بررسي تنوع ژنتيكي و پراكنش ويروس لكه سياه برگ چغندرقند (Beet black scorch virus) در برخي استان هاي ايران

Evaluation of Genetic Diversity and Distribution of Beet Black Scorch Virus (BBSV) in Some Provinces of Iran

ويروس لكه سياه برگ چغندر قند (BBSV) يكي از ويروس هاي خاكزاد جديد گزارش شده از مزارع چغندرقند در دنيا است كه به لحاظ داشتن علائم مشابه (ريشه ريشي) با ويروس رايزومانيا Beet necrotic yellow veinvirus و احتمالا برخي برهمكنش هاي متقابل با اين ويروس اهميت اقتصادي بالايي دارد. به منظور بررسي پراكنش BBSV در مزارع چغندرقند ايران در سال هاي زراعي ۱۳۹۳-۱۳۹۰ در مجموع ۳۰۹ مورد نمونه ريشه چغندرقند جمع آوري گرديد. بررسي نمونه ها با استفاده از آزمون RT-PCR نشان داد كه ۱۴۷ مورد از ۳۰۹ كل نمونه ها (۴۸ درصد) به اين ويروس آلوده بودند. همچنين اين ويروس داراي يك قطعه ماهوارهاي است كه در ۳۰ مورد (۱۰ درصد) از نمونه ها رديابي گرديد. بر اين اساس وجود ويروس BBSV در مزارع چغندر قند استان هاي خراسان رضوي، شمالي، جنوبي، لرستان، اردبيل، آذربايجان شرقي، آذربايجان غربي، قزوين، همدان، كرمان، كرمانشاه و زنجان تائيد گرديد. براساس اهميت پراكنش جغرافيائي مناطق چغندركاري و علائم ايجاد شده در گلخانه، ۵ جدايه شامل آذربايجان غربي، همدان، خراسان رضوي (مشهد)، خراسان شمالي (شيروان) و كرمانشاه براي بررسي تنوع ژنتيكي انتخاب گرديدند. دو ناحيه مهم شامل پروتئين پوششي و ۳''UTR اين پنج جدايه با يك جدايه چيني و يك جدايه آمريكائي مورد مقايسه قرار گرفت. در مجموع تحليل توالي اين دو ناحيه نشان داد كه جدايه چيني و آمريكائي به يكديگر شباهت بيشتري دارند تا با جدايه هاي ايراني. شباهت جدايه هاي ايراني با جدايه هاي چيني و آمريكائي در هر دو ناحيه حدود ۸۸ درصد بود. در بين جدايه هاي ايراني ميزان شباهت بالا و بين ۹۸ تا ۹۹ درصد بود و تنها جدايه خراسان شمالي (شيروان) با شباهت كمتر در دسته اي جدا قرار گرفت. به نظر مي رسد تفاوت ها در ناحيه ۳''UTR خيلي موثر نبوده و در تمام جدايه ها ژنوم ويروس به خوبي بيان و تكثير مي گردد. اما در ناحيه پروتئين پوششي تغييرات آمينو اسيدها بخصوص در ناحيه N-terminal در ايجاد علائم و بر همكنش با ساير ويروس هاي خاكزاد تاثير بسزائي داشته است.

ntroduction: Beet black scorch virus (BBSV) is a new soil-borne virus reported from sugar beet fields around the world that in terms of having similar symptoms (root beard) with Beet necrotic yellow vein virus and Probably some mutual interaction with that has high economic importance. BBSV is a sugar beet virus which was first reported in the late 1980s in China followed by identification in USA, Iran, and Europe. In China, the virus causes severe disease symptoms described as black scorched and inwardly-curled leaves of sugar beet (Beta vulgaris L.), and necrotic roots caused by sugar content reduction. The BBSV isolates reported from other parts of the world did not show any black scorching symptoms, but they showed proliferation of rootlets. Symptomless infections have been observed on sugar beet too. BBSV is belonging to the genus Betanecrovirus (Family Tombusviridae ). The virus is transmitted efficiently in the soil by zoospores of Olpidium brassicae. The virus comprises an icosahedral particle of 28 nm that encapsidates a positive-sense, single-stranded (ss) genomic (g) RNA of 3644 nt lacking a 5'-cap structure and 3'-poly(A) tail that encodes six functional protein. Because of lacking 3'-poly(A) tail in BBSV 3'UTR region act as a Barley yellow dwarf virus (BYDV) capindependent translation element probably acting as the promoter for the initiation of the negative strand synthesis. BBSV complete genome shares the highest sequence identity (61%) with Tobacco necrosis virus D (TNV-0). The 5' proximal p23 and p82 ORFs encode for produce replicas complex, as the P82 protein contains a GOD motif that is conserved in RNA-dependent RNA polymerases. The small P5', P7a and P7b central proteins that translated from sgRNA I arc required for the viral cell to cell movement. The 3'-proximal ORF encodes the 24 kDa viral coat protein (CP). BBSV CP is a multifunctional protein that is important in disassembly of parental virus and assembly of progeny virus. It also has a role in during infection cycle and virus-host interaction. Materials and Methods: For investigation of BBSV distribution in Iran's sugar beet farms a total of 309 samples were collected. These samples were transmitted to the lab and dried very well after washing carefully. Evaluation of test samples was done by RT-PCR using primers in 3'UTR and virus satellite genome. Five isolates for assessment of genetic diversity were selected based on geographical distribution and symptoms in greenhouse including West Azarbayjan, Hamedan, Khorasan Razavi (Mashhad), North Khorasan (Shirvan) and Kermanshah. These genome parts were propagated by Long PCR Master Mix of Promega. Gel band if isolates were extracted, purified and cloned to E.coli DH5a. Proper clones were propagated and sequenced in Sigma, Germany. Results were analyzed by BLASTn in Gene Bank and Mega6 software. Results and Discussion: BBSV were detected in 147 samples in total (48%). Also, BBSV has a satellite that in 30 cases (10%) of the samples was detected. According to this BBSV were detected in sugar beet farms of Khorasan Razavi, North Khorasan, South Khorasan, Lorestan, Ardabil, East Azarbayjan, West A?.arbayjan, Ghazvin, Hamedan, Kerman, Kermanshah and Zanjan Provinces. Two important parts including Coat protein and 3'UTR of these isolates were compared with Chinese -N and US -Co isolates. Sequence analysis indicated that US and Chinese isolates are more resemble together than to Iranians. The rresemblance of Iranian isolates to Chinese and US isolates were about 88% in both parts. Iranian isolates resembling is 98-99%. Only North Khorasan (Shirvan) isolate with less identical batch was separated. It seems that difTerences in 3'UTR are not efTective and in all isolates virus genome well-expressed and translated. But amino acid changes in CP especially N-terminal area are efl'ective in symptoms inducing and interaction with other soil-borne Viruses. Conclusion: BBSV is one of the new and so virulence sugar beet soil born viruses that distributed in almost all sugar beet important farms of Iran. Its symptoms so resemble to BNYVV the causal virus of Rhizomania disease. In this paper, we indicate that bearded root of sugar beet did not induce only by BNYVV but also by BBSV. This vast inlection area maybe is due to sugar beet cultivation for a long time in these areas that allow virus vector to be established in the soil and transmit it efficiently. In addition. traditional agricultural practices and cultivation of susceptible sugar beet varieties induced opportunity to BBSV population potentially increased and spread in these areas. Comparing of nucleotide sequences in 3'UTR and Coat proteins of BBSV Iranian isolates with US and Chinese isolates indicate that Chinese and the US isolates are more resemble together than Iranian isolates. Results of this study demonstrated that Iranian BBSV isolates are more diverse than other isolates available in GenBank. Almost BBSV isolates phylogenetic analysis are compatible with the geographical distribution of this viruses. Amino acid sequences in CP of different isolates show some important ditfcrences especially at N-terminal area of CP. May be next researches on these differences can explain the differences in symptoms and virus-host interactions among these seven isolates.