Molecular characterization of chilli veinal mottle virus infecting chilli (Capsicum annuum L.)

Abstract

Chili Veinal Mottle Virus (ChVMV) disease is emerging as major constraint in the production of chilli pepper (Capsicum annuum L.) in India and many other Asian countries. This is the first attempt in India and elsewhere in the world to study the molecular characterization and variability of ChVMV isolates. The survey results revealed the presence of ChVMV in all chilli Karnataka, Maharashtra, Andhra Pradesh, Tamil Nadu and Kerala and the incidence ranged from Zero to 87.9 per cent. Differentiation of 30 ChVMV isolates using 31 plant species was found difficult because of no significant differences in the symptoms produced on these test plants. However the new host plants identified in the study will be very useful for the management of the virus disease. The electron microscopy of purified virus preparations revealed flexuous rod shaped particles measuring 650-700X13 nm. Diagnostic tools RT-PCR, ELISA, DIBA, SDS-PAGE, western blot and RNA blot hybridization were found sensitive for detection of the virus. Polyclonal antiserum was raised in rabbit against purified native virus and its titer was equivalent to the commercially available antisera. A survey was undertaken in chilli growing areas of Southern India for the ChVMV incidence, the virus was confirmed by DAC-ELISA. During the survey, several ChVMV samples were collected, named as different isolates and were maintained on Datura metel in the glasshouse conditions. Biological differentiation of the 30 ChVMV isolates was carried out on 32 different host plants, indicating the similar type of reaction to the test plants with all the isolates. The Immunological differentiation of the ChVMV isolates were observed by performing ELISA by using ChVMV, PVBV, PVMV, PVY antisera. In the dot immuno assay, all the isolates showed positive reaction to the ChVMV polyclonal antisera. SDS-PAGE, RNA blot using the synthesized probe and Western blot were standardized for the detection of ChVMV and used for differentiation of isolates. All the above studies confirmed that the isolates were closely related. Analysis of 30 ChVMV isolates sequences revealed that the coat protein region is highly conserved and variation is present in the Nib region among the isolates. Further, the conserved regions present in the potyvirus genus were observed in all the isolates. The Phylogenetic analysis of ChVMV isolates along with the other members of family Potyvirdae revealed that these isolates are distinct from other viruses by forming a separate cluster. The ChVMV isolates were classified into five strainal groups based on the phylogenetic analysis and identity matrices of both nucleotide and deduced amino acid sequences which exhibit more than 5 per cent variation at nucleotide level and more than 3 per cent variation at amino acid level. The partial NIb region, complete coat protein region and 3’ UTR of 30 ChVMV isolates were sequenced. The comparison and phylogenetic analysis of ChVMV isolates nucleotide and deduced amino acid sequences with the other members of Potyviruses and Potyviridae revealed that the ChVMV isolates were distinct from all other viruses. The comparison and phylogenetic analysis among the ChVMV isolates indicated five groups, which may be considered as distinct strains as they exhibit more than five per cent variation at the nucleotide as well as more than three per cent at the amino acid levels. Screening of 87 chilli lines to ChVMV revealed that, 44 were immune, four highly resistant, four resistant, 14 moderately resistant, three susceptible and 18 highly susceptible. Some of the lines identified as immune were also the advanced breeding lines, which are ready to be used as varieties. Resistant source in chilli against ChVMV was identified by screening 84 germplasm lines using AUSPC (Area Under Symptom Progress Curve) criteria and utilized in the breeding programme.