تنوع ژنوتیپی جدایه‎های tumefaciens Agrobacterium و A. vitis از میزبان‌های مختلف در برخی از استان‌های غربی و جنوبی ایران با استفاده از نشانگر RAPD

نوع مقاله : مقاله کامل پژوهشی

نویسندگان

1 به ترتیب دانش آموخته کارشناسی ارشد بیماری شناسی گیاهی ،استادو استاد بیماری شناسی گیاهی دانشکده کشاورزی، دانشگاه شیراز

2 دانشیار پژوهشکده بیوتکنولوژی دانشکده کشاورزی، دانشگاه شیراز

چکیده

به منظور بررسی تنوع ژنتیکی گونه­های  tumefaciens Agrobacterium و A.vitis در سال­های 93-1392 از باغ‎های مو، گلخانه‌های پرورش گل رز و مزارع چغندر قند در استان­ جنوبی (فارس) ، جنوب غربی(کهکیلویه و بویراحمد) و غربی (کردستان و کرمانشاه)، از گیاهان دارای علائم گال طوقه و ریشه نمونه برداری صورت گرفت. بر اساس ویژگی‎های فنوتیپی، آزمون­های بیو‌شیمیایی، بیماری­زایی و همچنین با استفاده از آغازگرهای عمومی جنس (virD2A/virD2C) و اختصاصی در سطح گونه (VCF/VCR و PGF/PGR) بیمارگر به عنوان، tumefaciens A. و A.vitis  شناسایی شد. علاوه بر این با استفاده از جفت آغازگرهای  VisF/VisRو VisFF1/R2 نوع اوپین جدایه‌های A.vitis تشخیص داده شد. جهت بررسی تنوع ژنتیکی بین جدایه­های دو گونه tumefaciens. A و A.vitis در آزمون چندشکلی تصادفی قطعات تکثیر شده­ی دی­ان­اِی (RAPD) از 11 آغازگر تصادفی استفاده شد. در گونه tumefaciens. A جدایه‌های متعلق به میزبان‌های مختلف در سطح تشابه 73 درصد به سه گروه و جدایه‌های A.vitis به دو گروه  تقسیم شدند. نتایج نشان داد که جدایه‌های متعلق به دو گونه tumefaciens. A و A.vitis دارای تنوع زیادی بودند و جدایه­های به دست آمده از یک منطقه­ جغرافیایی در چند گروه مختلف قرار گرفتند. همچنین بین تنوع ژنتیکی جدایه­های مورد بررسی و منطقه­ جغرافیایی یا میزبان آن­ها ارتباطی وجود نداشت .با استفاده از آغازگرهای VisF/VisRو VisFF1/R2 مشخص شد  که 9/57 درصد از جدایه­های A.vitis دارای ژن کد کننده ویتوپین، 3/26 درصد از جدایه دارای ژن کد کننده اوپین اکتوپین / نوپالین  بودند و 8/15 درصد جدایه­ها قادر به تکثیر هیچ یک از قطعات ذکر شده نبودند

کلیدواژه‌ها

عنوان مقاله [English]

Genotypic Diversity among Agrobacterium tumefaciens and A. vitis Isolates From Different hosts in Some of Western and Southern Provinces of Iran using RAPD Marker*

نویسندگان [English]

  • H. Mafakheri 1
  • S. M. Taghavi 1
  • Z. Banihashemi 1
  • A. Niazi 2
1 Former M.Sc. Student, Prof and Prof of Plant Phathol., respectively, School of Agric., Shiraz University., Shiraz, Iran
2 Associate Prof. of Institute of Biotechnology, School of Agric., Shiraz Univ., Shiraz, Iran.
چکیده [English]

To assess the genetic diversity of Agrobacterium tumefaciens and A.vitis , during 2013 and 2014, samples of grapevine, rose and sugar beet with gall symptoms on crown and roots were collected from different areas of southern (Fars and kohgiluye and Boyerahmad) and western (Kurdistan and Kermanshah) provinces of  Iran. Based on phenotypic characteristics, biochemical and pathogenicity tests as well as using the generic primers virD2A/virD2C the isolates were identified as Agrobacterium. Furthermore, species-specific primer pairs VCF/VCR for A. tumefaciens, as well as PGF/PGR for A. vitis were used to determine the identity of the causal agent.For detection of opine type among A. vitis isolates, a PCR assay was carried out using primer pairs VisF/VisR and VisFF1/ R2. Genetic diversity of A. tumefaciens and A.vitis isolates were investigated by random amplified polymorphic DNA technique using 11 random primers. The A.tumefaciens isolates clustered in three groups at 73% level of similarity and A.vitis clustered in two  groups. Based on the results,  A. tumefaciens and A.vitis isolates showed high genetic diversity even in a same geographical region. In conclusion, no correlation was observed between genetic diversity and geographical origins or their host plants. For detection of opine genes among A. vitis isolates, a PCR assay was carried out using primer pairs VisF/VisR and VisFF1/R2. Our results revealed that 57.9% of A. vitis isolates produced vitopine and 26.3% of them produced octopine or nopaline, whereas 15.8% of isolates were unable to amplify specific fragment.

کلیدواژه‌ها [English]

  • Crown gall
  • Ti plasmid
  • Molecular marker

مراجع

Ale-Yasin S.K. and Banihashemi  Z. 1993. Isolation of causal agent of crown gall of grapevine in Fars and Kohgiluye and Boyerahmad provinces. Proceedings of the 11th Iranian Plant Protection Congress. Rasht, Iran. 215(Abst.).
Amani B. 1966. Crown gall of grapevine. Iranian Journal of Plant  Pathology. 3:12-20. (In Farsi with English Summary).
Arabi  F., Nikravesh  Z ., Rezaeian V. and  Rahimian  H. 2002. Bacterial leaf spot of Sisymbrium irio in Tehran province. Proceedings of the 16th Iranian Plant Protection Congress. University of Tabriz,p 253
Bini F., Kuczmog A ., Putnoky P., Otten L.,  Bazzi C. and  Szegedi  E. 2008. Novel pathogen-specific primers for the detection of Agrobacterium vitis and Agrobacterium tumefaciens. Vitis. 47:181-9.
Costechareyre  D., Rhouma  A., Lavire  C ., Portier  P ., Chapulliot  D ., Bertolla  F ., Boubaker A ., Dessaux  Y. and  Nesme  X. 2010. Rapid and efficient identification of Agrobacterium species by recA allele analysis: Agrobacterium recA diversity. Microbial Ecology60:862–872.
De Cleene  M. and  De Ley  J. 1976. The host range of crown gall. The Botanical Review 42: 389-466.
Fatehi R., Mohammadi M., Rahimian H., Sharifi-Tehrani A. and Zakeri Z. 1998 Identification and phenotypic characterization of Agrobacterium vitis the causal agent of crown gall disease of grapes in Karaj and Takestan regions. Proceedings of the 13th Iranian Plant Protection Congress. University of Karaj, p 250.
Haas  J.H., Moore  L.W., Ream  W. and  Manulis  S. 1995. Universal PCR primers for detection of phytopathogenic Agrobacterium strains. Applied and Environmental Microbiology 61:2879-84.
Irani  H. and  Ghasemi  A. 2004. Isolation of Agrobacterium tumefaciens biovar 3 crow gall disease agent and stady ok population in sap and infected vineyards soil. Proceedings of the 16th Iranian Plant Protection Congress .University of  Tabriz,p.358.
Irelan  N. A.  and  Meredith  C. P.  1996. Genetic analysis of Agrobacterium tumefaciens and A. vitis using randomly amplified polymorphic DNA. American Journal of Enology and Viticulture. 47:145-151.
Javaheri M., Mohammadi H., Rahimian H. and Sharifi-Tehrani. 2000. Detection of opines produced by tumergenic stains of Agrobacterium on grapevines in Iran. Proceedings of the 14th Iranian Plant Protection Congress.Univercity of Isfahan,p.343.
Kawaguchi  A., Sawada H. and  Ichinose Y. 2008.Phylogenetic and serological analyses reveal genetic diversity of Agrobacterium vitis strains in Japan. Plant  Pathology 57:747–753.
Knauf  V., Panagopoulos C. and  Nester E. 1982. Genetic factors controlling the host range of Agrobacterium tumefaciens. Phytopathology 72:1545-1549.
Kuzmanovic  N ., Ivanovic  M., Prokic  A., Gasic  K., Zlatkovic  N. and Obradovic  A. 2014. Characterization and phylogenetic diversity of Agrobacterium vitis from Serbia based on sequence analysis of 16S-23S rRNA internal transcribed spacer (ITS) region.European Journal of Plant Pathology 140:757–768.
Llop  P., Lastra  B., Marsal  H., Murillo  J. and López M.M. 2003. Tracking Agrobacterium strains by a RAPD system to identify single colonies from plant tumors. European Journal of Plant Pathology 109:381-389.
Mafakheri H, Taghavi  S. M, Banihashemi Z, Osdaghi E . and Lamichhane  J. R. 2016. 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.
Maki-Valkama T. and Karjalainen R.1994. Differentiation of Erwinia carotovora subsp. atroseptic and carotovora by RAPD-PCR. Annals of Applied Biology 125:301-309.
Mareft A.S. and  Hassanzadeh N. 1998. Bacterial crown gall in imported cherry and apple seeding . Proceedings of the 13th Iranian Plant Protection Congress . University of Tehran, 254-254.
Momol E., Burr T., Reid C., Momol M., Hseu S. and Otten L. 1998. Genetic diversity of Agrobacterium vitis as determined by DNA fingerprints of the 5′‐end of the 23S rRNA gene and Random Amplified Polymorphic DNA. Journal of Applied Microbiology 85: 685-92.
Moore L.W., Bouzar  H . and  Burr  T.J.  2001. Gram-negative bacteria:Agrobacterium. In: Schaad NW,Jones JB, Chun W, editors. Laboratory Guide for Identification of Plant Pathogenic Bacteria.St. Paul (MN). APS Press, 17–35.
Naghavi M. R., Ghariazi B. and Hosseini Salekdeh.2007. Molecular Marker. 2th ed. University of Tehran Press.334 p.
Niknezhad Kazempour M., Roustaei A. and  Rezaei M . 2007. Isolation of Agrobacterium tumefaciens, the causal agent of crown gall disease, from cypress in Kashan. Iranian Journal of Plant  Pathology. 43: 398-409.  (In Farsi with English Summary).
Pooler  M.R . and  Hartung  J.S . 1995. Genetic relationships among strains of Xylella fastidiosa from RAPD-PCR data. Current Microbiology, 31:134-137.
Puławska J. 2010.  Crown gall of stone fruits and nuts, economic significance and diversity of its causal agents: tumorigenic Agrobacterium spp. Journal of Plant Pathology .92:87-98.
Puławska J. and Kałużna M. 2011 . Phylogenetic relationship and genetic diversity of Agrobacterium spp. isolated in Poland based on gyrB gene sequence analysis and RAPD. European Journal of Plant Pathology 133:379–390.
Rademaker  J. L. W., and de Bruijn F. J. 1997. Characterization and classification of microbes by rep-PCR genomic fingerprinting and computer assisted pattern analysis, p. 151–171. In G. Caetano-Anolle´s and P. M. Gresshoff (ed.), DNA markers: protocols, applications, and overviews. J. Wiley and Sons, New York, N.Y.
Rouhrazi  K. and  Rahimian  H. 2012. Genetic diversity of Iranian Agrobacterium strains from grapevine. Annals of Microbiology 62:1661-1667.
Rouhrazia K. 2011. Determination of Rhizobium species and heterogeneity of strains causing grapevine crown gall disease in West Azarbayjan Province. MS Thesis. Sari Agricultural Sciences and Natural Resources University. P 121.
Safdari M, Rahimian H, Hejazi MA. and Barzegari  A . 2010 . A study on the genotypic diversity of the regional Agrobacterium by means of REP-PCR and BOX-PCR. Proc. 19th, Irania Plant Protection Congress. Iran. Research Institute of Plant Protection. Tehran. p 519.
Salahi Ardekani  A ., Taghavi S.M. and Banihashemi  Z. 2000. Distribution and identification of strains of the causal agent of crown gall of grapevine in Fars and Kohgiluye and Boyerahmad provinces. Iranian Journal of Plant  Pathology. 36: 313-328. (In Farsi With English Summary).
Salehi  S., Ghasemi A., Rahimian H. and Nazeriyan I. 2003.Occerence of aerial gall disease of Ficus benjamina caused by Agrobacterium larrymoorei in Iran. Iranian Journal of Plant  Pathology. 39:107-108.(In Farsi with English Summary).
Salehi  S., Rahimian H. and Ghasemi  A. 2006. Diversity of Agrobacterium tumefaciens strains in Iran. Iranian Journal of Plant  Pathology. 42:337-358. (In Farsi With English Summary)
Sawada  H. and  Ieki  H. 1992. Phenotypic characteristics of the genus Agrobacterium. Annals of the Phytopathological Society of Japan 58:37-45.
Schaad N.W., Jones J.B. and Chun W. 2001 . Laboratory Guide for Identification of Plant Pathogenic Bacteria. American Phytopathological Society Press,USA. 373p.
SefcK M., Lefort F., Grando S., Scott K.D., Steinkellner H. and Thomas M.R. 2001. Microsatellite markers for grapevine: A state of the art. in: Roubelakis. K. A. Molecular biology and biotechnology of grapevine. Kluwer Academic Publisher 433-444.
Shahabi Mohammad Abadi M., Taghavi S.M. and Djavaheri M. 2012. Phenotypic and genotypic characteristics of Agrobacterium isolates from different hosts in Fars and Kohgiluye andBoyerahmad provinces Iranian Journal of Plant  Pathology.  48:113 -115. (In Farsi With English Summary).
Shams M. 2012. Assessing the diversity of agrobacterial populations. PhD Thesis. Universite Claude Bernard Lyon 1.P 105Rouhrazia K. and Rahimian H.A. 2014.  Biochemical and genetic characterisation of Agrobacterium tumefaciens the causal agent of walnut crown gall disease in Iran. Archives of Phytopathology and Plant Protection. 47(20):2493–2500
Shams M., Vial  L., Chapulliot  D., Nesme  X. and  Lavire  C. 2013. Rapid and accurate species and genomic species identification and exhaustive population diversity assessment of Agrobacterium spp. using recA-based PCR. Systematic and Applied Microbiology. 351– 358.
Szegedi  E. and  Bottka S.  2002.  Detection of Agrobacterium vitis by polymerase chain reaction in grapevine bleeding sap after isolation on a semiselective medium. Vitis 41:37-42.
Szegedi E . 2003.  Opines in naturally infected grapevine crown gall tumors. Vitis42: 39-41
Taghavi  S.M. and Golkhandan E. 2005. Distribution and etiology of crown gall of sugar beet in Fars and Kohgiluye and Boyerahmad provinces. Iranian Journal of Plant  Pathology. 42:439-457. (In Farsi with English Summary).
Tzfira  T.  and Citovsky  V. 2008. Agrobacterium: from Biology to Biotechnology. Springer.
Xu, Y., M. Rong-Cai, H. Xie and C. Ming-Qing. 2004. Development of SSR markers for the philogenetic analysis of almond trees from China and  Mediterranean  region. Genome 47:1091-1104
Young  J.M., Park D.C . and  Weir  B.S . 2004. Diversity of 16S rDNA sequences of Rhizobium spp. implications for species determinations. FEMS Microbiology letters 238:125-31.
Young  J.M., Pennycook  S.R . and Watson D.R. 2006. Proposal that Agrobacterium radiobacter has priority over Agrobacterium tumefaciens.Request for an opinion. International Journal of Systematic and Evolutionary Microbiology 56:491–493.
بیماریهای گیاهی
دوره 53، شماره 2
شهریور 1396
صفحه 175-195

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