Document Type : Original Article
Authors
1 - M.Sc. in Plant Pathology, Department of Plant Protection, Faculty of Agriculture, University of Birjand, Iran.
2 Associate Professors of Plant pathology, Department of Plant Protection, Faculty of Agriculture, University of Birjand, Iran.
3 Associate Professor, Department of plant phatology, College of Agriculture, University of Birjand, Birjand, Iran.
Abstract
Introduction:Saffron (Crocus sativus L.) is a crucial crop in Iran, celebrated for its medicinal and economic value. Thriving in arid regions, it's a perennial herb with corms used in food and pharmaceuticals. As the world's priciest spice, it significantly impacts Iran's exports and industries, with a history in traditional medicine dating back 3000 years. Over 90% of global saffron comes from Iran, primarily Khorasan Razavi province (78%) and Khorasan Jonubi province (17%). Saffron is also grown in Fars, Isfahan, and Hamadan.
Boshrouyeh County in Khorasan Jonubi boasts saffron cultivation on 1,307 hectares in 2020-2021, yielding about 3.4695 tons, generating 70 billion Tomans. Regarding plant viruses, the Cucumber mosaic virus (CMV) is a significant threat, affecting various plants globally, including cucumbers, tomatoes, and more. In Iran, CMV has been reported in crops like bananas and melons, causing up to 20% yield loss.
This study focuses on CMV in saffron plants in Boshrouyeh County, using molecular methods for precise assessment due to its impact on saffron cultivation.
Materials and Methods:In the autumn of 2022, a survey was conducted in saffron fields to assess virus symptoms like mosaic, stunting, yellowing, wrinkling, and leaf deformities. A total of 148 samples were collected, dried, and stored for analysis. RNA was extracted from young plant leaves using a non-column RNA extraction kit. Specific viral protein coat primers were used for cDNA synthesis, followed by PCR amplification. The PCR reaction involved denaturation at 94°C for 2 minutes, followed by 40 cycles at 94°C for 40 seconds, 52°C for 30 seconds, and 72°C for 2 minutes, with a final extension at 72°C for 5 minutes. Electrophoresis confirmed three samples were infected with CMV. The amplified viral sequences were extracted and sequenced by a specialized company in South Korea. Sequence similarity was verified using BLAST tools on the NCBI database to confirm the presence of these virus strains and assess their genetic relatedness to global strains.
Results and Discussion: In collected plant samples, various symptoms such as curling, leaf complexity, leaf mosaic, yellowing, and discoloration were observed. To conduct phylogenetic studies, three isolates of Cucumber mosaic virus (CMV) from saffron fields in Raghe and Boshrouyeh were sequenced. Sequence comparisons using BLAST revealed all three isolates shared similarity with CMV. A phylogenetic tree based on the 657-nucleotide coat protein gene sequence was constructed. Analysis with MegaX software compared Iranian isolates with 17 gene bank isolates. The resulting tree contained twelve branches, with Iranian isolates showing the highest similarity to Syrian and Greek isolates. Specifically, Kho1 had the most significant similarity with 100% to ToCMV5-1 from Syria and 98% to PoCMV9-11, also from Syria. Kho2 had the highest similarity of 98% with CMV-GRcl1 from Greece. Kho3 showed 99% similarity to ToCMV5-1 and 99% to Kho1, both from different regions. These findings highlight the genetic diversity of CMV in saffron plants.
Conclusion: In this study, saffron fields in Boshrouyeh County were found to be infected with Cucumber mosaic virus (CMV) using a reverse transcription-polymerase chain reaction (RT-PCR) test with a specific CMV primer. Out of 148 saffron samples showing viral disease symptoms, three were confirmed to be infected with CMV, as evidenced by the successful amplification of a 657 base pair fragment using CMV-specific primers.
Genetic diversity was assessed based on the sequence of the coat protein of Iranian CMV strains and other strains available in the BLAST database. The results revealed the formation of twelve distinct branches, with Iranian strains falling within the first branch of the first group. The closest strains to Iranian ones were CMV-GRcl1 and ToCMV5-1 from Greece and Syria, respectively. The highest similarity was observed between two Iranian strains, Kho2 from Boshrouyeh and Kho3 from Raqqah, indicating geographical location did not play a significant role in viral classification.
While CMV infection in saffron had been reported using serological methods, this study emphasized the need for molecular identification and protein analysis. Notably, this research represents the first report of CMV presence in saffron fields in South Khorasan.
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