شناسایی فوزاریوم‏های پوساننده ریشه گل مریم در منطقه دزفول و تعیین تنوع ژنتیکی گونه غالب توسط گروه‏های سازگار رویشی و نشانگر مولکولی RAPD

نویسندگان

چکیده

جهت شناسایی گونه­ها و جدایه­های مختلف فوزاریوم عامل پوسیدگی ریشه گل مریم و بررسی تنوع ژنتیکی آنها در شهرستان دزفول نمونه‏برداری از گیاهان آلوده مزارع مختلف این شهرستان انجام گرفت. قارچ‏های وابسته به ریشه با استفاده از روش‏های معمول جداسازی در آزمایشگاه جداسازی گردیدند. جمعاً 143 جدایه از جنس فوزاریوم شامل 110 جدایه Fusarium oxysporum، 27 جدایه F. solani و 6 جدایه F. equiseti شناسایی گردید. تنوع ژنتیکی جمعیت F.oxysporum با استفاده از گروه‏های سازگار رویشی و روش RAPD مورد بررسی قرار گرفت. برای بررسی گرو‏ه‏های سازگار رویشی 45 جدایه به طور تصادفی انتخاب گردید. سپس جهش یافتگان نیت در محیطDox Agar Chlorate  C`zapeck و محیط حداقل (MMC) هر کدام با 3% کلرات پتاسیم تولید شدند.کلاس فنوتیپی جهش‏یافتگان نیت نیز روی محیط کشت پایه حاوی یکی از چهار منبع ازت (نیترات، نیتریت، هیپوزانتین و تارتارات آمونیوم) تعیین گردید. ازکل جهش‏یافتگان نیت تعداد ٣٠١، ١۷١ و ۷۵ عدد به ترتیب به  nit1، nit3 و NitM تعلق داشتند. آزمون مکمل سازی بین جهش‏یافتگان تمام جدایه‏های انتخابی انجام شد. درنهایت چهار گروه VCG مشخص گردید که VCG a شامل 28 جدایه، VCG b شامل 10جدایه، VCG c شامل سه جدایه و VCG d شامل چهار جدایه بود. همچنین تنوع ژنتیکی این جدا‏یه‏ها با استفاده از سیزده آغازگر تصادفی بررسی شد. تجزیه خوشه‏ای داده‏های RAPD با استفاده از روش‏های UPGMA، Single و Complete انجام گرفت. بهترین نتیجه با استفاده از روش UPGMA وضریب تشابه Dice در سطح تشابه 61% به‏دست آمد که بر این اساس شش گروه ژنتیکی مختلف تشخیص داده شد. گروه I شامل دو جدایه، گروه II حاوی 12جدایه، گروه III حاوی دو جدایه و گروه  IVو V تک عضوی و گروه VI نیز شامل دو جدایه بود. آزمون بیماریزایی که با استفاده از جدایه‏های انتخابی از گروه‏های مختلف VCG انجام گرفت نشان داد که تمام جدایه‏ها بیماریزا بودند. این مطالعه اولین گزارش از بررسی تنوع ژنتیکی F.oxysporum روی گل مریم در ایران می‏باشد.

کلیدواژه‌ها


عنوان مقاله [English]

Identification of Fusarium root rot from Tuberose (Polianthes tuberosa L.) in Dezful region and determination of genetic diversity of dominant species based on RAPD analysis and VCG groups

نویسندگان [English]

  • Vida Mahinpoo
  • Reza Farokhi Nejad
  • Hamid Rajabi Memari
  • Zaynab Bahmani
چکیده [English]

In this study, Fusarium root rot from Tubers in Dezful (Khuzestan, Iran) were identified and genetic diversity of domnnat species was determined. Root Associated fungi were isolated using common isolation technique in laboratory. Collectively 143 isolates of fungi including 110, 27, and 6 at 4 species of Fusarium oxysporum, F. solani, F.equiseti were recovered and identified respectively. Genetic diversity of the population of F.oxysporum was determined using VCG and RAPD techniques. For VCG method, 45 isolates of fungus were selected randomly. Then nit mutant were generated on MMC and Czapeck media each containing 3% KClO3. Phenotypic classes of nit mutants were determined according of the growth types on basal medium containing one of four nitrogen sources (Nitrate, Nitrate, Hypoxanthine, and Ammonium). From recovered nits, 301, 171, and 45 were nit 1, nit 3 and nit M respectively. Complementation test was conducted among different nit mutant of different isolates in all combinations. Results revealed that all isolates were placed into 4 VCG groups, the largest one containing 28 and the rest containing 10, 3 and 4 isolates respectively. In addition, genetic diversity of theses isolates was studied using thirteen primers. Cluster analysis of RAPD data was done using UPGMA, Single and Complete methods. The best results obtained by UPGMA and dice coefficient, which distinguished six main groups at 61% similarity level. In this grouping, group I, III and VI had 2 members each, group II had 12 members. Ten of that belonging to VCG a and groups IV and V had one member each. No close relation was observed between VCG and RAPD method results. Pathogenicity test that was conducted using selected isolates of different VCGs revealed that all were pathogenic to plant. This research is first report of genetic diversity of F. oxysporum on Tuberose in Iran.

کلیدواژه‌ها [English]

  • Heterokarion
  • Nit mutant
  • root rot
  • Molecular marker
  • Fusarium
  • Tuberose
Assigbetse, K. B., Fernandez, D., Dubois, M. P., & Geiger, J.P.1994. Differentiation of        Fusarium oxysporum f. sp. vasinfectum races123 on cotton by random amplified        polymorphic DNA (RAPD) analysis. Phytopathology, 84:622-626.

Belabid, L., Baum, M., Fortas, Z., Bouznad, Z. & Eujayl, I. 2004. Pathogenic and       genetic characterization of Algerian isolates of Fusarium oxysporum f. sp. lentis by

      RAPD and AFLP analysis. African Journal of Biotechnology, 3(1): 25-31. 

Burgess, L.W., Summerrell, B.A., Bullock, S., Gott, K.P. & Backhouse, D. 1994. Laboratory Manual for Fusarium Research. 3 th.ed, University of Sidney, Australia.

Correll. J.C., Klittich, C.J.R. & Leslie, J.F.1987. Nitrate nonutilizing mutants of Fusarium oxysporum and their use in vegetative compatibility tests. Phytopathology, 77: 1640-1646.

Dastjerdi, R., Mozafari, G., Falahatirastgar, M. & Jafarpour, B. 2005. Investigation on genetic diversity of vegetative compatibility groups of F.oxysporum f.sp. betae in sugar beet fields using RAPD marker in Khorasan province.Applied Entomology and Phytopathology, 1(72): 1-17.

Edwards, K., Johnstone, C. & Tompson, C.A. 1991. Simple and rapid method for preparation of plant genomic DNA for PCR analysis. Nucleic Acids Research, 19:1394.

Elias, W. H. & Cotty, P.J. 1994. Rose Bengal amended medium for selecting nitrate-metabolism mutant from fungi. Canadian Journal of Botany, 73: 680-682.

Grand, L.F. & Vernia, C. F. 2004. Fungi on plants in North Carolina. North Carolina State University. Available online from URL: http://www.cals.ncsu.edu. Course/pp 318/fungus.pdf.

Jacobson, D. J. & Gordon, T. R. 1988. Vegetative compatibility and self-incompatibility within Fusarium oxysporum f.sp. melonis. Phytopathology, 78:668-672.

Jelodar, A. 2009. Isolation and identification Fusarium species associated with Corn and investigation of genetic diversity among dominant species population using vegetative compatibility groups in Khuzestan province. M.Sc. Thesis, Shahid Chamran University, Ahvaz, Iran.

Leslie, J.F. & Sumerrell, B.A. 2006. The Fusarium Laboratory Manual. 3 th ed, Blackwell.

Lewinsohn, D., Nevo, E., Wasser, P.S., Hadar, Y. & Behara, V. 2001. Genetic diversity in population of the Pleurotus eryngii complex in Israel. Mycological Research, 105(8): 941-951.

Mahdaviamiri, M., Razavi, M., Sharifi, K. & Zare, R. 2009. Investigation on genetic diversity of Fusarium oxysporum causal agent of potato Fusarium wilt using pathogenecity test and RAPD marker. Iranian Journal of Plant Pathology, 45(1): 9-24.

Martin, M.G., Simon, C.J. & Nuehlbauer, F.J. 1993. Use of random amplified polymorphism DNA (RAPD) to characterize race 2 of Fusarium oxysporum f.sp pisi. Phytopathology, 83: 612-614.

Muthukumar, A. 2007. Occurrence of tuber rot (Fusarium oxysporum) of Tuberose (Polianthes tuberosa L.) in Tamil Nadu. Mycology and Plant Pathology, 37:1.

Muthukumar, A., Karthikeyan, G. & Prabakar, K. 2006. Management of tuber rot (Fusariumoxysporum) of Tuberose (Polinthes tuberosa L.). MadrasAgriculture Journal, 93(1-6):132- 134.

Natan, P. 2002. Evaluation of inoculation Methods to determine disease responses of cultivated and native cultivars to Fusarium oxysporum f.sp. vasinfectum. An Astralian National Botanic Gardens. Available online from URL:  http://www.anbg.gov.au.

Norasmofrad, N., Farokhi Nejad, R. & Alizadae, A. 2005. Genetic diversity in populations of Fusarium oxysporum f. sp. cumini, the causal agent of cumin wilt in Khorasan using vegetative compatibility groups. Plant Diseases, 41(1): 437-453.

Puhala, J.E.1985. Classification of strain of Fusarium oxysporum on the basis of vegetative compatibility. Canadian Journal of Botany, 63: 179-183.

Rahkhodayi, A. & Farokhi Nejad, R. 2007. Determination of vegetative compatibility groups among population of F.oxysporum f.sp. tuberosi and their pathogenicity on potato in Fars and Khuzestan provinces. Scientific Journal of Agriculture, 29(2): 43-53.

Reader, U. & Broda, P. 1985. Rapid preparation of DNA from filamentous fungi. Letter of Applied Microbiology, 1: 17-20.

Seifert, K. 1996. Fusarium Interactive Key. Agriculture & and Agri-Food Canada Available online from URL: http://res.agr:ca/brd/fusarium/home1.html.

Sing, A.K. 2006. Flower Crop: Cultivation and Management. New India Publishing. India.

Woo, S.L., Zonia, A., Sorbo, G., Lorito, M.D., Scalan, B.F. & Noviell, C. 1998. Charecterization of Fusarium oxysporum f.sp. phaseoli by pathogenic race, VCG, RFLP and RAPD. Phytopathology, 89: 966-973.

Xia, Z. & Achar, N. 2001. Random amplification polymorphic DNA and polymerase chain reaction markers for the different ion and detection of Stenocarpella maydis maize seeds. Phytopathology, 149: 35-44.

Zainadin, N.A., Ismail, N.A., Mohamadnor, N.M., Razak, A.A., Nordahliawate, S., Sedique, M. & Salleh, B. 2009. Nitrate nonutilizing mutant and vegetative compatibility groups of Fusarium proliferatum and Fusarium sacchari isolated from Rice in the Peninsular Malaysia and Kolimantan, Indonesia. Journal of Plant Protection, 49(2): 229-23.