Assessment of Genetic Diversity among Xanthomonas arboricola pv. pruni Strains Using gyrB Gene Sequencing and rep-PCR Genomic Fingerprinting in North Eastern Iran

Authors
E. Derakhshan
S. Baghaee-Ravari
E. Mahdikhani-Moghaddam
Department of Crop Protection, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Islamic Republic of Iran.
Abstract
In the current study, the phenotypic and molecular properties of twenty-five strains obtained from cankerous tissues or leaf necrotic lesions of different stone fruits were evaluated in north-east of Iran . All strains studied were identified as Xanthomonas arboricola pv. pruni (Xap) based on phenotypic assays and confirmed by means of specific PCR at species and pathovar levels. All obtained strains were pathogenic under artificial inoculation and exhibited brittle necrotic spots on plum leaves of cultivar Santa Rosa under lab conditions. Then, the pathogenic Xap strains were subjected to molecular assays. In a phylogenetic tree constructed with gyrB sequences, no polymorphism was observed in this gene and Iranian Xap strains were clustered with the reference one in a separate group. The ERIC, BOX and REP primer sets generated reproducible genomic PCR profiles in tested strains and, based on combined data for all primers, a low genetic diversity among Xap strains was revealed. In order to achieve results that are more accurate, application of Xap strains from all geographical regions of Iran will be needed to prove little polymorphism observed in Xap population. The current contribution is the first report of molecular homogeneity of Xap strains that were collected from northeastern Iran.

Keywords

Subjects

1. Ayers, S .H., Rupp, P. and Johnson, W. T. 1919. A Study of the Alkali-Forming Bacteria in Milk. United States Dept. Agr. Bull., 782: 1–39.
2. Barionovi, D. and Scortichini, M. 2008. Integron Variability in Xanthomonas arboricola pv. juglandis and Xanthomonas arboricola pv. pruni Strains. FEMS Microbiol. Lett., 288:19–24.
3. Bergsma-Vlami, M., Martin, W., Koenraadt, H., Teunissen, H., Pothier, J.F., Duffy, B. and van Doorn, J. 2012. Molecular Typing of Dutch Isolates of Xanthomonas arboricola pv. pruni Isolated from Ornamental Cherry Laurel. J. Plant Pathol., 94: S1.29–S1.35.
4. Boudon, S., Manceau, C. and Notteghem, J. L. 2005. Structure and Origin of Xanthomonas arboricola pv. pruni Populations Causing Bacterial Spot of Stone Fruit Trees in Western Europe. Phytopathology, 95:1081–1088.
5. Chen, W. W. C. 2002. Xanthomonadins, Unique Yellow Pigments of the Genus Xanthomonas. J. Plant hum. Health Institute, 4: 102-105.
6. EFSA PLH Panel (EFSA Panel on Plant Health). 2014. Scientific Opinion on Pest Categorization of Xanthomonas campestris pv. pruni (Smith) Dye. EFSA J., 12: 3857. doi: 10.2903/j.efsa.2014.3857.
7. EPPO (European and Mediterranean Plant Protection Organization). 2006. Xanthomonas arboricola pv. pruni. EPPO Bull., 36:129–133.
8. Essakhi, S., Cesbron, S., Fischer-Le Saux, M., Bonneau, S., Jacques, M.A. and Manceau, C. 2015. Phylogenetic and Variable-Number Tandem-Repeat Analyses Identify Nonpathogenic Xanthomonas arboricola Lineages Lacking the Canonical Type III Secretion System. Appl. Environ. Microbiol., 81:5395–5410.
9. Fischer-Le Saux, M., Bonneau, S., Essakhi, S., Manceau, C. and Jacques, M. A. 2015. Aggressive Emerging Pathovars of Xanthomonas arboricola Represent Widespread Epidemic Clones Distinct from Poorly Pathogenic Strains, as Revealed by Multilocus Sequence Typing. Appl. Environ. Microbiol., 81:4651– 4668.
10. Garita-Cambronero, J. Palacio-Bielsa, A. Lopez, M. M. and Cubero, J. 2016. Draft Genome Sequence for Virulent and Avirulent Strains of Xanthomonas arboricola Isolated from Prunus spp. in Spain. Stand. Genomic Sci. 11: 1-10.
11. Garita-Cambronero, J., Palacio-Bielsa, A., Lopez, M. M. and Cubero, J. 2017. Pan-genomic Analysis Permits Differentiation of Virulent and Non-Virulent Strains of Xanthomonas arboricola that Cohabit Prunus spp. and Elucidate Bacterial Virulence Factors. Front. Microbiol., 8: Article 573.
12. Garita-Cambronero., J. Palacio‐Bielsa. A. and Cubero., J. 2018. Xanthomonas arboricola pv. pruni, Causal Agent of Bacterial Spot of Stone Fruits and Almond: Its Genomic and Phenotypic Characteristics in The X. arboricola Species Context. Mol. plant pathol. 19:2053-2065.
13. Giovanardi, D., Dallai, D. and Stefani, E. 2017. Population Features of Xanthomonas arboricola pv. pruni from Prunus spp. Orchards in Northern Italy. Eur. J. Plant Pathol., 147:761-771.
14. Hajri, A., Pothier, J. F., Fischer-Le Saux, M., Bonneau, S., Poussier, S., Boureau, T., Duffy, B. and Manceau, C. 2012. Type Three Effector Gene Distribution and Sequence Analysis Provide New Insights into the Pathogenicity of Plant-Pathogenic Xanthomonas arboricola. Appl. Environ. Microbiol., 78: 371-384.
15. Jacques, M. A., Arlat, M., Boulanger, A., Boureau, T., Carrere, S., Cesbron, S., Chen, N. W. G., Cociancich, S., Darrasse, A., Denance, N., Fischer-Le Saux, M,, Gagnevin, L., Koebnik, R., Lauber, E., Nobel, L.D., Pieretti, I., Portier, P., Pruvos, O., Rieux, A., Robene, I., Royer, M., Szurek, B., Verdier, V. and Verniere, C. 2016. Using Ecology, Physiology, and Genomics to Understand Host Specificity in Xanthomonas. Annu. Rev. Phytopathol., 54: 163–187.
16. Jami, F., Kazempour, M. N., Elahinia, S. A. and Khodakaramian, G. 2005. First Report of Xanthomonas arboricola pv. pruni on Stone Fruit Trees in Iran. J. Phytopathol. 153:371-372.
17. Kawaguchi, A. 2014. Genetic Diversity of Xanthomonas arboricola pv. pruni Strains in Japan Revealed by DNA Fingerprinting. J. Gener. Plant Pathol., 80: 366–369.
18. Lamichhane, J. R. 2014. Xanthomonas arboricola Diseases of Stone Fruit, Almond, and Walnut Trees: Progress toward Understanding and Management. Plant Dis., 98: 1600-1610.
19. Lee, Y.A., Hildebrand, D. C. and Schroth, M. N. 1992. Use of Qquinate Metabolism as a Phenotypic Property to Identify Members of Xanthomonas campestris DNA Homology Group 6. Phytopathology, 82: 971–973.
20. Lopez-Soriano, P., Boyer, K., Cesbron, S., Morente, M. C., Penalver, J., Palacio-Bielsa, A., Verniere, C., Lopez, M. M. and Pruvost, O. 2016. Multilocus Variable Number of Tandem Repeat Analysis Reveals Multiple Introductions in Spain of Xanthomonas arboricola pv. pruni, the Causal Agent of Bacterial Spot Disease of Stone Fruits and Almond. PLoS One, 11: e0163729.
21. Loreti, S., Pucci, N., Perez, G., Catara, V., Scortichini, M., Bella, P., Ferrante, P., Giovanardi, D. and Stefani, E. 2015. Detection and Identification of Xanthomonas arboricola pv. pruni from Symptomless Plant Material: Results of an Italian Test Performance Study. OEPP/EPPO Bull., 45:41–51.
22. Louws, F. J., Fulbright, D. W., Stephens, C. T. and Bruijn, F. J. 1994. Specific Genomic Fingerprints of Phytopathogenic Xanthpmnas and Pseudomonas Pathovars and Strains Generated with Repetitive Sequences and PCR. Appl. Environ. Microbiol., 60: 2286-2295.
23. Mahmoudi, H., Rahnama, K., Rahimian, H., Nasrolahnejad, S. and Taghinasab, M. 2011. Investgation on Casual and Associated Agents with Bacterial Canker Stone Fruit Trees in Golestan Province. J. Plant Product., 18:1-14.
24. Morales, G., Llorente, I., Montesinos, E. and Moragrega, C. 2017. A Model for Predicting Xanthomonas arboricola pv. pruni Growth as a Function of Temperature. PLoS One, 12: e0177583.
25. Pagani, M. C. 2004. An ABC Transporter Protein and Molecular Diagnosis of Xanthomonas arboricola pv. pruni Causing Bacterial Spot of Stone Fruits. PhD. Thesis, Department of Plant Pathology, North Carolina State University, Raleigh, NC.
26. Palacio-Bielsa, A., Pothier, J. F., Roselló, M., Duffy, B. and López, M. M. 2012. Detection and Identification Methods and New Tests as Developed and Used in the Framework of COST 873 for Bacteria Pathogenic to Stone Fruits and Nuts; Xanthomonas arboricola pv. pruni. J. Plant Pathol., 94: 135- 146.
27. Palacio-Bielsa, A., Rosello, M., Cambra, M. A. and López, M. M. 2010. First Report on Almond in Europe of Bacterial Spot Disease of Stone Fruits Caused by Xanthomonas arboricola pv. pruni. Plant Dis., 94:786. http://dx.doi.org/10.1094/PDIS-94-6-0786C.
28. Parkinson, N., Cowie, C., Heeney, J. and Stead, D. 2009. Phylogenetic Structure of Xanthomonas Determined by Comparison of gyrB Sequences. Int. J. Syst. Evol. Microbiol., 59: 264–274.
29. Pothier, J. F., Pagani, M. C., Pelludat, C., Ritchie, D. F. and Duffy, B. 2011. A Duplex-PCR Method for Species and Pathovar-Level Identification and Detection of the Quarantine Plant Pathogen Xanthomonas arboricola pv. pruni. J. Microbial. Methods, 86: 16-24.
30. Rademaker, J. L. W., Hoste, B., Louws, F. J., Kersters, K., Swings, J., Vauterin, L., Vauterin, P. and de Bruijn, F. J. 2000. Comparison of AFLP and rep-PCR Genomic Fingerprinting with DNA-DNA Homology Studies: Xanthomonas as a Model System. Int. J. Syst. Evol. Microbiol., 50: 665-677.
31. Randhawa, R. S. and Civerolo, E. L. 1985. A Detached-Leaf Bioassay for Xanthomonas campestris pv. pruni. Phytopathology, 75: 1060–1063.
32. Rodriguez-R, L. M., Grajales, A., Arrieta-Ortiz, M. L., Salazar, C., Restrepo, S. and Bernal, A. 2012. Genomes-Based Phylogeny of the Genus Xanthomonas. BMC Microbiol., 12:43.
33. Rosello, M. Santiago, R. Palacio-Bielsa, A. Garcıa-Figueres, F. Monton, C. Cambra, M. and Lopez, M. 2012. Current Status of Bacterial Spot of Stone Fruits and Almond Caused by Xanthomonas arboricola pv. pruni in Spain. J. Plant Pathol. 94: 15–21.
34. Schaad, N. W., Jones, J. B. and Chun, W. 2001. Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3th Edition, USA, APS Press, 373 PP.
35. Scortichini, M., Rossi, M. P. and Marchesi, U. 2002. Genetic, Phenotypic and Pathogenic Diversity of Xanthomonas arboricola pv. corylina Strains Question the Representative Nature of the Type Strain. Plant Pathol., 51: 374-381.
36. Socquet-Juglard, D. Patocchi, A. Pothier, J. F. Christen, D. and Duffy, B. 2012. Evaluation of Xanthomonas arboricola pv. pruni Inoculation Techniques to Screen for Bacterial Spot Resistance in Peach and Apricot. J. Plant Pathol., 94(1): 91-96.
37. Stolp, H. and Starr, M. P. 1964. Bacteriophage Reactions and Speciation of Phytopathogenic Xanthomonads. Phytopathlogy, 51: 442–478.
38. Stefani, E. 2010. Economic Significance and Control of Bacterial Spot/Canker of Stone Fruits Caused by Xanthomonas arboricola pv. pruni. J. Plant Pathol., 92: 99–103.
39. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. and Kumar, S. 2011. MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Mol. Biol. Evol., 28: 2731–2739.
40. Thompson, J. D., Gibson, T. J., Plewniak, F. and Higgins, D. G. 1997. The CLUSTALX Windows Interface: Flexible Strategies for Multiple Sequence Alignment Aided by Quality Analysis Tools. Nuc. Acids Res., 24:4876–4882.
41. Vauterin, L., Yang, P., Alvarez, A., Takikawa, Y., Roth, D. A., Vidaver, A.K. Stall, R.E. Kersters, K. Swings J. 1996. Identification of Non-Pathogenic Xanthomonas Strains Associated with Plants. Syst. Appl. Microbiol., 19:96–105.
42. Versalovic, J., Koeuth, T. and Lupski, J. R. 1991. Distribution of Repetitive DNA Sequences in Eubacteria and Application to Fingerprinting of Bacterial Genomes. Nuc. Acids Res., 19: 6823-6831.
Journal of Agricultural Science and Technology
Volume 21, Issue 4
July and August 2019
Pages 1023-1034