Characterization of bud necrosis virus infecting tomato

Abstract

Studies were conducted to characterize the tospovirus causing the bud necrosis disease of tomato in Kerala. The characteristic symptoms observed were, necrotic ring spots on leaves and severe necrosis, death of the emerging buds, stem necrosis and concentric yellow colored rings on fruits. Host range studies were conducted and the virus was found to infect members of family Chenopodiaceae, Solanaceae, Fabaceae, and Cucurbitaceae. The virus was efficiently transmitted by mechanical means using 0.01 M phosphate buffer (pH 7.2) containing 0.1 per cent 2-mercaptoethanol. No seed transmission was recorded. However successful graft transmission was observed. The virus recorded a DEP in the range of 10-3 to 10- 4 , TIP of 50 oC to 55 oC, and LIV of 8 h at room temperature (28±2oC) and 24 h at 80C. The carbohydrate levels in inoculated plants were lower compared to the uninoculated tomato plants. Similarly the content of chlorophyll a, b and total chlorophyll were also lower in inoculated tomato plants. The phenol content was found to be more in inoculated plants. There was an increase in protein content in inoculated plants compared to the healthy plants. In case of inoculated plants the activity of the defense related enzymes were higher than the control plants. Protein profile of tospovirus infected tomato plants using SDS - PAGE showed three extra novel proteins with molecular weights of 28, 15 and 12 kDa respectively. Isozyme analysis of PPO produced three isoforms in both healthy and inoculated plants with relative mobility (Rm) values of 0.60 and 0.77. The activity of the two isoforms were more in the inoculated plants. The virus causing bud necrosis disease in tomato was confirmed as tospo virus by serological analysis such as ELISA and DIBA. The virus isolate showed close relationship with WSMV. The virus was also detected using PCR and an amplicon of size 800 bp was obtained using primer specific to tospovirus. The meristem from the infected tomato plants were regenerated into plantlets and were tested for the presence of the virus by subjecting it to DAC- ELISA. The absorbance of the plantlet regenerated from healthy and infected meristem were found to be 0.13 and 0.12 respectively which was on par with the healthy field sample but much lower than that of the infected field sample which was used as the positive control which recorded an absorbance of 0.81.