MSc

The present study entitled “Diversity of begomoviruses infecting major vegetable crops” was carried out at College of Agriculture, Vellayani during 2017-2019 with the objective of immunomolecular detection and genetic analysis of the begomoviruses viz., Bhendi yellow vein mosaic virus (BYVMV), Tomato leaf curl virus (ToLCV) and Pumpkin yellow vein mosaic virus (PYVMV). Plant samples showing the typical symptoms of bhendi yellow vein mosaic disease (BYVMD), tomato leaf curl disease (ToLCD) and pumpkin yellow vein mosaic disease (PYVMD) were collected from three taluks each of Thiruvananthapuram (Neyyattinkara, Nedumangad and Chirayinkeezhu) and Kollam (Kollam, Kottarakkara and Punalur) districts of Kerala during March-November, 2018. High disease incidence (DI) was observed in all the surveyed locations. High vulnerability index (V.I.) was recorded in BYVMD (86.6) and ToLCD (85) in Mukhathala of Kollam. Since PYVMD was observed only at Pappanchani of Thiruvananthapuram and Mukhathala of Kollam, the survey was extended to Tavanur of Malappuram and Pilicode of Kasaragod districts. High V.I. of PYVMD (72.8) was recorded in Tavanur.
Symptomatology of begomovirus infected crops revealed typical symptoms of BYVMD, ToLCD and PYVMD; like vein clearing, leaf curl and stunted growth. Vellayani and Neyyattinkara isolates of ToLCV produced additional symptoms viz., purplish discoloration, mottling with yellowing and cupping of leaves in tomato.
All the three viruses were transmitted by whiteflies (Bemisia tabaci Gen.) and through grafting whereas none of the viruses were seed transmitted. Cross infection of these three viruses using whiteflies did not produce infection in any one of the crops studied.
Immunodetection of the three viruses was confirmed using polyclonal antisera of other begomoviruses viz. Tomato leaf curl New Delhi virus (ToLCNDV) and Sri Lankan cassava mosaic virus (SLCMV) through TAS (Triple antibody sandwich)-ELISA and DAS (Double antibody sandwich)-ELISA and dot immunobinding assay (DIBA) in infected leaf samples whereas the seeds from infected plants did not produce any positive reaction.
The quality and quantity of isolated DNA using Qiagen’s DNeasy Plant DNA extraction Mini Kit was assessed using Bio-Spectrophotometer. The PCR based molecular detection using DENG and AV/AC primers specific to coat protein (CP) of begomovirus could yield amplicons of 520 bp and 575 bp respectively which confirmed the presence of the viruses in the infected leaf samples; whereas the seeds collected from infected plants did not yield any amplicons confirming the viruses were not seed transmitted.
The complete sequence of CP gene of the different isolates revealed maximum similarity of BYVMV isolates to Okra enation leaf curl virus (>99 %), ToLCV isolates to Tomato leaf curl Kerala virus (>99 %) and ToLCNDV (>96 %) but PYVMV isolates were more similar to Squash leaf curl China virus (SLCCNV) (>98 %). Further, phylogenetic analysis of the isolates of the three viruses revealed that they were closely related to each other as they were clustered together. But ToLCV isolates of Vellayani and Neyyattinkara were found to be closely related to isolates of PYVMV. Punalur and Tavanur isolates of ToLCV were more closely related to other BYVMV isolates. Genetic distance analysis among isolates of the viruses revealed that maximum variability was observed between Punalur isolate of BYVMV and Pappanchani isolate of PYVMV (0.528) while 100 per cent similarity was observed between ToLCV isolates of Punalur and Tavanur.
The full genome amplification and sequencing of Vellayani isolate BYVMV and ToLCV; and Mukhathala isolate of PYVMV were performed using three sets of overlapping degenerative primers. The blast analysis of full genome revealed that Vellayani isolate of BYVMV was closely related to other BYVMV (95.04 %); Vellayani isolate of ToLCV to ToLCNDV (92.89 %); and Mukhathala isolate of PYVMV to SLCCNV (98.09 %).
Phylogenetic analysis of the isolates of three viruses with other isolates of similar viruses from neighboring state and countries retrieved from NCBI database revealed that Vellayani isolates of BYVMV was closely related to other BYVMV isolates as they were grouped in a clad while Vellayani isolate of ToLCNDV was distantly related to other ToLCNDV isolates. ToLCNDV isolates were clustered in both BYVMV and SLCCNV suggesting that ToLCNDV may be the connecting link between BYVMV and SLCCNV which were grouped in different clades. The genetic distance analysis confirmed least divergence of evolution between ToLCNDV-Vellayani and SLCCNV-Mukhathala and greater divergence of evolution between BYVMV-Vellayani and SLCCNV-Mukhathala isolates. Among the three viruses, ToLCNDV is more related to SLCCNV compared to BYVMV. Therefore, phylogenetic analysis using whole genome is more reliable to decipher genetic relationship and diversity among viruses.
Specific primers for detection and identification of BYVMV, ToLCNDV and SLCCNV were developed through in silico analysis of whole genome sequences. The present study clearly demonstrates that PYVMD is caused by Squash leaf curl China virus.