شناسایی و ردیابی مولکولی Phytophthora melonis بر‌اساس ژنوم هسته‌ای و میتوکندریایی*

نوع مقاله: مقاله کامل پژوهشی

نویسندگان

1 دانش‌آموخته کارشناسی ارشد

2 استاد بیماری شناسی گیاهی و دانشیار اصلاح نباتات، دانشکده کشاورزی دانشگاه شیراز

3 دانش آموخته کارشناسی ارشد دانشکده کشاورزی دانشگاه شیراز

چکیده

گونه­ی بیمارگر گیاهی Phytophthora melonis از نظر ریخت­شناختی به برخی گونه­های بدون پستانک جنس Phytophthora به­خصوص P. drechsleri شباهت دارد وبنابراین تفکیک این آرایه­های هم­گرا دشوار است. این پژوهش به منظور طراحی آغازگرهای اختصاصی P. melonis بر اساس ژنوم هسته­ای و میتوکندریایی، بررسی اختصاصیت آن­ها در برابر سایر گونه­های همگرا، و بهینه­سازی استفاده از این آغازگرها برای ردیابی P. melonis انجام شد. برای طراحی آغازگر­های اختصاصی گونه­ی P. melonis نُه ژن­ هسته­ای و چهار ژن میتوکندریایی از نظر تفاوت نوکلئوتیدی مناسب بودند، بنابراین سیزده آغازگر اختصاصی طراحی و خصوصیات آن­ها بهینه‌سازی گردید. ردیابی P. melonis با استفاده از پنج جفت از آغازگر­های اختصاصی در بافت مایه­زنی شده­ی گیاهان میزبان آن شامل خیار، خربزه، هندوانه، چغندر­قند و پسته­ انجام شد. با استفاده از آغازگرهای طراحی شده در واکنش زنجیره‌ای پلیمراز تودرتو، تا سطح یک درصد مایه­ی بیماری­زا­ در خاک آلوده و زئوسپورهای بیمارگر تا غلظت­10 زئوسپور در میلی­لیتر در آب آلوده ردیابی شدند. با بررسی اختصاصیت و حساسیت آغازگر­های طراحی شده، کارامد­ترین آن­­­ها مجموعه­ی ITS-M2 (ترکیب آغازگرهای ITS-MF1 و ITS-MR2) در نظر گرفته شد. دمای هم­جوشی بهینه‌سازی شده برای این مجموعه 68 درجه­ی سلسیوس بود. به نظر می‌رسد استفاده از واکنش زنجیره­ای پلیمراز تودرتو با استفاده از مجموعه­ی ITS-M2 به همراه آغازگر­های عمومی ITS4 و ITS6 در نقش آغازگر­های خارجی در حدود 106 برابر حساس­تر از واکنش زنجیره­ای پلیمراز مستقیم است. ­آزمون زنجیره­ای پلیمراز چندگانه‌ی طراحی شده، قادر به ردیابی هم­زمان سه گونه­ی بیمارگر شامل P. melonis، P. drechsleri و P. nicotianae بود.آزمایش‌ها نشان داد که آغازگر­های طراحی شده ابزاری کارآمدی برای ردیابی P. melonis در بافت گیاه، خاک و آب آلوده هستند.

کلیدواژه‌ها


عنوان مقاله [English]

Molecular Identification and Detection of Phytophthora melonis Based on Nuclear and Cytoplasmic Genome

نویسندگان [English]

  • S. M. Zolanvari 1
  • R. Mostowfizadeh-Ghalamfarsa 2
  • A. Dadkhodaie 3
چکیده [English]

The plant pathogen Phytophthora melonis is morphologically similar to some other non-papillate Phytophthora spp. especially P. drechsleri and therefore it is difficult to discriminate these convergent taxa. This study was performed to design specific primers based on nuclear and cytoplasmic genome, examine their specificity against other convergent species, and optimize the specific primers’ conditions to detect P. melonis. Nine nuclear and four cytoplasmic genes were appropriate to design thirteen specific primers based on their nucleotide polymorphism. PCR conditions were optimized. Phytophthora melonis were detected by specific primers in inoculated plants such as cucumber, watermelon, melon, sugar beet and pistachio. All specific primers detected pathogen in 1:100 (pathogen: soil) inoculated soil.Up to 10 zoospores per milliliter were detected using nested polymerase chain reaction. ITS-MF1 and ITS-MR2 (ITS-M2 set, from internal transcribed spacers of rRNA gene) were selected as the most efficient primers based on their specificity and sensitivity. The optimized annealing temperature for this primer set was 68 °C. It seemed that nested PCR by ITS-M2 primer set together withthe universal primers ITS6 and ITS4 as external primers is at least 106 times more sensitive than simple PCR. Multiplex polymerase chain reaction simultaneously detected the three cucurbits’ pathogens including P. melonis, P. nicotianae and P. drechsleri. This study showed that the designed primers could be effective tools for detection of P. melonis isolates from infected tissues, and infested water and soil.

کلیدواژه‌ها [English]

  • Detection
  • identification
  • Oomycota
  • Root rot
  • specific primers
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