org/10.1111/j.1364-3703.2012.00804.x

Oguniobi, A. A., Fagade, O. E., and Dixon, A. G. 2010. Comparative analysis of genetic variation among Xanthomonas axonopodis pv. manihotis isolated from the western states of Nigeria using RAPD and AFLP. Indian Journal of Medical Microbiology 50(2):132–138. https://doi.org/10.1007/s12088-010-0037-6

Pappa, O., Mandilara, G., Vatopoulos, A., and Mavridou, A. 2013. Typing of Pseudomonas aeruginosa strains isolated from Greek water samples by three typing methods: serotyping, Random Amplified Polymorphic DNA (RAPD) and Pulsed Field Gel Electrophoresis (PFGE). Water Science and Technology 67(6):1380–1388. https://doi.org/10.2166/wst.2013.678

Pruvost, O., Magne, M., Boyer, K., Leduc, A., Tourterel, C., Drevet, C., Ravigne, V., Gagnevin, L, Guerin, F., Chiroleu, F., Koebnik, R.,Verdier, V., and Verniere, C. 2014. A MLVA genotyping scheme for global surveillance of the citrus pathogen Xanthomonas citri pv. citri suggests a worldwide geographical expansion of a single genetic lineage. PLoS ONE 9(6):e98129. https://doi.org/10.1371/journal.pone.0098129

Pruvost, O., Vernière, C., Vital, K., Guérin, F., Jouen, E., Chiroleu, Ah-You, N., and Gagnevin, L. 2011. Insertion sequence- and tandem repeat-base genotyping techniques for Xanthomonas citri pv. mangiferaeindicae. Phytopathology 101(7):887–893. https://doi.org/10.1094/PHYTO-11-10-0304

Terletskiy, V., Kuhn, G., Francioli, P., and Blanc, D. 2008. Application and evaluation of double digest selective label (DDSL) typing technique for Pseudomonas aeruginosa hospital isolates. Journal of Microbiological Methods 72:283–287. https://doi.org/10.1016/j.mimet.2007.12.006

Terletsky, V. P., Tyshchenko, V. I., Novikova, I. I., Boykova, I. V., Tyulebayev, S. D., and Shakhtamirov, I. Ya. 2016. An effective method of genetic certification of Bacillus subtilis strains – promising producers of biological preparations. Microbiology 85(1):50–55. (In Russian) https://doi.org/10.7868/S0026365616010134

Ziebell, K., Chui, L., King, R., Johnson, S., Boerrlin, P., and Johnson R. P. 2017. Subtyping of Canadian isolates of Salmonella Enteritidis using Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) alone and in combination with Pulsed-Field Gel Electrophoresis (PFGE) and phage typing. Journal of Microbiological Methods 139:29–36. https://doi.org/10.1016/j.mimet.2017.04.012