Characterization of different viruses infecting small Cardamom (Elettaria cardamomum Maton) and production of disease free plants

Abstract

The study entitled “Characterization of viruses infecting small cardamom (Elettaria cardamomum Maton) and production of disease free plants” was conducted at Department of Plant Pathology, College of Agriculture, Vellayani during 2017-2019, with the objective to study the occurrence and distribution of viruses infecting small cardamom, molecular characterization of the viruses and elimination of viruses through meristem culture for the production of disease free planting material. Survey was conducted in Kattappana, Pampadumpara and Ambalapara panchayats of Kattappana block; and Nedumkandam, Thukkupalam and Chembalam panchayats of Nedumkandam blocks of Idukki district during November 2018 – May 2019. The incidence of katte disease caused by Cardamom mosaic virus (CdMV) was present in all the panchayats surveyed and it ranged from 3.75 to 43.0 per cent in Kattappana block and 5.0 to 31.33 per cent in Nedumkandam block. Disease incidence for chlorotic streak caused by Banana bract mosaic virus (BBrMV) was recorded from Kattappana (41%), Pampadumpara (30%) and Nedumkandam (8.33%) panchayats. The aphids infestation was absent in all the surveyed plots. Colocasia spp. and Alpinia spp. were the major plants observed in and around the cardamom fields and were not having visible symptoms of the viral infections. The virus inoculums were maintained under insect proof net house at Cardamom Research Station, Pampadumpara. Katte disease produced slender chlorotic flecks developing into pale green discontinuous stripes running parallel to veins from midrib to leaf margin of the infected leaves. Mosaic mottling and chlorotic specks were seen on the infected leaves and young pseudostems. In case of severe infection, plants produced stunted tillers. Chlorotic streak disease was characterised by continuous and discontinuous chlorotic streaks along veins and midribs of the infected leaves and green discontinuous spindle streaks on pseudostem. CdMV (a potyvirus) and BBrMV in cardamom was detected using polyclonal antibodies of Potato virus Y (PVY) and BBrMV respectively procured from DSMZ, Germany by direct antigen coating- Enzyme linked immunosorbent assay (DAC-ELISA) and Dot immunobinding assay (DIBA).The highest virus titre of CdMV and BBrMV was obtained in samples collected from Pampadumpara and Kattappana respectively. Molecular detection of the viruses was carried out using reverse transcriptase - polymerase chain reaction (RT-PCR) with specific primers for CdMV and BBrMV; and obtained amplicons of expected size of 879-905 bp for CdMV- and 625-633 bp for BBrMV- infected samples. The sequences of the isolates of CdMV from Kattappana, Pampadumpara and Nedumkandam were subjected to BLAST analysis and found to be similar to Indian cardamom mosaic virus isolates from Thalathamane and Appangala with > 96 per cent similarity. The BBrMV in cardamom from Kattappana, Pampadumpara and Nedumkandam was similar to Banana bract mosaic virus (BBrMV) CdM isolate of Karnataka (91.01%), Coimbatore (90.29%) and Thrissur (95.76%) respectively. Phylogeny tree constructed in MEGA 6.0 software differentiated CdMV and BBrMV into four clades, in which CdMV Kattappana and Nedumkandam isolates were clustered together whereas CdMV Pampadumpara isolate was in separate clade. Similarly, BBrMV isolates of Pampadumpara and Nedumkandam clustered together while BBrMV Kattappana was in separate clade. Meristem of 2 mm size separated from infected plants were grown in Murashige and Skoog medium supplemented with 3 mg benzyl amino purine (BAP), 1.5 mg indole acetic acid (IAA) and 0.8 mg kinetin expressed direct organogenesis but multiple shoots were not produced. The TC plants were subjected to DAC-ELISA with the specific polyclonal antibodies and PCR with specific primers of the viruses and confirmed that the plants produced from meristems were free of both the viruses. Thus, the present study revealed that two viral diseases viz., katte and chlorotic streak affecting small cardamom in Idukki. Serologically and molecularly it was detected that katte disease was caused by Cardamom mosaic virus (CdMV) and chlorotic streak disease was caused by Banana bract mosaic virus (BBrMV), and the viruses could be eliminated from the infected plants through meristem tip culture to produce the diseases free plants.