Development of RT-PCR degenerate primers for the detection of two mandariviruses infecting citrus cultivars in India.

Indian citrus ringspot virus (ICRSV) and Citrus yellow vein clearing virus (CYVCV) are the mandariviruses infecting various citrus cultivars in India and around the world. In the fields, it was observed that citrus plants infected by both the viruses and frequently expressed only ringspot symptoms. The ICRSV-specific polyclonal-antibody used in immuno-sorbent electron microscopy (ISEM) and enzyme linked immuno-sorbent assay (ELISA) could detect only ICRSV in mixed infections. Therefore, the conserved sequences of the RNA dependent RNA polymerase (RdRP) gene of the alphaflexiviruses were exploited for developing a RT-PCR based assay for detection of both the mandariviruses simultaneously, if present. A degenerate primer pair was designed to amplify a ∼435bp fragment by multiple alignments of the RdRP gene sequences of the members of genera Mandarivirus, Potexvirus and Allexivirus. The developed RT-PCR assay was validated for detecting both, CYVCV and ICRSV in mixed infections as well as in single virus-infected citrus plants. The presence of ICRSV or CYVCV or both of them together in such plants were confirmed by using primer pair specific to each of these viruses. Further, the identity of the amplicons was confirmed by sequencing and the virus species were determined with BLASTN analysis. The degenerate primers also amplified the corresponding target sequences of an allexivirus and a potexvirus from the respective infected garlic/ onion and tobacco plants. The use of the degenerate primers for the detection of these virus species of the genus Mandarivirus will be useful in citrus certification programmes.