Purification of Some Legume Carlaviruses

VEERISETTY, V., and M. K. BRAKKE. 1978. Purification of some legume carla viruses. Phytopathology 68:59-64. Purification schemes were developed which yielded 0.7 to single nucleoprotein component in rate-zonal sucrose and 1.0 mg of alfalfa latent virus (ALV) and pea streak virus equilibrium cesium chloride density gradient centrifugation. (PSV) and 0.1 to 0.3 mg of red clover vein mosaic virus The virus preparations did not contain detectable impurities. (RCVMV) per gram pea cullivar Lincoln ) plant tissue The ALV, had a sedimentation coefficient of 161 + 1.5 S, a (excluding roots). The freezing of the tissue and the use of an value similar to other members of the carlavirus group. Both appropriate extraction buffer were crucial. Virus from sap ALV and PSV multiplied and accumulated when they were was precipitated by 6%(w/v) polyethylene glycol (PEG, MW inoculated to the same host plant, thus supporting the 6,000) and concentrated by two cycles of differential previous evidence that they are indeed different viruses. centrifugation. Partially purified virus preparations had a Viruses of the carnation latent virus group characterization studies are undertaken. In this report we (carlaviruses) (6) have been difficult to purify in sufficient describe purification methods that gave higher yields of quantity because of their low concentration in plant virus with no detectable contamination by nonviral RNA extracts and their tendency to aggregate (5, 7, 10). Alfalfa or protein. latent virus (ALV) was isolated from alfalfa and was established as a new member of the carlavirus group (14). MATERIALS AND METHODS In repeated trials using the published purification procedures for other legume carlaviruses, we failed to Viruses and host plants.-Alfalfa latent virus (ALV) obtain appreciable quantities of ALV. However, we was isolated from alfalfa (Medicago sativa L.) and later found numerous virus particles of ALV and also pea maintained in broad bean (Viciafaba L.) and pea (Pisum streak virus (PSV) and red clover vein mosaic virus sativum L. 'Lincoln') (14). Initially the virus was purified (RCVMV), other legume carlaviruses in leaf-dip from broad bean leaf tissue but later from pea cultivar preparations of infected Lincoln peas, which suggested Lincoln because the whole pea plant (excluding roots) that most of the virus was not extracted by the usual could be extracted. The isolates of PSV and RCVMV buffers during purification. It also indicated that virus is (ATCC/PV 110) were gifts from D. J. Hagedorn and M. either adsorbed to the host components or aggregated to A. Khan, respectively, University of Wisconsin, Madison, itself in extracts of infected plants, and requires special and also were maintained in Lincoln pea. Tobacco mosaic treatments and extraction buffers for its release. virus (TMV) and southern bean mosaic virus (SBMV) Therefore, purification procedures were investigated in strains were those maintained in this laboratory. an attempt to improve the yields. The inoculated and systemically infected leaves of Physico-chemical characterization of flexuous viruses broad bean showing typical symptoms (11) were is useful for their classification and also for determining harvested 15 days after inoculation. Whole pea plants, the virus structure (11, 12, 13). However, the viruses must excluding roots, were harvested 20 days after inoculation. be obtained in a pure state and their purity should be Since ALV was symptomless in peas, usually a single assessed by reliable methods before any chemical plant was checked by leaf dip electron microscopy. If 0032-949X/78/000009 $03.00/0 virus was found, it was assumed that all inoculated plants Copyright © 1978 The American Phytopathological Society, 3340 were infected. They were harvested and stored at -20 C Pilot Knob Road, St. Paul, MN 55121. All rights reserved, before purification. 59 Published in PHYTOPATHOLOGY 68 (1978), pp. 59-64.