Preponderance of mixed infection of Cucumber mosaic virus and Candidatus Phytoplasma australasia on brinjal in India.

Cucumber mosaic virus (CMV) has broad host range by infecting major stable food crops and causes heavy loss especially in brinjal. In major brinjal growing tracts of Tamil Nadu, Krishnagiri recorded the highest combined infection of CMV and Candidatus Phytoplasma australasia (Ca. P. australasia) with 26%. The symptoms ranged from mild to severe mosaic, mottling, filiformity of leaves and little leaf. The virus was successfully transmitted to cowpea cv. CO7 and ridge gourd through mechanical inoculation and the presence of virus was detected both by DAC-ELISA and RT-PCR. Electron microscopy of CMV exemplified isometric particles with 28-35 nm under TEM and phytoplasma with 700-820 nm in SEM analysis. Among the different test hosts, Luffa acutangula was found to be the best indicator host for brinjal CMV isolate as it requires shorter period (4-5DPI) to express symptoms with good virus titer (A405nm 2.318). The genome characterization of CMV TNB isolate revealed that the RNA1, RNA2 and RNA3 have 97, 96 and 99% homology with other 1B sub group CMV isolates, respectively. Recombination analysis of RNA2 of CMV TNB has tomato Egyptian isolate (KT921315) as major parent and black pepper Indian isolate (KU947030) as minor parent at the conserved region (52-805nt). The characterization of phytoplasma using iphy classifier reveled Ca. P. australasia belonging to 16SrIID subgroup was present along with CMV infection. In addition, the Solanum torvum grown in and around brinjal ecosystem showed severe mosaic and exhibited 99% nucleotide identity with CMV TNB isolate and these plants also act as inoculum source during the on and off cropping season in India. To our knowledge this is the first record of mixed infection of CMV and Ca. P. australasia in brinjal and first record of CMV infection in S. torvum in India.

Production of polyclonal antibodies for Capsicum chlorosis virus (CaCV) infecting chilli in India through recombinant nucleocapsid protein expression and its application.

Bud necrosis and chlorotic spots causing virus affecting chilli crop in Tamil Nadu (India) was identified as Capsicum chlorosis virus (CaCV). Specific primers were used for amplification and sequencing of the nucleocapsid protein (NP) gene. Polyclonal antibody against the bacterially expressed NP from the CaCV-TN-CBE isolate was produced using recombinant DNA technology. NP gene was subcloned into the pET-28a (+) vector and expressed by transformation in BL21 (DE3) pLysS. The expressed protein was about ∼34 kDa and was confirmed through western blot analysis using Groundnut bud necrosis virus (GBNV) polyclonal antiserum from ICRISAT, India. The purified recombinant protein was used to immunize rabbits to generate CaCV-specific polyclonal antiserum. The sensitivity levels of polyclonal antiserum thus raised was assayed through indirect ELISA or direct antigen coating (DAC)-ELISA using the recombinant protein as antigen. The recombinant antiserum produced in this study successfully detected the natural infection of CaCV on chilli plants collected from the field as well as on cowpea plants artificially inoculated with CaCV by using DAC-ELISA, DIBA and western blotting.