Development of a graft inoculation method and a real-time RT-PCR assay for monitoring Tomato chlorosis virus infection in tomato

A graft inoculation method coupled with RT-qPCR was developed for monitoring ToCV infection in tomato plants. Ten seed-grown tomato seedlings were graft inoculated with phloem tissue-containing stem segments from a ToCV-infected tomato plants. Another group of tomato seedling were grafted with similar stem segments from a healthy tomato plant as mock inoculated control. The CP gene of ToCV was cloned under the control of T7 promoter and in vitro synthesized RNA was used as a standard for quantification. Total RNA was isolated from leaf samples of ToCV-inoculated and mock-inoculated control plants before the inoculation and 1-60 days post inoculation (dpi). The presence and the titer of ToCV were determined from all ToCV-inoculated or mock-inoculated control plants by RT-qPCR. After 15 dpi, ToCV was detected in 20-30% of graft-inoculated plants. The infection rate then increased progressively and reached to 70-80% by 60 dpi. Titer of ToCV was at the detectable level at 15 dpi and increased and reached to maximum level by 40 dpi and then started to decrease. The results showed that patch 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 the laboratories. The patch grafting could be combined with RT-qPCR and used for infecting and quantitative monitoring of ToCV or other phloem-limited viruses in tomato or in other plants.