ردیابی ویروس سوختگی سیاه برگ چغندر (Beet black scorch virus) و سایر ویروس‌های خاک‌زاد همراه و تعیین برخی خصوصیات مولکولی آن‌ها

نوع مقاله : مقالات پژوهشی

نویسندگان

1 فردوسی مشهد

2 دانشگاه فردوسی مشهد

3 موسسه تحقیقات اصلاح و تهیه بذر چغندرقند

4 عضو هیئت علمی مرکز تحقیقات کشاورزی و منابع طبیعی

چکیده

تاکنون در ایران ویروس‌های خاک زاد متعددی از جمله ویروس رگبرگ زرد نکروتیک چغندرقند (Beet necrotic yellow vein virus; BNYVV)، ویروس سوختگی سیاه برگ چغندر (Beet black scorch virus; BBSV)، ویروس خاک زاد چغندر (Beet soil-borne virus; BSBV) و ویروس Q چغندر (Beet virus Q; BVQ) روی ریشه چغندرقند شناسایی‌شده است. ویروس رگبرگ زرد نکروتیک چغندرقند، ویروس اصلی عامل ایجاد سندروم ریشه ریشی (ریزومانیا) است. با اینکه در بررسی‌های انجام‌شده در بعضی از نمونه­های دارای علائم ریشه ریشی چغندرقند، هر سه ویروس مذکور و یا تعدادی از آن‌ها همراه BNYVV یافت شده، اما تاکنون علائم سوختگی روی برگ چغندرقند در هیچ­یک از مزارع کشت چغندرقند گزارش نشده است. اخیراً علائم سوختگی روی برگ­ها و علائمی مشابه ویروس عامل بیماری ریزومانیا روی ریشه برخی از ارقام چغندرقند در مزارع چغندرقند استان خراسان رضوی مشاهده‌شده است. به‌منظور بررسی این موضوع، چغندرقندهای دارای علائم ریشه­ریشی و یا سوختگی برگ از مزارع مناطق مختلف استان شامل: مشهد، فریمان، چناران، جلگه رخ و جوین جمع‌آوری و پس از استخراج ریبونوکلئیک‌اسید (RNA) کل آن‌ها، دئوکسی ریبونوکلئیک اسید مکمل (cDNA) سنتز شد. با استفاده از واکنش پلیمراز با ترانویسی معکوس در همه نمونه‌ها BBSV ردیابی گردید، اما از سه ویروس خاک‌زاد دیگر تنها BNYVV در نمونه‌های مشهد، جلگه رخ و جوین ردیابی شد. از بین سه تیپ مختلف BNYVV شناسایی‌شده در دنیا، جدایه های مورد مطالعه از نوع تیپ A تشخیص داده شد. نوع تتراد ویروس ریزومانیا در جدایه­های مشهد و جلگه رخ از نوع ACHG بوده اما در جدایه جوین، AHHG بود که در اسیدآمینه شماره 68 آن از سیستئین (C) به هیستیدین (H) در پروتئین p25 تغییر یافته بود که می‌توان جدایه را به‌عنوان یک جدایه شکننده مقاومت این ویروس محسوب نمود. درنتیجه کشت متوالی ارقام مقاوم چغندرقند دارای ژن مقاومت Rz1 با تغییرات در اسیدآمینه‌های ناحیه تتراد پروتئین p25 ویروس، جدایه های شکننده مقاومت جدیدی از ویروس ایجاد می‌کند که کشت چغندرقند را در این مزارع در سال‌های آتی با مشکل جدی مواجه خواهد نمود؛ بنابراین تحقیقات بیشتر در مورد بیماری­زایی BNYVV و کنترل آن از طریق استفاده از منابع مقاومت جدید و روش‌های کنترل تلفیقی ضروری به نظر می­رسد. از طرف دیگر اینکه، BBSV در حضور BNYVV موجب تشدید ریشه­ریشی، نکروز آوندی و سوختگی برگ می­گردد حال‌آنکه به‌تنهایی موجب نکروز آوندی در ریشه و سوختگی برگ می‌شود. این مطلب می‌تواند در تشخیص بیماری بر اساس علائم و جلوگیری از خطا در ارزیابی مقاومت ارقام چغندرقند در مزرعه بسیار حائز اهمیت باشد.

کلیدواژه‌ها

عنوان مقاله [English]

Detection of Beet black scorch virus and other Associated Soil-borne Viruses and Determining some of their Molecular Aspects

نویسندگان [English]

  • J. Soltani Idliki 1
  • M. Mehrvar 2
  • M. Zakiaghl 2
  • S.B. Mahmoudi 3
  • Mansour salati 4
1 Ferdowsi University of Mashhad
2 Ferdowsi University of Mashhad
3 Agricultural Research, Education and Extension Organization )AREEO(, Karadj, Iran
چکیده [English]

Introduction: Soil-borne sugar beet viruses are responsible for the destructive diseases of sugar beet. They can cause significant yield losses worldwide. Four of the soil-borne sugar beet viruses consisted of Beet necrotic yellow vein virus (BNYVV), Beet soil-borne mosaic virus (BSBMV), Beet soil-borne virus (BSBV) and Beet virus Q (BVQ) are transmitted by the protis Polymyxa betae, a common root infecting parasite which ensures the long-term persistence of the viruses in the soils and one consisted of Beet black scorch virus (BBSV), is transmitting by Olpidium brassicae. So far, four out of five aforementioned viruses have been reported (except for BSBMV) in Iran. BNYVV is the causal agent of the rhizomania disease which has been reported in mixed-infections with BSBV, BVQ and/or BBSV. In previous studies, BSBV, BVQ and BBSV have been found alongside the BNYVV in some root samples. Nevertheless, up to now, no field has been spotted with black scorch symptoms of sugar beet leaves. Recently, we observed symptoms of burns on leaves and similar signs of rhizomania disease in the roots of some sugar beet cultivars in the fields of Khorasan Razavi province. The purpose of this study was to detect soil-borne viruses of sugar beet and determine some of their molecular aspects.
Materials and Methods: In this study, sugar beet with bearded roots and black scorching of leaves symptoms were collected from Mashhad, Fariman, Chenaran, Jolge-Rokh, and Jovein sugar beet fields. Total RNA was extracted from 100 mg of rootlets using RNeasy Mini Kit (Qiagen-Germany) based on the manufacturer's protocol. The presence of three viruses of BSBV, BVQ and BNYVV were analyzed by multiplex reverse transcription-polymerase chain reaction (mRT-PCR). However, For BBSV detection, simplex RT-PCR was used to detect of the 3′-UTR of the genomic RNA. For the molecular characterization of the BNYVV isolates, the BNYVV type (A or B) was determined with duplex RT-PCR (dRT-PCR), using A/B-type-specific primers pairs for the triple gene block (TGB) gene in RNA-2 and the partial p25 gene of the RNA-3 segment of the virus. RT-PCR was done using the PrimeScript™ Reverse Transcriptase kits and the PrimeSTAR GXL DNA Polymerase (Takara, Japan). The PCR products were cloned in pGEM®-T Easy Vector (Promega-USA). The recombinant plasmids were extracted using PrimPrep Plasmid DNA isolation kit (GenetBio-Korea) and then sequenced (Macrogene, South Korea). Nucleotide sequences data were analyzed using Chromas (version 1.45) and MEGA7 softwares.
Results and Discussion: The results showed that in the three samples with bearded root symptoms (Mashhad, Jovein, and Jolge-rokh), only BNYVV (A-type) was present and there were no BSBV and BVQ in the tested samples. In addition, in the two samples (Fariman and Chenaran), none of the three viruses was detected. The results showed that the two BNYVV isolates had ‘ACHG’ (Mashhad and Jolge-Rokh isolates) or ‘AHHG’ (Jovein isolate) residues in the tetrad position. So that the amino acid cysteine (C) in a68 position was converted to histidine (H). Although this A-type tetrad has been previously reported by Mehrvar et al. (2009) in Khorasan Razavi and Northern Khorasan, Semnan, Qazvin, Zanjan, Ilam, Hamedan, and West Azarbaijan provinces. In this study, BBSV was detected in all samples. In Mashhad, Jolge-rokh, and Jovein samples, BNYVV was present accompanied by BBSV. However, BBSV was detected alone from Fariman (holding black scorching of the leaves and vascular necrosis of root symptoms) and Chenaran (black scorching of the leaves) samples. These results are consistent with the results of other researchers from Spain and the United States who reported the presence of rhizomnia symptoms in the BBSV infected roots.
Conclusion: While most of  the farmers in Khorasan province cultivate resistant cultivars of sugar beet carrying the Rz1 gene for successive years in a field, the breakdown of the resistance and emerging of new resistance breaking (RB) variant of the virus have occurred via amino acid changes. However, more research on BNYVV pathogenicity by the use of additional sources of resistance and alternative disease control majors is needed to have a suitable conclusion. In addition, the results of this study showed that the presence of both viruses (BBSV and BNYVV) together could exacerbate the Rhizomania syndrome symptoms while single infection by BBSV could just cause vascular necrosis in the root and black scorching symptoms of the leaves. This could be very important in symptoms based diagnosing of the disease and preventing errors in evaluating the resistance of sugar beet cultivars in the field.

کلیدواژه‌ها [English]

  • Beet necrotic yellow vein virus
  • Beet black scorch virus
  • Sugar beet

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