واکاوی تبارزائی سه جدایه ویروس همراه با پیچیدگی برگ مو شماره یک (glrav-1) از تاکستان‌های استان خراسان رضوی براساس توالی نوکلئوتیدی بخشی از ژن cpm2

انگور (vitis vinifera l.) به‌عنوان یک محصول کشاورزی مهم در جهان، میزبان طیف وسیعی از ویروس‌های گیاهی از جمله 86 گونه ویروس شناخته شده است. یکی از این ویروس‌ها، ویروس همراه با پیچیدگی برگ مو شماره یک (grapevine leafroll-associated virus 1, glrav-1) است که به جنس ampelovirus و خانواده closteroviridae تعلق دارد. با توجه به تغییرات اخیر در نام‌گذاری گونه‌های ویروسی، نام این گونه ampelovirus univitis شده است. در این مطالعه در بهار سال 1399، آلودگی به glrav-1 در تاکستان‌های شهرستان کاشمر، استان خراسان رضوی مورد بررسی قرار گرفت. علائم مشاهده‌شده در تاک‌های آلوده شامل زردی خفیف، تاخوردگی حاشیه برگ به پشت و قرمزی پهنک همراه با رگبرگ‌های سبز بود. 70 نمونه از تاک‌های مشکوک به آلودگی جمع‌آوری و مورد آزمایش قرار گرفتند. نتایج نشان داد که 22 نمونه (31.4 درصد از نمونه‌ها) به ویروس glrav-1 آلوده بودند. توالی‌یابی ژن پروتئین پوششی فرعی 2 (orf cpm2) ویروس از سه نمونه انجام شد. توالی‌های به‌دست آمده با توالی‌های موجود در genbank مقایسه شدند و نتایج نشان داد که توالی نوکلئوتیدی جدایه‌های این مطالعه، شباهت 87.7 تا 77.6 درصدی با توالی‌های glrav-1 موجود در genbank داشتند. درخت تبارزایی براساس بخشی از توالی نوکلئوتیدی ژن cpm2 بازسازی شد. جدایه‌های شمال شرق ایران با جدایه‌های سایر کشورها در یک گروه قرار گرفتند که نشان‌دهنده عدم ارتباط خوشه‌بندی تبارزایی با منشا جغرافیایی می‌باشد. این یافته نشان می‌دهد که ویروس glrav-1 می‌تواند به‌راحتی در بین مناطق مختلف جغرافیایی جابه‌جا شود و تهدیدی جدی برای تاکستان‌ها در سراسر جهان محسوب می‌شود.

phylogenetic analysis of three isolates of grapevine leafroll-associated virus 1 (glrav-1) from vineyards in khorasan razavi province based on the nucleotide sequence of a portion of the minor capsid protein 2 (cpm2)

introductiongrapevines, as a vital agricultural product, face significant challenges from pests and diseases that adversely affect both the quality and quantity of the crop. among these, viruses are particularly detrimental, with 86 different species identified in grapevines. the viruses associated with grapevine leafroll disease (gld) are considered the most prevalent in vineyards. specifically, six species of grapevine leafroll-associated viruses (glravs), belonging to the family closteroviridae, are linked to this disease. these viruses typically enter vineyards through infected cuttings and are disseminated by vectors, notably mealybugs. the symptoms of gld manifest as downward rolling of leaf margins, leaf interveinal reddening, and leaf interveinal chlorosis across various grapevine varieties. these symptoms negatively impact the quality of the berries, ultimately affecting yield and marketability. the glrav-1 genome is composed of positive-strand rna and contains ten open reading frames (orfs), which play crucial roles in its biology and pathogenicity. in iran, glrav-1 has been identified as one of the most widespread grapevine viruses, exhibiting high genetic diversity that complicates management and control efforts. this study focuses on the identification of glrav-1 and the phylogenetic analysis of its isolates in the vineyards of razavi khorasan province. by understanding the genetic relationships and variability of glrav-1 isolates, this research aims to provide insights which could be useful for designing effective management strategies and improving the overall health of grapevine in the region. materials and methodsin the spring of 2020, a study was conducted in the kashmar region of khorasan razavi province, focusing on grapevine plants exhibiting distinct symptoms of viral infection. a total of 70 samples were collected from affected plants. the rna was extracted from these samples to facilitate the detection of grapevine leafroll-associated virus 1 (glrav-1). the initial identification process involved reverse transcription-polymerase chain reaction (rt-pcr). this method confirmed the presence of glrav-1 in 22 samples, marking a crucial step in understanding the viral infection’s prevalence. to ensure the accuracy of the rt-pcr results, sanger sequencing was used on the amplified fragments from three selected samples. this technique provided definitive confirmation of the viral identity by analyzing the nucleotide sequences, thereby validating the initial findings from the rt-pcr. data analysis included several critical components. nucleotide sequence similarity analysis was performed to compare the identified glrav-1 isolates against known sequences in databases, assessing their similarity and identifying potential genetic variants. following this, multiple sequence alignments were conducted using mafft software, arranging the sequences to highlight similarities and differences among the isolates. the phylogenetic tree was constructed using the maximum-likelihood method with mega7 software. results and discussionthe samples collected from black grape vineyards in kashmar exhibited mild yellowing and downward rolling of leaf margins, symptoms commonly associated with increased anthocyanin levels in red grape varieties. in samples infected with glrav-1, a fragment of the minor capsid protein 2 (cpm2) gene was successfully amplified, while no such fragments were detected in healthy plants. this indicates the presence of glrav-1, a virus recognized as one of the common pathogens affecting grapes across various regions of iran.