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Rice Yellow Mottle Virus (RYMV)
1. The morphology of Rice yellow mottle virus
2. The virus is stable in vitro and easy to purify (Bakker, 1970, 1974). Fresh or deep-frozen young rice leaves, harvested 10-12 days after infection are best for purification.
3. Homogenize small pieces in 0.1 M phosphate buffer pH 5.0 + 0.2% 2-mercaptoethanol (1 g of leaves/20 ml of buffer). Squeeze through cloth. Emulsify extract with 0.5 vol chloroform (5 min) and centrifuge at low speed.
4. To each 100 ml of the aqueous phase add 20 g (NH4)2SO4 while stirring, and centrifuge at low speed. Repeat this procedure with the supernatant fluid but this time retain the pellets after low speed centrifugation.
5. Resuspend them in a small amount of buffer pH 5.0 and dialyse against the same buffer. Clarify by one cycle of differential centrifugation (20 min at 6600 g, 100 min at 78,500 g).
6. Suspend the sediment from high speed centrifugation in 0.01 M phosphate buffer pH 7.0. Dialyse against the same buffer and centrifuge at low speed.
7. Work at room temperature during precipitation of the virus with (NH4)2SO4 and during the first dialysis, otherwise at 4°C. Yield is about 1 mg virus/g of leaves.