Development of real time pcr method for determination of Tswv isolates breaking tomato Sw-5 resistance

In tomato, resistant to Tomato spoted wilt virüs (TSWV) is controlled by the interaction of Sw-5 dominant gene in plant with the NSm cell-to-cell movement protein of the virus. In recent years, TSWV isolates breaking the Sw-5 resistance by interfering this interaction have been identified. It has been reported that two point mutations in the NSm gene were identified by comparison of the NSm gene of the resistance breaking (RB) and non-resistance breaking (NRB) isolates are responsible for the breaking the Sw-5 resistance. In this study, 57 leaf or fruit samples were collected from tomato production areas and local market in Antalya and Isparta province and samples were tested by PCR, RT-PCR and/or real-time RT-PCR methods. The results showed that 46 of the 57 suspected samples were infected with TSWV and four of the infected samples were taken from tomato cultivars with Sw-5 resistance gene. As many as 13 different potential RB isolates were identifed by mechanaical inoculation to resistant and susseptibe tomato cultivars. To determine possible mutations in the NSm gene, this gene was amplified from six selected RB isolates and it was cloned and sequenced. Comparison of amino acid sequences showed that while the NSm gene of Turkish isolates were 96-99% identical, they showed 95-100% sequence identity with world isolates. Analysis also determined that the previously identified point mutations were not present in the NSm genes of RB isolates identifed in this study. However, a glutamic acid (E) to aspartic acid (D) or glycine (G) point mutation was identied in amino acid 287 of the NSm genes of RB isolates identifed in this study. Comparison of the NSm genes of these isolates with the that of world isolates in the GenBank showed that these mutations were only present in Turkish isolates identified in this study. A SYBR Green based real-time RT-PCR method was developd for amplification of the the region of the NSm gene containing the identifed mutations. The amplification and melting analysis of the 3' endof the NSm gene by real-time RT-PCR enabled differentiation of point mutataions identified in this study and showed that TSWV isolates carring these mutations can more quickly and enonomicaly be distinguded from wild type isolates without sequencing.