Standardization of mechanical sap inoculation technique and in vitro longevity studies of Peanut bud necrosis virus (PBNV) in groundnut and cowpea under protected conditions

Abstract

Peanut bud necrosis disease, caused by Peanut bud necrosis virus (PBNV), is one of the most destructive viral diseases affecting Arachis hypogaea in India and other parts of South and Southeast Asia. The present study was conducted to standardize a mechanical sap inoculation technique for PBNV using two indicator hosts, groundnut cv. TMV-2 and cowpea cv. C-152, under protected glasshouse conditions. Virus inoculum was prepared from symptomatic groundnut leaves using 0.05 M phosphate buffer and mechanically inoculated onto seedlings dusted with carborundum. Different sap dilution ratios and seedling ages were evaluated to determine optimal conditions for successful transmission. In cowpea, typical symptoms including chlorotic spots, necrotic ring spots and systemic crinkling appeared within 4-8 days after inoculation. The highest infection rate was obtained with a 1:1 sap dilution on 7-day-old seedlings, while higher dilutions failed to produce symptoms. In groundnut, chlorotic and necrotic ring spots appeared at 7-8 days post inoculation, with the highest symptom expression observed at a 1:10 dilution on 7-9-day-old seedlings. The in vitro longevity study indicated that infected leaf tissues retained infectivity up to 10 days at 4-6 °C and up to 60 days at −80 °C. The standardized protocol developed in this study provides a reliable bioassay system for PBNV, facilitating plant-virus interaction studies and resistance breeding programs.