Some Properties of Chrysanthemum Mild Mottle Virus, and Comparision of This Virus with Cucumber Mosaic Virus

Abstract

A virus which is widespread in chrysanthemum in Japan has been identified by Inoue et al. with tomato aspermy virus. In the present study, this virus was found to be serologically unrelated to cucumber mosaic virus (CMV), closely resembling Lawson's chrysanthemum virus. It is considered adequate to separate this virus from tomato aspermy virus of Blencowe type, as suggested by Lawson, and a tentative name chrysanthemum mild mottle virus (CMMV) is proposed.
The virus was purified from inoculated leaves of tobacco (Ky-57) by the following procedure: young tobacco plants were inoculated with leaf extract of systemically infected tobacco, ground in 0.1M phosphate buffer (P.B.) containing 0.02M Na₂SO₃. The inoculated leaves were harvested 3-5 days after inoculation. The leaves were ground in 1 vol. each of 0.15M Na₂HPO₄ containing 0.01M Na-EDTA and 0.1% thioglycollic acid (pH 7.6), and chloroform-butanol mixture (9:1 vol.), subjected to two cycles of differential centrifugation (9, 000×g15min., and 80, 000×g 150min. or 90, 000×g90min.), and then to density gradient centrifugation in 10-40% sucrose at 24, 000rpm for 180-210min., at 4-6°C. 0.05M P.B. containing 0.005M Na-EDTA was used to resuspend the pellets. The yield of the final preparation was about 30-40mg per 100g of inoculated leaves. For comparision, CMV-Y (yellow strain) was purified by a procedure modified from Scott and Takanami et al. 0.5-1 vol. of 0.005M borate buffer containing 0.005M Na-EDTA pH 8.6-8.9 was used to prepare leaf extract, instead of dialysis (Scott) or treatment with Sephadex (Takanami et al.). Borate buffer containing Na-EDTA was used as the resuspending medium. The yield was about 60mg per 100g of inoculated leaves.
The absorbancy of CMMV preparation at 260mμ was 5.6cm²/mg. The UV absorption spectrum between 230 and 300mμ was typical of a nucleoprotein (max. at 260-261, min. at 241-242mμ), and showed 260/280=1.70-1.78 and max./min.=1.46-1.50. CMMV resembles CMV-Y in the UV absorption spectrum, but the ratio of 260/280 showed a slightly larger value in CMMV than in CMV-Y. CMMV preparation yielded about 100 lesions per cowpea leaf at 4.5μg/ml, and about 200 lesions per leaf of Chenopodium amaranticolor at 1.8μg/ml.
The purified virus was treated by phenol saturated with 0.1M P.B. containing 0.001M Na-EDTA (pH 7.2) and 5mg/ml bentonite. Sodium-acetate and ethanol were added to the aqueous phase, and the pellet was resuspended in 0.01M P.B. pH 7.2, and ethanol was removed under reduced pressure. Infectivity of the preparation was tested before and after the addition of 0.01 or 0.001μg/ml of RNase-A. The non-RNase preparation yielded more than 250 lesions per cowpea leaf, while the RNase-treated preparation completely lost infectivity.
CMMV preparation was examined under electronmicroscope, after being fixed in 2% neutralized-formalin and stained with 1% phosphotungstic acid, or shadowed with Pt-Pd. The virus particles were found to be spherical and about 25-30mμ in diameter (Fig. 2). The sedimentation coefficient of the virus was determined by ultracentrifugation using schlieren optical system, at different virus concentrations (1.2-3.6mg/ml). The value of the sedimentation coefficient of the virus extrapolated to zero concentration was S_{20, w}=97. The mixture of CMMV and CMV-Y showed a single symmetrical schlieren peak (Fig. 3).
CMMV preparation (25μg/ml virus dissolved in 0.05M P.B. pH 7.8), completely lost infectivity by 10min. treatment at 55°C, while there was a considerable loss of infectivity by treatment at 50°C (Table 1).
Serological comparisons between CMMV and CMV-Y were made in agar gel doublediffusion tests at 4-8°C (1% agar, 0.85% NaCl, 0.0025M Na-EDTA, 0.01% sodium azide, and 0.02M P.B., pH 7.2-7.4). CMMV (0.5-5mg/ml) and CMV-Y