تنوع ژنتیکی و صفات فیزیولوژیکی استرین‌های Ralstonia solanacearum مزارع سیب زمینی استان همدان

نوع مقاله : مقاله پژوهشی

نویسندگان

1 دانشجوی سابق کارشناسی‌ارشد بیماری‌شناسی گیاهی، دانشکده کشاورزی، دانشگاه بوعلی‌سینا، همدان

2 استاد بیماری‌شناسی گیاهی، گروه گیاه‌پزشکی، دانشکده کشاورزی، دانشگاه بوعلی‌سینا، همدان

چکیده

بیماری پژمردگی باکتریایی ناشی از Ralstonia solanacearum از بیماری­های مهم سیب­زمینی در استان همدان است. برای بررسی تنوع ژنتیکی جدایه‌های R. solanacearum نمونه‌های گیاهی مشکوک به آلودگی در بهار و تابستان سال 1389 از مناطق مختلف این استان جمع‌آوری گردید. کشت اندام‌های آلوده گیاهی (غده و ساقه) روی محیط نوترینت آگار (NA) حاوی تری فنیل تترازولیوم کلراید و بررسی خصوصیات بیوشیمیایی و فیزیولوژیکی، منجر به شناسایی 52 استرین R. solanacearum بیوار A2 گردید. برای تشخیص دقیق­تر جدایه­ها واکنش زنجیره­ای پلیمراز با استفاده از آغازگرهای ps96H و ps96I انجام شد. نتایج PCR نشان داد که نمونه­ها همراه با نمونه استاندارد یک باند مشابه 148جفت بازی تولید نمودند. تنوع ژنتیکی 26 جدایه انتخابی با آغازگرهای BOX و ERIC مورد بررسی قرار گرفت. طول قطعات تکثیری با استفاده از آغازگر BOX A1R در محدوده 100 تا bp1500 و آغازگر ERIC از200 تا bp1200 متغیر بود. گروه­بندی جدایه­های به‌دست آمده با روش UPGMA و براساس ضریب جاکارد با استفاده از داده­های هر دو آغازگـر، جـدایه­ها را در سطح تشابه 74 درصـد به 5 گروه اصـلی منتسب کرد. نتایج حاصل نشان‌دهـنده‌ی آن است که استرین­های R. solanacearum جمع­آوری شده خصوصیات فنوتیپی و ژنتیکی متفاوتی داشتند، هرچند ارتباط مشخصی بین منطقه جغرافیایی و خصوصیات استرین­ها به‌دست نیامد. این بررسی کارآیی روشPCR  با آغازگرهایBOX  و ERIC را برای نشان دادن چندشکلی ژنتیکی در جمعیت­های R. solanacearum نشان داد.

کلیدواژه‌ها

موضوعات


عنوان مقاله [English]

Diversity and Physiological Characteristics of Ralstonia solanacearum from Potato Fields in Hamedan Province

نویسندگان [English]

  • Somayeh Esfandiari 1
  • Gholam Khodakaramian 2
1 M. Sc. Former Student of Plant Pathology, Department of Plant Protection, Faculty of Agriculture, University of Bu-Ali Sina, Hamedan
2 Professor of Plant Pathology, Department of Plant Protection, Faculty of Agriculture, University of Bu-Ali Sina, Hamedan
چکیده [English]

Bacterial wilt disease caused by Ralstonia solanacearum is an important disease on potato in Hamedan province. During spring and summer 2011 infected plants were collected from potato fields to investigate the genetic diversity among R. solanacearum strains. A total of 52 strains were isolated on NA medium containing triphenyl tetrazolium chloride (TTC) from tubers and stems and their phenotypic characteristics revealed that they were belong to biovar 2A. PCR using ps96h and ps96i primers produced 148 bp bands in tested and standard strains. Genetic diversity of 26 selected strains were analyzed by PCR fingerprinting using, ERIC and BOX primers. The length of produced fragments ranged from 200bp to 1200bp in BOX primer and 200bp to 1500bp in rep primer. Tested bacterial strains were grouped based on the obtained molecular DATA from PCR using UPGMA method and Jaccard`s coefficient. Results showed five distinguished clusters. R. solanacearum strains were genetically divers although no direct connection was observed between geographic locations and PCR groups. The results of this study indicated that rep-PCR technique with two ERIC and BOX primers is capable to show genetic polymorphism among different populations of R. solanacearum.

کلیدواژه‌ها [English]

  • Biovar 2A
  • BOX
  • ERIC
  • Potato wilt disease
  • Hamedan province
Ausoble, F. M., Brent, R.., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. and Struhl, K. 2002. A compendium of method from current protocols in molecular biology. John Wiley and Sons, 900 pp.
Bahar, M., Nouri, S. H., Tasaodian, F., Samavatian, H. and Mortazavi, B. 2004. Comparison of different methods for detection of Ralstonia solanacearum in soil. Proceeding 16th Iran. Plant Protection. Congress. Tabriz. Iran. 226.
Bahar, M. and Danesh, D. 1988. Etiology of potato wilt in Iran. Plant Pathology, 24: 1-11.
Buddenhagen, I. W. and Kelman, A. 1964. Biological and Physiological aspects of bacterial wilt caused by Pseudomonas solanacearum. Phytopathology, 2: 203-230.
Debrujin, F. J. 1992. Use of repetitive (repetitive extragenic palindromic and enterobacter repetitive intergenic consensus) sequence and the polymerase chain reaction to fingerprint the genomes of Rhizobium meliloti isolates and other soil bacteria. Applied and Environmental Microbiology, 58: 2180-2187.
Cook, D., Barlow, E. and Sequeira, L. 1989. Genetic diversity of Pseudomonas solanacearum: Detection of restriction fragment length polymorphism with DNA probes that specify virulence and the hypersensitive response. Molecular Plant Microbe Interaction, 2: 113-121.
Fahy, P. C. and Haywerd, A. C. 1983. Media and methods for isolation and diagnostic tests. P. 337-378. In: Fahy, P. C. and Persley, G. J. (eds.). Plant Bacterial Disease, A Diagnostic Guide. Academic Press, Sydeny, Australia.
Fegan, M. and Prior, P. 2005. How complex is the Ralstonia solanacearum species complex. in: Bacterial wilt disease and the Ralstonia solanacearum Species Complex. Allen, C., Prior, P. and Hayward, A. C. eds. American Phytopathological Society, 26: 449-461.
Fouche-Wiech, J., Poussier, S., Trigalet-Demery, D., Berger, D. and Countinho, T. 2006. Molecular identification of some African strains of Ralstonia solanacearum from eucalyptus and potato. Genetic Plant Pathology, 72: 369-373.
Jaunet, T. X. and Wang, J. F. 1998. Variation in genotype and aggressiveness of Ralstonia solanacearum Race 1 isolated from tomato in Taiwan. Phytopathology, 89: 320-327.
Hayward, A. C. 1991. Biology and epidemiology of bacterial wilt caused by Pseudomonas solanacearum. Phytopathology, 29: 65-87.
Hayward, A. C. and Hartman, G. L. 1994. Bacterial wilt: The disease and its causative agent, Pseudomonas solanacearum. CAB International, 272 pp.
Hayward, A. C. 1995. Systematics and Phylogeny of Pseudomonas solanacearum and related bacteria. In: Horita, M. and Tsuchiya, K., (eds). Genetic diversity of Japanese strains of Ralstonia solanacearum. Phytopathology, 91: 399-407.
Horita, M. and Tsuvhya, K. 2001. Genetic diversity of Japanese strains of Ralstonia solanacearum. Phytopathology, 91: 399-407.
Horita, M., Tsuvhya, K. and Ooshiro, A. 2005. Characteristics of Ralstonia solanacearum Biovar N2 strains in Asia. Phytopathology, 153: 209-213.
Irandoost, H., Niknam, G. H., Ghasemi, A., Taghavi, M. and Torabi, A. 2008. Evaluation of Genetic Diversity in East Azarbaijan and Fars Provinces Among Ralstonia solanacearum isolates by rep-PCR. Agricultural Sciences, 18: 234-242.
Izadian, M. and Taghavi, M. 2010. Genetic diversity of Ralstonia saolancearum biovars from same potato-growing region of Iran. Proceeding. 19th Iran. Plant Protection. Congress. Tehran. Iran. 403.
Karimi, M. and Harighi, B. 2010. Reaction of several potato cultivars to Ralstonia solanacearum, the causal agent of potato bacterial wilt in Kurdestan and Maekazi provinces. Proceeding. 19th Iran. Plant Protection. Congress. Tehran. Iran. 456.
Kelman, A., Hartman, G. L. and Hayward, A. C. 1994. Introduction. In: Bacterial wilt: the disease and its causative agent, Pseudomonas solanacearum (Ed. by Hayward, A. C. and Hartman, G. L.), pp. 1-7. CAB international, Wallingford, UK.
Khakvar, R., Sijam, K., Muiyan, M., Radu, S. and Kwaie, D. 2008. Improving PCR-based method for identification of Ralstonia solanacearum in natural sources of west Malaysia. Phytopathology, 3: 490-493.
Louws, F. J., Fulbright, D. W., Taylor-Stevens, C. and Debrujin, F. J. 1994. Specific genomic fingerprints of phytopathogenic Xanthomonas and Pseudomonas pathovars and strains generated with repetitive sequence and PCR. Applied and Environmental Microbiology, 60: 2286-2295.
Melanie, L., Brian, B., Gardner, M., Opina, N. and Miller, A. 2007. Diversity of Ralstonia solanacearum infecting eggplant in the Philippines. Phytopathology, 97: 1467-1475.
Mohamadi, S. A. and Parasanna, B. M. 2003. Analysis of genetic diversity in crop plants: Salient statistical tools and considerations. Crop Sciences, 43: 1235-1248.
Norman, D., Zapata, M., Gabrial, D., Duan, Y. and Yuene, J. 2009. Genetic diversity and host range variation of Ralstonia solanacearum strains entering North America. Phytopathology, 99: 1070-1077.
Nouri, S. H., Bahar, M. and Sharifnabi, B. 2004. Assess the genetic diversity of bacteria Ralstonia solanacearum race 3 isolates collected from potato areas of Iran. Proceeding 16th Iran. Plant Protection. Congress. Tabriz. Iran. 225.
Poussier, S., Trigalet, D., Vandewalle, P., Bruno, J. and Trigalet, A. 2000. Genetic diversity of Ralstonia solanacearum as assessed by PCR-RFLP of the hrp gene region AFLP and 16S rRNA Sequence analysis and identification of an African subdivision. Microbiology, 146: 1679-1692.
Prior, P. and Fegan, M. 2005. How complex is the Ralstonia solanacearum species complex. Pp. 449-461. In: Allen, C., Prior, P. and Hayward, A. C. (eds.). Bacterial Wilt Disease and the Ralstonia solanacearum Species Complex. APS, Press, St Paul, Minnesota USA, 510 pp.
Prior, P., Allen Ass, C. and Elphinstone, J. 1998. Bacterial Wilt Disease: Molecular and Ecological Aspects, Paris: INRA Editions.
Poussier, S., Trigalet, D., Vandewalle, P., Bruno, J. and Trigalet, A. 2000. Genetic diversity of Ralstonia solanacearum as assessed by PCR-RFLP of the hrp gene region AFLP and 16S rRNA Sequence analysis and identification of an African subdivision. Microbiology, 146: 1679-1692.
Robertson, AE., Fortnum, BA., Wood, TC. and Klueofel, DA. 2001. Diversity of Ralstonia solanacearum in the Southeastern United States. Beitrage zur Tabakforschung International, 19 (7): 323-331.
Schaad, N. W., Jones, J. B. and Chun, W. 2001. Laboratory Guide for the Identification of Plant Pathogenic Bacteria. 3rd Edn. Phytopathology, St. Paul, MN. pp: 398.
Seal, S. E., Jackson, L. A., Young, J. P. W. and Daniels, M. J. 1993. Differentiation of Pseudomonas solanacearum, Pseudomonas syzygii, Pseudomonas pickettii and the blood disease bacterium by partial 16S rRNA sequencing: constrution of oligonucletide primers for sensitive detection by polymerase chain reaction. Journal of General Microbiology,139: 1587-1594.
Smith, J. J., Offord, L. C., Holderness, M. and Saddler, G. S. 1995. Genetic diversity of Burkholderia solanacearum (Synonym: Pseudomonas solanacearum) race 3 in Kenya. Applied Environmental Microbiology, 61 (12): 4263-4268.
Taghavi, M., Hayward, C., Sly, L. and Fegan, M. 1996. Analysis of the phylogenetic relationships of strains of Burkholderia solanacearum, Pseudomonas syzygii and the blood disease bacterium of banana based on 16S rRNA gene sequences. Bacteriology, 46: 10-15.
Van Der Wolf, J. M., Bonants, P. J. M., Smith, J. J., Hagenaar, M., Nijhuis, E., Van Beckhoven, J. R. C. M., Saddler, G. S., Trigalet, A. and Feuillade, R. 1998. Genetic diversity of Ralstonia solanacearum race 3 in Western Europe determined by AFLP, RC-PFGE and Rep-PCR. Pp. 44-49. In: Prior P, Allen Ass. C. and Elphinstone, J. (eds). Bacterial Wilt Disease: Molecular and Ecological Aspects, Paris: INRA Editions.
Yabbuchi, E., Kosako, Y., Yan, I., Hotta, H. and Nishiuch, Y. 1995. Transfer of two Bukholderia and Alcaligenes species to Ralstonia gen. nov.: Proposal of Ralstonia pockettii (Ralston, Palleroni and Doudoroff, 1973) comb. nov. Rolstonia solanacearum (Smith, 1896) comb. nov. and Ralstonia eutropha (Divis, 1969) comb. nov. Microbiology and Immunology, 39 (11):897-904.