1Plant Virology Unit, Division of Plant Pathology, Indian Agricultural Research Institute New Delhi-110012
2AICRP on Tropical Fruits (Citrus) Tirupati-517502, India.
Survey of sathgudi sweet orange was done in the Nagri village of Chittor district in the AP state during November 2006. Incidence of mosaic disease was observed in a large number of citrus plants. A few plants showed unusual symptoms of vein clearing and mosaic. Two types of virus particles were seen in electron microscopy in samples of the diseased citrus plants showing vein clearing and mosaic. One particle was bacilliform and the other was flexuous. ISEM studies indicated that the baciliform virus particles of Citrus mosaic virus (CMBV) while flexuous particles were of Indian citrus ring spot virus. Since ISEM can not be used for routine detection, duplex PCR for detection of both the viruses were standardized. The total DNA and total RNA were isolated separately from the leaves of infected citrus plants using commercial Kits. cDNA for ICRSV was prepared using a reverse primer designed coat protein region of ICRSV. cDNA and total DNA from the infected citrus plant were used for standardization of duplex PCR for simultaneous detection of ICRSV and CMBV using primers of these virus providing amplification product of ∼1100 bp for ICRSV and ∼500 bp for CMBV. The sequencing of PCR product confirmed that the fragments amplified were of ICRSV and CMBV. The duplex PCR provide can a useful and rapid method for detection of two viruses. The technique should prove highly useful in disease surveys, nursery certification and quarantine applications.
