Immunomolecular detection and characterisation of potyviruses infecting cowpea (Vigna unguiculata (L.) Walp.) and papaya (Carica papaya L.)

Abstract

The study entitled “Immunomolecular detection and characterisation of Potyviruses infecting cowpea (Vigna unguiculata (L.) Walp) and papaya (Carica papaya L.)” was conducted at Department of Plant Pathology, College of Agriculture, Vellayani, Thiruvananthapuram during 2013-2015 with the objective to study the symptomatology and transmission of Potyviruses infecting cowpea and papaya, along with their biological, immunological, molecular detection and characterisation. Symptoms caused by Blackeye cowpea mosaic virus (BlCMV) in cowpea include mosaic and vein banding and that of Papaya ring spot virus (PRSV) in papaya showed mosaic and shoe string on leaves and ring spot on fruits and leaves. BlCMV and PRSV were mechanically transmitted (80 and 100 per cent respectively). Seed transmission (48.5 per cent) was recorded for BlCMV in cowpea. PRSV was not transmitted through seeds. BlCMV was transmitted by Aphis craccivora Koch and Aphis gossypii Glover (30 per cent respectively). Papaya mealy bug (Paracoccus marginatus Williams and Garnara de Willink) (80 per cent) was the most efficient vector of PRSV followed by Aphis gosssypii Glover (40 per cent) and Aphis craccivora Koch (30 per cent). Host pathogen interaction studies in resistant and susceptible genotypes of the cowpea (CO6 and Vellayani Jyothika) and papaya (Pusa Nanha and local variety) indicated a significant reduction in total carbohydrates and chlorophyll contents in susceptible genotypes on inoculation with Potyviruses, whereas resistant genotypes showed no significant difference. However, phenol, protein and defense related enzymes showed a significant increase in resistant genotypes on inoculation, compared to the susceptible genotypes. Sodium dodecyl sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE) and native-PAGE were conducted for the comparison of proteins and isozymes respectively, in resistant and susceptible genotypes. Eleven novel proteins were induced in CO6 whereas ten new proteins were observed in Vellayani Jyothika. Nine and eight induced proteins were identified in Pusa Nanha and local variety of papaya respectively. Three isoperoxidases with Relative mobility (Rm) values of 0.13, 0.20 and 0.27 were unique for CO6. There was no unique isozyme on inoculation in Vellayani Jyothika. Isoperoxidases with Rm values of 0.08 and 0.23 were unique for Pusa Nanha. However no specific isozyme was observed in local variety of papaya. Chenopodium amaranticolor and Chenopodium quinoa were identified as indicator plants for the biological detection of BlCMV and PRSV. Immunological studies using Direct antigen coating-Enzyme linked immunosorbent assay (DAC-ELISA) and Dot immunobinding assay (DIBA) and molecular detection viz. Reverse transcription-Polymerase chain reaction (RT-PCR) identified the Potyviruses as Bean common mosaic virus (BCMV) strain Blackeye and PRSV respectively. Comparative amino acid sequence alignment revealed a 95 and 100 per cent homology with coat proteins of BCMV and PRSV respectively. The transmission studies revealed that Potyviruses were transmitted mechanically and by insects. Present study also revealed that BlCMV was transmitted through seeds while, PRSV was not seed transmitted. The host pathogen interaction studies identified phenol, protein and defense related enzymes along with induced proteins and isoforms of peroxidase could be used as biochemical markers to identify resistance and susceptibility in plants. The present molecular study confirmed that BlCMV infecting cowpea was closely related to BCMV, a strain of Blackeye. PRSV in the present investigation was found related to PRSV isolates reported from other parts of India. Comparative sequence analysis of BlCMV and PRSV Potyviruses showed that the sequences were entirely different and showed only a 51.12 per cent similarity at nucleotide levels. Serological and sequence studies thus suggest that BlCMV caused the mosaic disease in cowpea and PRSV, the ring spot disease in papaya.