Molecular cloning and characterization of virus causing leaf curl disease of capsicum spp.

Abstract

Chilli is one of the most important crops cultivated across the globe, as vegetable, spice and for industrial purposes. According to the statistics of National Horticulture Board (2017), the crop covers an area of 1860 ha in the state of Kerala with an average production of 12470 tonnes. During the last decade, the threats posed by the emerging begomoviruses infecting solanaceous crops have affected the economic cultivation of chilli. Chilli leaf curl disease caused by Chilli leaf curl virus belonging to the genus Begomovirus and family Geminiviridae is a serious constraint to chilli production in India which causes upto 100 per cent yield loss especially when infected at an early stage of the crop. Considering the importance of the disease, the present study was undertaken with the objective to study the incidence and symptomatology of chilli leaf curl disease and to characterize and clone the coat protein gene of the Chilli leaf curl virus isolates. The project initiated with purposive sampling surveys conducted in eleven different locations of Thrissur district, Kerala to document the incidence and symptomatology of leaf curl disease on chilli plants. The disease incidence recorded during the survey ranged from 43.30 to 85.00 per cent under open field conditions and from 45.75 to 79.40 per cent under protected conditions while the disease severity ranged from 43.60 to 81.54 per cent and from 49.40 to 87.50 per cent, respectively under open field conditions and protected conditions. The symptomatology of chilli leaf curl disease on different parts of the plant such as leaves, internodes, fruits and the whole plant under natural conditions was documented during the survey. The symptoms observed on the leaves of infected chilli plants under natural conditions include upward curling, crinkling, puckering, vein banding, interveinal chlorosis, size reduction of leaf lamina and leaf malformation. The fruits produced by the infected plants showed size reduction and deformation. The infected plants were stunted and bushy in appearance. The transmission of the virus by insect vector, Bemisia tabaci and grafting was studied and the symptoms were documented. The newly emerged leaves after artificial inoculation expressed symptoms such as curling, puckering and crinkling along with stunting of plant growth.

Molecular characterization of the four virus isolates collected from various locations of Thrissur district viz., VKA1 VKA2, KAR1 and KOD4 and two isolates viz., VLNY1 and PKD1 collected from Vellayani, Thiruvanathapuram district and from Vithinasseri, Palakkad district, respectively were undertaken. The total genomic DNA from virus infected chilli leaf samples was isolated and subjected to PCR amplification of viral coat protein gene to confirm the presence of virus infection. PCR amplification of the isolated DNA was carried out using two Begomovirus specific degenerate (universal) primers, namely, AV494 / AC1048 (Wyatt and Brown, 1996) and Deng 540 / 541 (Deng et al., 1994). The amplicons of size 550 bp were obtained and were sequenced. The partial coat protein gene of size 550bp were also cloned into the vector pTZ57R/T and transformed into DH5α strain of Escherichia coli and the true recombinants with desirable insert were confirmed by colony PCR. The sequence data obtained in the study were subjected to in silico analysis to assess the diversity of the isolates. The nucleotide BLAST (BLASTn) analysis revealed more than 90 per cent sequence identity with Chilli leaf curl Vellanad virus isolate (Accession No. NC038442.1) from Vellanad region of Thiruvanathapuram district, Kerala. The translated nucleotide - protein BLAST (BLASTx) analysis of the viral sequences revealed more than 96 per cent sequence identity with Chilli leaf curl Vellanad virus coat protein sequence (accession no. YP_009506391.1). The coat protein sequences of all the six isolates were translated into corresponding amino acid sequence by ExPASy Translate tool and were used for further analysis and interpretation. The phylogenetic analysis revealed that, the isolates VKA2, KAR1 and KOD4 had very distinct sequence alignment when compared to other Chilli leaf curl virus isolates from India. The results indicated that, the three isolates viz., VKA2, KAR1 and KOD4 could be new strains of Chilli leaf curl virus infecting chilli. Three, possibly new strains of Chilli leaf curl virus infecting chilli have been identified and hence the study highlights the need for monitoring the emergence of new strains of plant viruses especially begomoviruses infecting solanaceous crops grown in Kerala. As this disease is one of the most important challenges to chilli cultivation, the information generated from the study could also be applied for the timely detection and effective disease management