Optimization of tissue and time for rapid serological and molecular detection of Apple stem pitting virus and Apple stem grooving virus in apple’

Majority of the apple trees are known to be infected by two latent viruses, Apple stem grooving virus (ASGV) and Apple stem pitting virus (ASPV). The importance of ASGV and ASPV is due to their non expression of symptoms, worldwide occurrence and wide host range on pome and stone fruits. Due to their latent nature in apple, early and rapid diagnostics plays important role for production of virus free quality planting material. The present investigation was conducted to detect and quantify ASPV & ASGV from different plant parts (spatial) in apple trees during different seasons (temporal) for optimisation of tissue and time for their rapid and early detection. Detection and relative quantification using immuno-molecular diagnostic techniques like, Double Antibody Sandwich-ELISA, Reverse Transcription-PCR and Real Time RT-PCR in various plant parts (leaf, whole flower, sepal, petal, anther, stigma with style, bark, fruit, seed and root) during different seasons was done. The DAS-ELISA based detection revealed infection in all plant parts except root and fruit with ASGV and ASPV, showing more expression in leaves followed by bark and whole flower. Similar results were also observed on RT-PCR based detection. Quantitative real time PCR analysis showed variation in expression of ASGV and ASPV in different parts during different seasons. Results confirmed that the ASGV and ASPV expression is higher in leaves followed by bark and whole flower. Periodic detection of these viruses in different plant parts during all the four seasons revealed varied virus titer from one season to another in the same plant. During all the seasons, both ASPV and ASGV were detected in bark in measurable titer using immuno-molecular detection tools, however via DAS-ELISA, ASGV remained undetected during dormant season. Hence leaves and bark except leaf during fall, can be directly used as detection material for their early and rapid detection leading to production of virus free planting material.