شناسایی فوزاریومهای پوساننده ریشه گل مریم در منطقه دزفول و تعیین تنوع ژنتیکی گونه غالب توسط گروههای سازگار رویشیو نشانگر مولکولی RAPD
محورهای موضوعی : گیاه پزشکیویدا مهین پو 1 , رضا فرخی نژاد 2 , حمید رجبی معماری 3 , زینب بهمنی 4
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کلید واژه: Tuberose, فوزاریوم, پوسیدگی ریشه, Fusarium, root rot, گل مریم, Molecular marker, هتروکاریون, جهش یافتگان نیت, نشانگر مولکولی, Heterokarion, Nit mutant,
چکیده مقاله :
جهت شناسایی گونه ها و جدایه های مختلف فوزاریوم عامل پوسیدگی ریشه گل مریم و بررسی تنوع ژنتیکی آنها در شهرستان دزفول نمونه برداری از گیاهان آلوده مزارع مختلف این شهرستان انجام گرفت. قارچ های وابسته به ریشه با استفاده از روش های 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 گروه های سازگار رویشی و روش RAPD مورد بررسی قرار گرفت. برای بررسی گرو ه های 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 جهش یافتگان نیت نیز روی محیط کشت پایه حاوی یکی از چهار منبع ازت (نیترات، نیتریت، هیپوزانتین و تارتارات آمونیوم) تعیین گردید. ازکل جهش یافتگان نیت تعداد ٣٠١، ١۷١ و ۷۵ عدد به ترتیب به nit1، nit3 و NitM تعلق داشتند. آزمون مکمل سازی بین جهش یافتگان تمام جدایه های 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 جدا یه ها با استفاده از سیزده آغازگر تصادفی بررسی شد. تجزیه خوشه ای داده های RAPD با استفاده از روش های UPGMA، Single و Complete انجام گرفت. بهترین نتیجه با استفاده از روش UPGMA وضریب تشابه Dice در سطح تشابه 61% به دست 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 جدایه های انتخابی از گروه های مختلف VCG انجام گرفت نشان داد که تمام جدایه ها بیماریزا بودند. این مطالعه اولین گزارش از بررسی تنوع ژنتیکی F.oxysporum روی گل مریم در ایران می باشد.
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.
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