شناسایی و بررسی خصوصیات مولکولی جدایه‌های ویروس کوتولگی زرد پیاز (onion yellow dwarf virus) در برخی از مناطق کشت سیر در ایران

ویروس کوتولگی زرد پیاز (onion yellow dwarf virus, oydv)، یک پوتی ویروس مهم و خسارت‌زا در سیر بوده که باعث ظهور رگه‌های کلروتیک خفیف تا زرد روشن در برگ‌های آلوده می‌شود. به دنبال گسترش علائم، کاهش رشد و اندازه قپه‌های سیر نیز رخ می‌دهد، اما به طور کلی اصلی‌ترین علامت آلودگی oydv هم در سیر و هم در پیاز وقوع کوتولگی در میزبان آلوده است. به منظور شناسایی و تعیین برخی از خصوصیات مولکولی و بیولوژیکی oydv در کشور، در سال‌های 1397-1396 تعداد 68 نمونه برگی مشکوک به آلودگی از مزارع سیر در استان‌های خراسان رضوی، مازندران، لرستان، کرمان و خوزستان جمع آوری شد. در واکنش زنجیره‌ای پلیمراز با انجام نسخه‌برداری معکوس (reverse transcriptionpcr, rtpcr) با استفاده از یک جفت آغازگر دژنره مربوط به ژن ci، قطعه 700 جفت بازی در 54 نمونه تکثیر شد. جهت انجام آنالیز ژنومی و تعیین روابط فیلوژنتیکی در جدایه‌های ایرانی، تعداد 7 نمونه برگی دارای علائم مشخص بیماری به عنوان نماینده از هریک از مناطق نمونه‌برداری انتخاب شدند. این جدایه‌ها پس از همسانه سازی در ناقل ptg19t، به شرکت ماکروژن کره جنوبی جهت توالی یابی ارسال شدند. مقایسه نتایج توالی‌یابی جدایه‌های ایرانی با سایر جدایه های موجود در بانک ژن، نشان دهنده درصد تشابه نوکلئوتیدی جدایه‌های ایرانی oydv با یکدیگر و با سایر جدایه‌های موجود در بانک ژن به ترتیب (94.44-79.89) و (75.04-99.86) بود. همچنین دندروگرام حاصل از مطالعه تبارزایی بر اساس ژن ci، جدایه‌های ایرانی را در دو گروه مجزا از هم نشان داد. بررسی وقوع نوترکیبی در میان جدایه‌های ایرانی نشان دهنده عدم وجود هر گونه نوترکیبی در این ناحیه از ژن ci بود. لازم به ذکر است این پژوهش اولین گزارش از وجود oydv در برخی از مناطق عمده کشت سیر در ایران، بر اساس ژن ci ویروسی می‌باشد.

Identification and Molecular Characterization of Onion yellow dwarf virus Isolates in some Garlic Growing Areas of Iran

Introduction: Garlic (Allium sativum L.) is one of the medicinal and economically important crop plants belongings to the Amaryllidaceae and is well known for its worldwide applications. It is widely used as a vegetable. In Iran despite the agricultural potential, we are still facing a garlic shortage in the market, mainly due to the high prevalence of plant diseases particularly viral infections. Most garlic plants are infected by several viruses belonging to different taxonomic groups known as “garlic viral complex”. However, Onion yellow dwarf virus (OYDV) acts as a major element of this complex. This virus is an important and damaging potyvirus in garlic which causes mild chlorotic to bright yellow stripes on infected leaves. OYDV survives in bulbs and sets and therefore can be transmitted during vegetative reproduction. Also, the green peach aphid, Myzus persicae, as well as other aphids, spreads the virus from plant to plant in a nonpersistent manner.  OYDV, RTPCR technique is an effective analytical tool for this purpose. In Iran, for the first time, Shahraeen et al. (2008) were reported OYDV by the serological method in garlic. Afterward, Baghalian et al. (2010), studied the molecular structure of Iranian OYDV based on the coat protein gene analysis. The current study was intended to investigate the partial spreading, genetic diversity and phylogenetic analysis of the different OYDV isolates from garlic and comparing them with other sources of OYDV isolates available in the GenBank. In this study, we present for the first time the genome sequence of Iranian isolates of OYDV based on the CI gene. Besides, the recombination pattern of the CI gene in Iranian isolates was analyzed. Materials and Methods: During two consecutive growing seasons of 20162017 and 20172018, a survey was conducted in some of the major areas under the cultivation of garlic in Iran (Khorasan Razavi: Mashhad, Golbahar and kadcan, Mazandaran: Sari and Babolsar, Kerman: Jiroft, Lorestan: Aleshtar and Khuzestan: Shoshtar). Leave samples with yellow striping; crinkling; dwarfing and mosaic symptoms were collected and taken to the laboratory for both sap (mechanical) and molecular tests (RTPCR/PCR), to identify the disease cause. Total RNA was extracted from the symptomatic leaf samples using the RNeasy Mini Kit (Qiagen, Germany) and used for the reverse transcription (RTPCR) test. Primary detection was done by RTPCR using degenerate primer pairs (CIF/R). After performing PCR, products with 700 bp length, separated by electrophoresis in 1% agarose gel extracted and purified with the Qiaquick Gel Extraction Kit (Qiagen, Germany). Then PCR products were cloned in pTG19T Vector (Vivantis, Malaysia). The recombinant plasmids were extracted using the plasmid DNA isolation kit (Denazist, Iran) followed by sequencing (Macrogene, South Korea). Nucleotide sequence data were analyzed using Clustal Omega, MEGA 6, and RDP 4 software. Results and Discussion: Among Iranian OYDV garlic isolates which detected in this study, seven isolates from Golbahar, kadcan, Sari, Jiroft, Aleshtar and Shoshtar with severe mosaic symptom and dwarfing were selected for sequencing and determination of their molecular and biological characteristics. Comparative analyses of the partial CI gene sequences showed that seven Iranian isolates shared 75.0499.86% and 90.22100% sequence identities at the nucleotide and amino acid level respectively, with different OYDV isolates available in the GenBank. Phylogenetic tree based on partial CI gene placed Iranian OYDV isolates in two varying evolutionary groups; I and II. Group, I consisted of OYDV isolates from Argentina (KF632714/KF632715), Japan (AB219833/AB219834), Australia (HQ258894/JN127342), China (AJ510223), Spain (JX429964), India (KJ451436) alongside Iranian isolates. This group is geographically heterogeneous. All these isolates in group I are isolated from the garlic host. Three Iranian isolates, IRKh1, IRKh2, and IRKh16, which isolated from garlic placed in subgroup III, along with isolates from Spain (JX429964), Australia (HQ258894), and India (KJ451436). It indicates the high degree of genetic relationship among Iranian isolates in this subgroup. However, group II, which is also divided into two subgroups, includes four Iranian garlic isolates, IRKh33, IRKh54, IRKh23, and IRKh48, and are closely related to the German (JX433020)  and Argentine (JX433019) onion isolates. Recombination analysis among Iranian and other isolates in the CI gene showed that there was no recombination in this part of the genome. Conclusion: Based on the results of this study, the presence of OYDV isolates from some garlic areas of Iran was confirmed and for the first time, the molecular characteristics of the virus based on CI gene were determined. Results showed that OYDV is a dominant pathogen in the garlic field, which is highly prevalent in most sampled areas. Considering the importance of garlic in the food, pharmaceutical, and medical industries, determining the molecular characteristics of its viruses is highly important to determine an efficient control program.