شناسایی ژنوتیپی و بررسی تنوع درون گونهای جدایههای بیماری¬زای زانتوموناس از میزبانهای لوبیا و یونجه در استانهای آذربایجانشرقی و غربی
فردین نصرت نژاد 1 , سویل نعمت اللهی 2 , کیومرث روح رضی 3
1 - آزاد تبریز
2 - دانشگاه
3 - دانشگاه آزاد
کلید واژه: آذربایجان شرقی, آذربایجان غربی, لوبیا, یونجه, زانتوموناس,
چکیده مقاله :
طی سالهای 1398 و 1399 به منظور جداسازی باکتریهای بیماریزا از میزبانهای لوبیا و یونجه دارای علائم بیماری لکهبرگی، پژمردگی و بلایت که از برخی مزارع و باغات استانهای آذربایجان شرقی و غربی نمونه برداری شده بودند، در مجموع 140 جدایه باکتریایی روی محیط کشت نوترینت آگار به صورت جدایههای اولیه، کشت و خالصسازی گردیدند. براساس رنگ و شکل ظاهری کلنیها جدایههایی که به باکتری زانتوموناس مورد نظر ما نزدیک بودند انتخاب شده و پس از کشت دوباره و خالص سازی در مجموع 62 جدایه بدست آمد. برای اثبات بیماریزایی جدایهها، هر یک از آنها به صورت جداگانه به میزبان خود تلقیح شده و در شرایط گلخانهای به مدت دو هفته نگهداری شدند که 33 استرین توانستند روی میزبان خود بیماریزایی ایجاد کنند. در این استرینها قطعه 239 جفت بازی ژن اختصاصی زانتوموناس که بخشی از ژن ITS است تکثیر شد. بنابراین این استرینها به عنوان جنس زانتوموناس درنظر گرفته شدند. براساس انگشت نگاری ژنتیکی به روش rep-PCR استرینهای جداشده از لوبیا و یونجه نشان داد که استرینهای لوبیا به چهار خوشه و استرینهای یونجه به سه گروه تقسیم بندی شدند که نتایج حاکی از تنوع ژنتیکی بین استرینها میباشد. براساس توالی یابی ژن های 16S rRNA، gyrB و rpoD،، استرینهای یونجه X. alfalfae subsp. alfalfa و استرینهای جداشده از میزبان لوبیا به عنوان گونه X. phaseoli pv. pahseoli شناسایی شدند. با بررسی منابع موجود، تحقیق حاضر اولین گزارش از جداسازی، شناسایی و بررسی تنوع ژنتیکی این استرینها از میزبانهای لوبیا و یونجه در استانهای آذربایجان شرقی و غربی میباشد.
In order to isolate pathogenic bacteria from the hosts of bean and alfalfa, 140 bacterial isolates were cultured and purified on NA medium in East and West Azerbaijan provinces. Based on the color and appearance of the colonies, the isolates that were close to the desired Xanthomonas bacteria were selected. After re-cultivation and purification, a total of 62 isolates were obtained. To perform the pathogenicity test of the isolates, each of them was inoculated individually to its host and kept in greenhouse conditions for two weeks. 33 strains were able to cause pathogenicity on the host. The strains were able to amplify the 239 bp fragment of Xanthomonas specific gene, which is part of the ITS gene. Therefore, these strains were considered as Xanthomonas bacteria. Based on genetic fingerprinting by rep-PCR method, the strains isolated from bean and alfalfa was divided into four and three groups, respectively, which results indicated genetic diversity between the strains. Based on the sequencing of 16S rRNA, gyrB and rpoD genes, the strains isolated from alfalfa were identified as X. alfalfae subsp. alfalfae and bean strains were identified as X. phaseoli pv. pahseoli. By reviewing the available sources, the present research is the first report of the isolation, identification and genetic diversity of these strains from bean and alfalfa hosts in East and West Azerbaijan provinces.
لک، م. ر.، شمس¬بخش، م. و بهار، م. 1372. وقوع بیماری سوختگی باکتریایی معمولی لوبیا در استان مرکزی. خلاصه مقالات چهاردهمین کنگره گیاهپزشكی ایران. دانشگاه صنعتی اصفهان. صفحه 995.
لک، م. 1397. بیماری سوختگی باکتریایی معمولی لوبیا و روش¬های مدیریت آن. دستور¬العمل فنی. مؤسسه تحقیقات گیاهپزشکی کشور. 16 صفحه.
یاری¬پور، ز.، تقوی، م.، اسداغی، ا. و زارعی، ص. 1397. اولین گزارش از وقوع لکه¬باکتریایی یونجه ناشی از Xanthomonas alfalfae subsp. alfalfae در ایران. بیست و سومین کنگره گیاهپزشکی ایران. شیراز. صفحه 238.
Adriko, J., Mbega, E.R., Mortensen, G., Wulff, E., Tushemereirwe, W., Kubiriba, J. and Lund, O. 2014. Improved PCR for identification of members of the genus Xanthomonas. European Journal of Plant Pathology 138: 293-306.
Agrios, G.N. 2005. Plant Pathology, 5th edition. Academic Press, 635 pp.
Alavi, S.M., Sanjari, S., Durand, F., Brin, C., Manceau, C. and Poussier, S. 2008. Assessment of the genetic diversity of Xanthomonas axonopodis pv. phaseoli and Xanthomonas fuscans subsp. fuscans as a basis to identify putative pathogenicity genes and a type III secretion system of the SPI-1 family by multiple suppression subtractive hybridizations. Applied and Environmental Microbiology 74(10): 3295–3301.
Almeida, N.F., Yan, S., Cai, R., Clarke, C.R., Morris, C.E., Schaad, N.W., Schuenzel, E.L., Lacy, G.H., Sun, X., Jones, J.B., Castillo, J.A., Bull, C.T., Leman, S., Guttman, D.S., Setubal, J.C. and Vinatzer, B.A. 2010. PAMDB, a multilocus sequence typing and analysis database and website for plant-associated microbes. Phytopathology 100: 208–215.
Alexander, B.C., Mkandawire, R.B., Mabagala, P.G., Gepts, P. and Robert, L. 2004. Genetic diversity and pathogenic variation of common blight bacteria (Xanthomonas campestris pv. phaseoli and X. campestris pv. phaseoli var. fuscans) suggests pathogen coevolution with the common bean. Bacterilogy 19: 27-35.
Bradbury, J.F. 1981. Xanthomonas campestris pv. alfalfae. IMI Description of Fungi and Bacteria. CAB International. https://doi.org/10.1079/DFB/20056400698.
Barionovi, D. and Scortichini, M. 2008. Integron variability in Xanthomonas arboricola pv. juglandis and Xanthomonas arboricola pv. pruni strains. FEMS Microbiology Letters 288: 19–24.
Chen, N.W.G., Ruh, M., Darrasse, A., Foucher, J., Briand, M., Costa, J., Studholme, D.J. and Jacques, M.A. 2021. Common bacterial blight of bean: a model of seed transmission and pathological convergence. Molecular Plant Pathology 22: 1464–1480.
Gaudin, C., Gihaut, C., Briand, M., Marques, S.A., Marisa, A.S.V. and Chen, W.G. 2023. Whole genome sequences of nine Xanthomonas strains responsible for common bacterial blight of bean. Microbiology Resource Announcements 12(3): e01259-22.
Gilbertson, R.L. and Maxwell, D.P. 1992. Common bacterial blight of bean. Pp: 18-39. In: Chaub, H.C., Singh, U.S. and Mukhopadhay, A.N. (eds.). Plant Diseases of International Importance, Vol. II. Diseases of Vegetables and Oil Seed Crops. Prentice Hall, New Jersey, USA.
Giovanardi, D., Dallai, D. and Stefani, E. 2017. Population features of Xanthomonas arboricola pv. pruni from Prunus spp. orchards in northern Italy. European Journal of Plant Pathology 147: 761-771.
Heidari, A. and Khodakaramian, G. 2011. Detection and identification of Clavibacter michiganensis subsp. insidiosus inducing alfalfa wilt disease using specific PCR and physiological methods in Iran. Research Journal of Chemistry and Environment 15: 486–491.
Ivanović, Z., Popović, T., Janse, J., Kojić, M., Stanković, S., Gavrilović, V. and Fira, D. 2015. Molecular assessment of genetic diversity of Xanthomonas arboricola pv. juglandis strains from Serbia by various DNA fingerprinting techniques. European Journal of Plant Pathology 141: 133–145.
Jacques, M.A., Bolot, S., Charbit, E., Darrasse, A., Briand, M., Arlat, M., Gagnevin, L., Koebnik, R., Noël, L.D., Portier, P., Carrère, S. and Boureau, T. 2013. High-quality draft genome sequence of Xanthomonas alfalfae subsp. alfalfae strain CFBP 3836. Genome Announcements 1(6): e01035–13.
Jiang, S., Balan, B., Assis, R.A.B., Sagawa, C.H.D. and Wan, X. 2020. Genome-wide profiling and phylogenetic analysis of the SWEET sugar transporter gene family in walnut and their lack of responsiveness to Xanthomonas arboricola pv. juglandis infection. International Journal of Molecular Science 21(4): 1251.
Leyns, F.M.D., Cleene, J. and Swings, J.D. 1984. The host range of the genus Xanthomonas. Botany Review 50(3): 308-353.
López, R., Asensio, C. and Robert, L. 2006. Phenotypic and genetic diversity in strains of common blight bacteria (Xanthomonas campestris pv. phaseoli and X. campestris pv. phaseolivar. fuscans) in a secondary center of diversity of the common bean host suggests multiple introduction events. Bacteriology 4: 23-35.
Mafakheri, H., Taghavi, S.M., Banihashemi, Z., Osdaghi, E. and Lamichhane, J.R. 2017. Pathogenicity, host range and phylogenetic position of Agrobacterium species associated with sugar beet crown gall outbreaks in Southern Iran. European Journal of Plant Pathology 147: 721–730.
Martins, L., Fernandes, C., Blom, J., Dia, N.C., Pothier, J.F. and Tavares, F. 2020. Xanthomonas euroxanthea sp. nov., a new xanthomonad species including pathogenic and non-pathogenic strains of walnut. International Journal of Systematic and Evolutionary Microbiology 70: 6024–6031.
Mhedbi-Hajri, N., Hajri, A., Boureau, T., Darrasse, A., Durand, K., Brin, C., Fischer-Le Saux, M., Manceau, C., Poussier, S., Pruvost, O., Lemaire, C. and Jacques, M.A. 2013. Evolutionary history of the plant pathogenic bacterium Xanthomonas axonopodis. PLoS ONE 8(3): e58474.
Osdaghi, E., Taghavi, S.M., Fazliarab, A., Elahifard, E. and Lamichhane, J.R. 2015. Characterization, geographic distribution and host range of Curtobacterium flaccumfaciens: An emerging bacterial pathogen in Iran. Crop Protection 78: 185–192.
Parkinson, N., Aritua, V., Heeney, J., Cowie, C., Bew, J. and Stead, D. 2007. Phylogenetic analysis of Xanthomonas species by comparison of partial gyrase B gene sequences. International Journal of Systematic and Evolutionary Microbiology 57: 2881–2887.
Parraguirre-Lezama, C., Arenas O.R., Valencia de Ita, M.A., Rivera, A., Villa-Ruano, N. and Gutiérrez, A.M. 2021. First report of Xanthomonas campestris pv. campestris as the causal agent of necrotic leaf spot in Phaseolus vulgaris at Puebla, Mexico. Plant Disease 105: 12-15.
Rademaker, J.L., 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. International Journal of Systematic and Evolutionary Microbiology 50(2): 665-677.
Rohlf, F.J. 1993. Numerical Taxonomy and Multivariate Analysis System NTSys-PC Version 1.80 Exeter Software. New York.
Rouhrazi, K. and Rahimian, H. 2012. Characterization of Iranian grapevine isolates of Rhizobium (Agrobacterium) spp. Journal of Plant Pathology 94: 555–560.
Saitou, N. and Nei, M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4: 406–425.
Schaad, N.W., Jones, J.B. and Chun, W. 2001. Laboratory Guide for Identification of Plant Pathogenic Bacteria. American Phytopathology Society Minnesota, 373 pp.
Schaad, N.W., Postnikova, E., Lacey, G., Sechler, A., Agarkova, I., Stromberg, P.E., Stromberg, V.K. and Vidaver, A.K. 2007. List of new names and new combinations previously effectively, but not validly, published. International Journal of Systematic and Evolutionary Microbiology 57: 893–897.
Stutevllle, D.L. and Sorensen, E.L. 1966. Distribution of leaf spot and damping-off (Xanthomonas alfalfae) of alfalfa in Kansas, and new hosts. Plant Disease Reporter 50: 731–734.
Tamura, K., Stecher, G., Peterson, D., Filipski, A. and Kumar, S. 2013. MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Molecular and Biology Evolution 30: 2725-2729.
Taran, B., Michaels, T.E. and Pauls, K.P. 2001. Mapping genetic factors affecting the reaction to Xanthomonas axonopodis pv. phaseoli in Phaseolus vulgaris L. under field conditions. Genome 44: 1046-1056.
Timilsina, S., Potnis, N., Newberry, E.A., Liyanpathirange, P., Bocardo, F.I., White, F.F., Goss, E.M, and Jones, J.B. 2020. Xanthomonas diversity, virulence and plant–pathogen interactions. Natural Review Microbiology 18: 415–427.
Weisburg, W.G., Barns, S.M., Pelletior, D.A., and Lanem, D.J. 1991. 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology 173: 697–703.
Vancheva, T., Bogatzevska, N., Moncheva, P., Mitrev, S., Vernière, C. and Koebnik, R. 2021. Molecular epidemiology of Xanthomonas euvesicatoria strains from the Balkan Peninsula revealed by a new multiple-locus variable-number tandem-repeat analysis scheme. Microorganisms 9(3): 536.
Vauterin, L., Hoste, B., and Korstersk, S.L. 1995. Reclassification of Xanthomonas. International Journal of Systematic Bacteriology 45: 472-489.
Versalovic, J., Koeuth, T., and Lupski, J.R. 1991. Distribution or repetitive DNA– sequences in eubacteria and application of fingerprinting of bacterial genomes. Nucleic Acid Research 19: 6823–6831.
Versalovic, J., Schneider, M., de Bruijn, F.J. and Lupski, J.R. 1994. Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction. Methods in Molecular Cell Biology 5: 25–40.
Zamani, Z., Morid, B., Jacques, M., Lak, M. and Akhavan, A. 2011. Genetic diversity of the common bacterial blight pathogen of bean, Xanthomonas axonopodis pv. phaseoli, in Iran revealed by rep-PCR and PCR–RFLP analyses. World Journal of Microbiology and Biotechnology 27: 2371–2378.
Yamamoto, S., Kasai, H., Arnold, D.L., Jackson, R.W., Vivian, A. and Harayama, S. 2000. Phylogeny of the genus Pseudomonas: intrageneric structure reconstructed from the nucleotide sequences of gyrB and rpoD genes. Microbiology (Reading) 146(10): 2385-2394.
Yaripour, Z., Taghavi, M., Osdaghi, E. and Lamichhane, J. 2018. Host range and phylogenetic analysis of Xanthomonas alfalfae causing bacterial leaf spot of alfalfa in Iran. European Journal of Plant Pathology 150: 267-274.
Young, J.M., Dye, D.W., Bradbury, J.F., Panagopoulos, C.G. and Robbs, C.F. 1978. A Proposed Nomenclature and Classification for Plant Pathogenic Bacteria. New Zealand Journal of Agricultural Research 21: 153–177.