Phylogenetic Relation of Watermelon chlorotic stunt virus (WmCSV) Isolates of Sistan and Baluchestan Province by Sequencing of Coat Protein Gene

Introduction: Cucurbits are among the main vegetable crops that are extensively grown in commercial greenhouses, plastic tunnels, and open farms in many areas of Iran. Many begomoviruses are known to cause serious damages in economically important cucurbit crops such as watermelon and melon. Among these viruses, WmCSV has been quoted as one of the major limiting factors for cucurbit production throughout the south of Iran. Begomovirus genus is a member of Geminiviridae family that has the most species in comparison to other viral genus infecting plants. Begomoviruses are notorious pathogens infecting dicotyledonous plants globally. Thereare transmitted exclusively by the whitefly, Bemisia tabaci. Whitefly is one of the most important and prevalent pests in the warm south and southeast provinces of Iran and infects cucurbits fields in Sistan and Baluchestan province with a widespread population. Begomoviruses are single-stranded circular DNA viruses and have been observed in a wide host range of plants. There are different reports of infectious Begomoviruses species such as Watermelon chlorotic stunt virus (WmCSV) from a world that can damage the cucurbits. The purpose of this study is to find the phylogenetic relation of WmCSV isolates from Sistan and Baluchestan province, which is located southeast of Iran, together and other isolates from the world, too. Because of this province is the largest area, with about 11.4% of the total area of Iran. Materials and Methods: To identify the WmCSV infecting cucurbits in the southeast of Iran, several surveys in different regions of Sistan and Baluchestan were selected for the study, including Saravan, Iranshahr, Nikshahr, Sarbaz, Konarak, Chabahar, Zabol, Zahak, Hamoon, Hirmand, Nimroz, and Zahedan. Sampling has been taken from different cucurbits, such as different kinds of melons, watermelon, and squash, in 2015-2017. The samples were taken from plants showing viral symptoms such as vein yellowing, stunting, chlorotic, and mottling, deformation and severe reduction of fruit size and co-infection with whiteflies. The collected samples were transformed into the laboratory and the total DNA was extracted. To identify WmCSV were used from specific primers, Gem-CP-V-5' and Gem-CP-V-3' detecting Begomovirus genus in cucurbits. PCR products have been sequenced and blasted in the GenBank NCBI database and submitted there. Then, carried out the Alignment of acid nucleotide sequences and description of the phylogenetic tree by MEGA5 software. The Identity and differences percentage of Watermelon chlorotic stunt virus nucleic acid sequence from Sistan and Baluchestan province isolates with together and other areas of Iran carried out by MegAlign software too. Results and Discussion: All areas of sample collection of Sistan and Baluchestan province were infected with WmCSV in this study. Positive PCR watermelon samples have been infected to WmCSV, severity. The expected PCR products, 550 bp size, from 16 amplified partial CP genes from different areas of Sistan and Baluchestan province in Iran were done sequencing. Phylogenic sequencing analysis results of isolates from other countries and other regions of Iran also WmCSV isolates in this research from Sistan and Baluchestan province, classified them in two apart groups involves A and B. The A group was divided into 2 groups, that were named AI and AII. Isolates of Sistan and Baluchestan province from this research involved both of them. These findings seem to indicate Sistan and Baluchestan as of the possible origins of WmCSV in Iran. There was no relationship between geographical source and host in this province. Some isolates of this province had a near relationship to Oman and Saudi Arabia isolates. Conclusion: WmCSV can be considered as a serious disease threatening watermelon production, in Sistan and Baluchestan province in the southeast of Iran as severe symptoms. On another hand, individually or in interaction with other begomoviruses, WmCSV can cause severe damage in farms infected by whiteflies. Probably, the spread of isolates belong Sistan and Baluchestan province and Oman or Saudi Arabia has been taken by whiteflies that transformed via between Iran and these countries, because of the most relationships between the WmCSV isolates of these countries. More importantly, performing collaborative studies with neighboring countries such as Pakistan and India, seems necessary. Also, further studies on Iranian isolates of WmCSV that occurred on either cultivated or wild plants are essential to have a better understanding of the viral epidemics in the country. The results of sequencings will be used to design constructs inducing RNA silencing as a resistant strategy, against WmCSV isolates that were classified in both two groups.