Molecular Characterization of Bean Common Mosaic Virus Infecting Phaseolus vulgaris L. in Kashmir

Extensive survey of commercial bean growing areas of Kashmir conducted during kharif 2009 and 2010 revealed only 13 out of 100 surveyed locations free from Bean Common Mosaic Virus (BCMV) disease. The disease incidence was found to range from 0.50 to 85.00 per cent, with the maximum incidence recorded in Srinagar district followed by that in district Anantnag and Bandipora. The samples of BCMV from infected bean plants showing characteristic mosaic, leaf distortion and stunting type of symptoms were categorized into 21 isolates based on the symptomatology and the representative commercial bean growing areas of the Valley. Confirmation of the association of BCMV with each isolate was made by mechanical transmission and DAC-ELISA tests. The test isolates in DAC-ELISA test reacted positively with only BCMV polyclonal antisera, confirming the association of BCMV, which was further established through RT-PCR amplification of coat protein gene using BCMV specific primers. The strain spectrum of BCMV, discerned by inoculation separately of 21 BCMV isolates on international differentials, revealed the existence of three pathogroups - PG I, PG II and PG VII- based on the reaction of isolates on host group 1 to 5 of differential varieties having recessive resistance genes. The pathogroups PG I and PG II were further divisible into four strain groups viz., Ia (i.e., NL-1), Ib (i.e., NL-1n) and IIa (i.e., NL-7), IIb (i.e., NL-7n) , based on the capability to induce veinal necrosis at high temperature (>300C) on differential cultivar ‘Jubila’ possessing dominant gene. The prevalence of pathogroup PG VII (i.e., strain NL4) in the cooler regions of Kashmir recorded during the present studies is the first report of its occurrence in India. In order to characterize viral isolates, molecular characterization of coat protein gene of NL-1, NL-4 and NL-7 strains of BCMV was made using standard methods of molecular biology. RT-PCR of the test strains using specific primer pair amplified a product of ~860 bp. Purified PCR product (DNA) was ligated in PGEM-T easy vector system and transformed in E. coli strain DH5α. The cloned product revealed 860 bp sequences, which were submitted to NCBI gene bank Nucleotide Database under accession number JN692256, JN692258 and JN692257, respectively. The pair-wise homology between the nucleotide sequence of NL-1 and NL-4 was 95 per cent, whereas the homology between NL-1 and NL-4 and between NL-4 and NL-7 was 93 per cent, respectively. The nucleotide sequence of NL-4 from Kashmir showed maximum homology of 96 per cent with BCMV strains EU492546 and AF328753 from India and Mexico, respectively. Phylogenetic analysis of CP sequence of NL-1, NL-4 and NL-7 from Kashmir were most closely related to BCMV strains from H.P. (India), USA and other countries. In all the three strains, DAG motif was present at a/a position 13 of CP which is important for aphid transmission in potyviruses. BCMV specific conserved motif MVWCIDN was also present in all the three strains. Further, eighty-five accessions were evaluated under controlled glass-house conditions against all the three pathogroups (I, II and VI) of BCMV prevalent in Kashmir to find out the sources of resistance against BCMV. Only 17 genotypes were found resistant to the test virus strains. Some of the resistant accessions/cultivars include R-40, R-155, R-3, B-6, Pencil white, SKAU-B-02, SKAU-B-01, Pointed lady, TO, P1207262, TU, KRC-5, R-14, Jubila, Contender and Monroe