ردیابی هم‌زمان دو گونه ویروس رگبرگ زرد نکروتیک چغندرقند و ویروس سوختگی سیاه چغندرقند با استفاده از واکنش زنجیره‌ای پلی‌مراز دوگانه

نوع مقاله : علمی پژوهشی-فارسی

نویسندگان

1 استادیار، بخش تحقیقات چغندرقند، مرکز تحقیقات و آموزش و منابع طبیعی خراسان رضوی، سازمان تحقیقات ،آموزش و ترویج کشاورزی، مشهد، ایران

2 دانشیار، بخش گیاه‌پزشکی،دانشکده کشاورزی دانشگاه فردوسی ، مشهد، ایران

چکیده

تاکنون ویروس‌های رگبرگ زرد نکروتیک چغندرقند (Beet necrotic yellow vein virus, BNYVV) و سوختگی سیاه چغندرقند (Beet black scorch virus, BBSV) به‌طور مجزا در ریشه‌های آلوده از مزارع چغندرقند ایران ردیابی شده‌اند. هدف از این مطالعه ردیابی هم­زمان دو ویروس ذکرشده به‌منظور صرفه‌جویی در زمان و انرژی در فرآیند تشخیص آلودگی بود. بدین منظور از سه روش استفاده شد. در روش اول (روش رایج): ویروس‌ها با استفاده از واکنش زنجیره‌ای پلی‌مراز با ترانویسی معکوس (RT-PCR) به‌وسیله آغازگرهای اختصاصی مربوط به هر کدام و مجزا از یکدیگر روی ریشه آلوده ردیابی شدند. در روش های دوم و سوم، این دو ویروس به ترتیب با استفاده از واکنش زنجیره‌ای پلی‌مراز با ترانویسی معکوس به‌صورت تکی (Simplexrt-PCR) اما هم‌زمان در یک ترموسایکلر و به صورت دوگانه (DuplexRT-PCR) با به­ کارگیری دو جفت آغازگر اختصاصی ردیابی شدند. نتایج در روش های دوم و سوم نشان داد که با بهینه‌سازی دمای اتصال آغازگرهای اختصاصی (56 درجه سانتی‌گراد) مبتنی بر ناحیه ژن کد کننده پروتئین پوششی، به ترتیب قطعات هدف 391 و 453 جفت بازی از ژنوم BNYVV و BBSV تکثیر می‌یابند. در نتیجه این دو روش جهت ردیابی هم‌زمان مناسب بوده و ازنظر صرفه‌جویی در زمان و انرژی مقرون‌به‌صرفه است و همچنین قابلیت استفاده از آن‌ها به‌منظور تهیه نقشه پراکنش در مناطق کشت چغندرقند و نیز ارزیابی و شناسایی لاین­ های مقاوم چغندرقند در برنامه‌های به نژادی در شرایط گلخانه وجود دارد.

کلیدواژه‌ها

عنوان مقاله [English]

Simultaneous detection of Beet necrotic yellow vein virus (BNYVV) and Beet black scorch virus (BBSV) using Duplex Reverse Transcription-PCR method

نویسندگان [English]

  • J. Soltani Idliki 1
  • M. Mehrvar 2
1 Assistant Professor, Sugar Beet Research Department, Khorasan Razavi Agricultural and Natural Resources Research and Education Center, AREEO, Mashhad, Iran
2 Associate Professor, Department of Plant Pathology, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran
چکیده [English]

Background and Objectives
Beet necrotic yellow vein virus (BNYVV) and Beet black scorch virus (BBSV) have been observed in infected sugar beet roots from Iranian fields. This study aimed to detect the two viruses simultaneously to save time and energy during the infection detection process.
Materials and Methods
Three methods were utilized in this study. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect two viruses following total RNA extraction from the farm sample. In the first method (the standard method), the mentioned viruses were identified by amplifying 545 bp fragments from the TGB region of the RNA2 genome of the BNYVV virus and 305 bp fragments from the 3'UTR region of the BBSV virus using specific primers. Viruses on an infected root were identified using RT-PCR with primers specific to each virus and performed independently. In the second and third methods, we employed simplex and duplex RT-PCR gradient methods to simultaneously identify two viruses at annealing temperatures of 52, 54, and 56 °C. To this end, virus-specific primers (designed from a part of the gene encoding the coat protein) were used separately but simultaneously with a common PCR program in a thermocycler. These specific primers amplified 391 and 453 bp fragments of the BNYVV and BBSV coat proteins, respectively.
Results
The study's findings show that by performing simplex and duplexRT-PCR simultaneously and optimizing the annealing temperature of primers from the gene encoding the protein coat, target fragments of 453 and 391 bp from BBSV and BNYVV, respectively, are amplified. This study found that an annealing temperature of 56 °C is the optimal temperature for duplex RT-PCR to separate the two viruses. Consequently, simultaneous identification of the BNYVV and BBSV was achieved through this method.
Discussion
The present study demonstrates that simplex and duplexRT-PCR are suitable for simultaneous detection and are cost-effective in terms of saving time and energy. They can also be used to prepare distribution maps in sugar beet cultivation areas and to evaluate and identify resistant sugar beet lines in breeding programs under greenhouse conditions.

کلیدواژه‌ها [English]

  • Primer
  • Rhizomania
  • Beet black scorch virus
  • sugar beet
  • RT-PCR

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 © 2023 by the authors. Licensee SCU, Ahvaz, Iran. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0 license) (http://creativecommons.org/licenses/by-nc/4.0/.
گیاه پزشکی
دوره 46، شماره 1
اردیبهشت 1402
صفحه 9-23

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