Partial characterization and development of sensitive and reliablediagnostic for the detection of cucumber mosaic virus

Abstract

A survey on Cucumber mosaic virus (CMV) was conducted on 21 banana orchards in three bananas producing states: Karnataka, Maharashtra, and Uttar Pradesh. Sampling was done during winter and autumn and all samples were initially examined for the presence of CMV using DAS-ELISA. Out of 300 tested samples, the presence of CMV was observed in 13. The lowest rate of infection was determined in Uttar Pradesh followed by Maharashtra, while high infections were observed in samples from Karnataka. Three CMV infected samples represented in each state were amplified through RT-PCR using the movement protein (MP) gene specific primers and cloned in the pGEMT essay vector. The RT-PCR products of amplified samples were sequenced. Gene sequences of CMV-KAR, CMV-MR, and CMV-UP isolates under study coding for the movement protein revealed 99.76%-99.88% and 99.28%-99.64% sequence identity at the nucleotide and amino-acid level. These isolates were compared with 29 CMV isolates reported from various plants around the world, which formed three distinct subclades: -IA, -IB, and -II. These isolates were most closely related to subgroup-I isolates, sharing up to 95.81% and 96.84% sequence identity at nucleotide and amino acid, respectively. However, these isolates shared 81.79%-81.90% and 82.80%-83.15% nucleotide and amino acid identity with subgroup II isolates. The isolates under study clustered with the CMV subgroup-IB as confirmed by phylogenetic analysis. Further, we standardized the minimum limit of RNA transcript for the sensitivity of RT-PCR with respect to the coat protein (CP) gene and movement protein (MP) gene. The results of RT-PCR showed that the CP gene was more sensitive than the MP gene for the detection of CMV as it amplified the gene product up to 0.02 ng RNA. The CP gene based RT-PCR assay may be a more sensitive, reliable, and convenient molecular tool for detection of the CMV pathogen, and can be used in quarantine, eradication, and tissue culture certification programs.