Development of a single-leaflet graft inoculation method and a nested PCR assay for monitoring tomato chlorosis virus infection in tomato

Tomato chlorosis virus (ToCV), a member of the Crinivirus genus, has caused an epidemic disease that affects tomato production worldwide including Azerbaijan. The whitefly-transmitted ToCV is a phloem-limited virus and Bemisia tabaci plays a crucial role in ToCV transmission. In this study, experimental propagation of ToCV has been performed primarily by using the single-leaflet grafting inoculation method. To develop a simple and effective method for transmission of ToCV, we investigated grafting single-leaflets from tomato plants infected with ToCV to recipient tomato seedlings. Total RNA was isolated from leaf samples of ToCV-inoculated and mock-inoculated plants before the inoculation and 1-60 days post-inoculation (dpi). The presence of ToCV was determined from all virus-inoculated or mock-inoculated (control) plants by nested PCR. After 15 dpi, ToCV was detected in 18-25% of graft-inoculated plants. The infection rate then increased progressively and reached 30-65% by 30 dpi and finally, 75-80% by 60 dpi. In total, fifty (50) tomato seedlings were tested and forty-five (45) were grafted successfully with single-leaflets infected with ToCV. In this study, we showed that single-leaflet grafting could provide a sufficient amount of inoculum for the transmission of ToCV to the grafted tomato seedlings. The results manifested that single-leaflet grafting is a simple and efficient method for experimental inoculation of ToCV and can be used as an alternative and/or complementary to vector transmission in laboratories. The single-leaflet grafting could be combined with nested PCR and used for monitoring of ToCV or other phloem-limited viruses in tomatoes also in other plants.