ردیابی و شناسایی Candidatus phytoplasma trifolii از درختان سیب در ایران

نوع مقاله : مقاله پژوهشی

نویسندگان

1 گروه گیاهپزشکی، دانشکده کشاورزی، دانشگاه زابل

2 2بخش تحقیقات گیاه پزشکی، مرکز تحقیقات و آموزش کشاورزی و منابع طبیعی استان خراسان رضوی، سازمان تحقیقات، آموزش و ترویج کشاورزی، مشهد، ایران.

3 گروه گیاهپزشکی، دانشکده کشاورزی، دانشگاه زابل.

4 گروه گیاهپزشکی، داﻧﺸﮕﺎه ﻋﻠﻮم ﻛﺸﺎورزی و ﻣﻨﺎﺑﻊ ﻃﺒﻴﻌﻲ ﮔﺮﮔﺎن.

5 کارشناس بخش مدیریت آب و خاک، سازمان جهاد کشاورزی استان خراسان رضوی- مشهد

چکیده

چکیده
در بازدیدهای بهار و تابستان سال 1397 از باغات درختان سیب در استان خراسان رضوی، علائم مشکوک به بیماری فیتوپلاسمایی مانند زردی، ریزبرگی، و همچنین برگ­ هایی که گوشوارک در آن به طور غیرعادی کشیده شده بود در سطح باغات مشاهده شد و 33 نمونه از برگ درختان جمع­ آوری گردید. با استفاده از آغازگرهای عمومی P1 / P7 و آغازگرهای آشیان ه­ای R16F2n / R16R2، fU5 / rU3 و R16MF2 / R16MR2 در واکنش زنجیره­ای پلیمراز (PCR) از کلیه نمونه­های دارای علائم، قطعات 1800 جفت باز در PCR عمومی و در PCRهای آشیان ه­ای به ترتیب قطعات 1250، 876 و 1400 جفت باز تکثیر شدند. در نتایج حاصله مشخص گردید 18 نمونه آلوده به فیتوپلاسما مثبت بودند. محصول PCR دومرحله­ای با جفت آغازگرهای R16F2n / R16R2، همسانه­سازی و تعیین ترادف شده و در بانک جهانی ژن ثبت گردید. مقایسه توالی­ های به دست آمده با توالی ­های موجود در NCBI توسط نرم افزار BLAST و همچنین بررسی چند شکلی طولی قطعات برشی (RFLP) حقیقی با محصول PCR دومرحله­ ای با استفاده از آنزیم ­های AluI،  RsaIو Tru1I (MseI)، نشان داد که فیتوپلاسمای همراه درختان سیب بیشترین شباهت را با Candidatus Phytoplasma trifolii مربوط به گروه افژولش شبدر (Clover proliferation group, 16SrVI) دارند. آنالیز توالی­ ها با استفاده از نرم افزار iPhyClassifier، تعیین میزان تشابه نوکلئوتیدی و آنالیز تبارزایی با استفاده از ترادف کامل ژن RNA ریبوزومی 16S نیز تعلق فیتوپلاسمای همراه درختان سیب در استان خراسان رضوی را در گروه 16SrVI-A تایید کرد. این اولین گزارش از سیب­ های آلوده به Ca. Phytoplasma trifolii در ایران و سایر نقاط جهان است.
 

کلیدواژه‌ها

عنوان مقاله [English]

Detection and identification of Candidatus phytoplasma trifolii on apple trees in Iran

نویسندگان [English]

  • Reza Barazandeh Aghkariz 1
  • Naser Radman 1
  • Mahmoud Reza Karimi Shahri 2
  • Mahdi Pirnia 3
  • Abdolhossein Taheri 4
  • Seyed Mahdi Fatemi 5
1 Department of Plant Protection, Faculty of Agricultural, University of Zabol.
2 Plant Protection Research Department, Khorasan Razavi Agricultural and Natural Resources Research and Education Center, Mashhad, Iran.
3 Department of Plant Protection, Faculty of Agricultural, University of Zabol.
4 Department of Plant Pathology, Agricultural and natural science University Gorgan.
5 Working in Water and Soil Expert Department of Agricultural Jehad Organization of Khorasan Razavi Province, Mashhad. Iran.
چکیده [English]

Abstract
During spring and summer of 2018, suspected symptoms of the phytoplasma disease such as leaf shrinkage and yellowing as well as leaves with abnormally-enlarged stipules were observed in apple orchards in Khorasan Razavi province and 33 leaf samples were collected. Samples of symptomatic apple tree were assayed for infection with phytoplasma, using universal primer pairs P1 / P7 and nested PCR using primer pair R16F2n / R16R2, fU5 / rU3 and R16MF2 / R16MR2 in Polymerase Chain Reaction (PCR). The universal PCR products were 1800 bp fragments and the nested-PCR products were 1250, 876 and 1400 bp fragments, respectively, which were amplified by primer pairs. Of 33 leaf samples from apple, 18 samples were tested positive for phytoplasmas. The R16MF2/R16MR2 primed nested PCR products were cloned, sequenced and submitted into GenBank. Comparison of the obtained sequences with the sequences of NCBI using BLAST software and Restriction Fragment Length Polymorphism (RFLP) analysis of the nested PCR products using the enzymes AluI, RsaI and TruI (MseI) indicated that phytoplasma in apple trees in Razavi Khorasan province was most similar to Candidatus Phytoplasma trifolii related to Clover Proliferation (CP) 16SrVI group. Analysis of sequences using iPhyClassifier software, determination of nucleotide similarity, and phylogenetic analysis of full-length 16S rDNA showed that phytoplasma in apple tress of Razavi Khorasan province belongs to 16SrVI-A subgroup. This is the first report on Ca. Phytoplasma trifolii infections in apple trees in Iran and other regions across the world.
 

کلیدواژه‌ها [English]

  • Keywords: Phylogenetic analysis
  • Restriction enzyme
  • RFLP
  • Yellowing
  • 16SrVI group

مراجع

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پژوهش های کاربردی در گیاهپزشکی
دوره 10، شماره 3
مهر 1400
صفحه 41-50

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  • تاریخ دریافت: 05 مرداد 1400
  • تاریخ پذیرش: 05 مرداد 1400

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