1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)
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Item Cloning and characterization of coat protein gene of Tomato leaf curl virus infecting tomato and its phylogenetic relationship with other members of geminiviridae(Department of Plant Biotechnology, College of Agriculture , Vellayani, 2022-10-27) Athira ,S M; Ayisha ,RThe study entitled ‘Cloning and characterization of coat protein gene of Tomato leaf curl virus infecting tomato and its phylogenetic relationship with other members of Geminiviridae’ was carried out at College of Agriculture, Vellayani during the year 2021- 2022. The objective of the study was molecular characterization and cloning of coat protein gene of Tomato leaf curl virus (ToLCV) infecting tomato and its phylogenetic analysis with other members of Geminiviridae. Symptomatology of virus infected tomato plants was studied. Infected plants were collected from different regions of Vellayani campus, Kerala Agricultural University and the virus was maintained by graft inoculation. The virus was serologically characterized using DAS-ELISA (Double Antibody Sandwich-ELISA) and DIBA (Dot Immuno Binding Assay) using ToLCNDV (Tomato leaf curl New Delhi virus) antisera and higher viral titer was shown by plants with severe reduction in leaf size (8-fold absorbance value). Genomic DNA was extracted from the infected samples, and coat protein (CP) gene-specific primers were used for molecular detection. PCR yielded expected amplicon size of 500bp and 600bp and were sequenced. The BLAST analysis of the sequence revealed similarities with Tomato leaf curl Palampur Virus (ToLCPMV) and ToLCNDV of 95% and 94%, respectively. Both bipartite virus and monopartite virus with a satellite DNA were detected by rolling circle amplification (RCA), which was followed by Restriction Fragment Length Polymorphism (RFLP). PCR was done using RCA fragments as template with DNA A specific primers and the amplicons obtained were cloned. Sequencing of cloned genes followed by BLAST analysis showed 98.61 per cent similarities with ToLCPMV isolates. Phylogenetic analysis of partial DNA A gene of Vellayani isolate with other strains of ToLCV showed close relation to ToLCPMV infecting cucurbits. Comparitive analysis of partial DNA A sequence with other viruses in genera Begomovirus showed closest relation with Melon leaf curl virus and Cotton leaf curl virus from Pakistan. Comparison of partial amino acid sequence of CP of ToLCV Vellayani isolates with other mono and bipartite begomoviruses revealed a maximum of 99.11 per cent similarity with pre coat protein genes of ToLCPMV. According to the current investigation, the Begomovirus that infects tomatoes in the Vellayani region is bipartite as well as monopartite with a satellite DNA. The CP and DNA A genome phylogenetic analyses revealed a strong relationship between the Vellayani isolate and the ToLCPMV isolates infecting cucurbitsItem In-vitro chemotherapy for inducing tolerance towards Tomato leaf curl New Delhi virus (ToLCNDV)in bitter gourd (Momordica charanita L.)(Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, 2023-12-12) Adarsh, M V.; Smitha BhasiThe study entitled “In-vitro chemotherapy for inducing tolerance towards Tomato Leaf Curl New Delhi virus in bitter gourd (Momordica charantia L.)” was conducted at the Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram during 2022-23. The objective of the study was the evaluation of in-vitro treatment with antiviral compounds, viz., Ribavirin, Virus–Ex and extracts of Bougainvillea spectabilis for inducing tolerance towards Begomovirus- ToLCNDV in bitter gourd (Momordica charantia L.). The explants namely seed of bitter gourd variety Preethi was collected from, Instructional farm, College of agriculture, Vellayani. Seeds were subjected to hot water treatment at 55°C for 15 minutes, followed by washing in 0.2% bavistin for 15 minutes and kept in dark for inducing germination. 91.2% germination was noticed in 3 days. The germinated seeds were grown in Murashige and Skoog (MS) medium supplemented with 2ppm BA and 20ppm concentration of various antiviral compounds viz., Ribavirin (Treatment 1), Virus-Ex (Treatment 2), extract of Bougainvillea spectabilis (Treatment 3) along with control. Percentage of culture establishment was found to be maximum (65.25%) in ribavirin treated plants followed by virus ex and bougainvillea. 20 days old in-vitro grown plants were hardened in coir pith compost and the biometric observations were taken and compared. Significant increase in plant height and number of leaves over control was noticed in treatment using ribavirin and virus ex respectively. DNA was isolated from 20 day old (hardened) treated and control plants prior to whitefly transmission and were confirmed to be virus free using PCR. Whitefly mediated artificial inoculation of virus in hardened plants (25 day old) was done and the plants were maintained inside insect proof cage for symptom development. Symptoms were observed in control plants after 10 days of whitefly transmission. Further PCR was performed to confirm the presence/absence of Begomovirus. Absence of virus in treatments with ribavirin and virus ex were 59 confirmed in PCR. Treatment using extracts of bougainvillea did not show any symptoms but confirmed positive in molecular detection. The control plants which developed symptoms was found positive in PCR. Peroxidase assay (Lobenstein and Linsey method) showed maximum increase in activity (3-fold) in plants treated with ribavirin followed by extracts of bougainvillea (2-fold) three days after whitefly transmission. Biometric observations on 35th day viz., plant height, number of leaves and leaf area were found higher in all the treatments compared to control, out of which treatment using ribavirin was found to be highly significant for all the parameters. To conclude, the present study in bitter gourd could confirm the antiviral activity of ribavirin and virus ex towards Begomovirus through whitefly mediated transmission test and molecular detection using PCR