Abstract
A purification procedure for watermelon mosaic virus (WMV) was developed. After initial clarification of expressed infected pumpkin leaf sap with 15% carbon tetrachloride in 0.3M potassium phosphate solution containing 0.01M DIECA, 0.1% ME, pH 8.8, addition of 2% Triton X-100 to the extract was effective for the removal of green host materials. WMV was concentrated by two cycles of differential centrifugation utilizing 20% sucrose cushion and further purified by rate zonal sucrose density gradient centrifugation. The ultraviolet absorption spectrum of the purified WMV preparation was typical of other potyviruses with a maximum at 260nm and a minimum at 246nm. The yield of purified WMV was 2 to 3.5mg per 100g of infected pumpkin leaves. Precipitation with polyethylene glycol MW 6, 000 (PEG) was not advisable, because of close aggregation of WMV particles, difficulty of subsequent dispersion, and greatly reduced yield. Enzyme-linked immunosorbent assay was found to be a rapid and valuable technique for comparing the various methods in each step of the purification procedure.
