Optimization of a more efficient protocol for mechanical inoculation for watermelon bud necrosis orthotospovirus and its validation with different watermelon genotypes

Watermelon bud necrosis orthotospovirus (WBNV) is one of the emerging and most serious pathogens affecting watermelon cultivation in India. In the present study, the effect of different inoculation methods and buffer formulation on virus transmission was studied and validated in 17 watermelon genotypes. Among various inoculation methods used, maximum transmission efficiency 60-100% was obtained in 4-10 days post-inoculation (DPI) with pin-pricking and rubbing together, followed by rubbing (85-100%; 20-25 DPI) and pin-pricking (10-32%; 10-25 DPI) alone with the sodium buffer (PBS) having pH 7.0 supplemented with sodium sulfite (SS) and 2-mercaptoethanol (2-ME). Similarly, among the various abrasives used, maximum transmission efficiency 82-100% was obtained at 10-35 DPI with Celite and Carborundum together, followed by Celite (64-100%; 10-20 DPI) and Carborundum (72-100%; 20-40 DPI) alone. Among various additives used, maximum transmission efficiency 82-100% was obtained in 10 DPI with PBS supplemented with SS and 2-ME followed by PBS supplemented with SS alone (85-100%; 20-40 DPI) and PBS with no additive (76-85%; 20-35 DPI). The 15-20 days old watermelon plants were found suitable for the efficient mechanical transmission (100%) and to obtain maximum virus titre (Absorbance at 405 nm) i.e. 1.52-2.50 at 8-25 DPI. The 21-60% and 100% transmission was obtained at 4 and 10 DPI, respectively using the same buffer composition as mentioned above. For validation of optimized sap inoculation protocol for WBNV, 17 watermelon genotypes were inoculated under glasshouse conditions and the percent transmission ranged from 80 to 100%. Besides symptomatology, virus transmission was further confirmed by DAC-ELISA, RT-PCR and sequencing of the partial N gene. The O. D. at 405 nm ranged from 0.38 to 0.9 in naturally infected watermelon plants whereas in the glasshouse inoculated watermelon plants it was ranged from 0.30 to 0.73 at 15 DPI. This is the first effort on optimization of more efficient sap inoculation protocol for WBNV and it validated to 17 watermelon genotypes.