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 S20, 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
Effect of the continuous light exposure with white fluorescent lamp on sporulation of Alternaria kikuchiana Tanaka depended more or less on the kinds of media. On V-8 juice medium, pear leaf juice medium and dry apricot-V-8 juice medium, the spore production of the fungus were increased by the light exposure. Stimulately or inhibitory effect of spore production of the fungus on the dry apricot juice medium, which produced the most abundant spores among the media in darkness, were not observed when exposed to continuous light by white fluorescent lamp. Effect of the light exposure on sporulation of the fungus depend also on temperature and the distance from lamp to mycellium. In high temperature and long distance from lamp to mycellium, spore production of the fungus were decreased and the good growth of airial mycellium. On the other hand, in low temperature and short distance from lamp, the spore production of the fungus were increased. The spore formation of the fungus stimulated with black light blue fluorescent lamp, pure blue fluorescent lamp and sterile lamp among the lampes, in low temperature and short distance from the lampes on the all media except dry apricot juice medium. The spore formation of the fungus stimulated when exposed to continuous light with the glass filters which transmitted mainly 340mμ and 365mμ light in low temperature and long or short distance from the lampes on dry apricot-V-8 juice medium. On the other hand, spore formation of the fungus inhibited by 400mμ-500mμ, especially 450mμ light.
A yellow ringspot disease of aster was noticed in Saitama Prefecture for the first time in 1967. The symptoms were brilliant yellow rings, which were identical with those of aster ringspot disease described by Anderson (1954). From these asters, a virus was isolated by juice inoculation, and was identified to be tobacco rattle virus as designated by Corbett (1967). The virus in electronmicroscopy by dip method showed a bimodal length distribution of rigid rod-like particles. The shorter particles had a normal length of 70-80mμ; the longer particles a normal length of 170-180mμ. It was found that the host plants of the virus were aster, tobacco, cucumber, plantago, torenia, bean, cowpea, spinach, etc. The symptoms on these plants were similar to those described for tobacco rattle virus by many workers. The virus was not transmitted by Myzus persicae, nor through seed, but soil-transmission was obvious. Thermal inactivation point in crude juice was 70-75°C (10min.), dilution end point 1:106, and longevity in vitro 91-98 days at 20°C. This aster isolate of tobacco rattle virus causes different symptoms on aster from those caused by HSN isolate of the same virus isolated from tobacco (Tomaru et al., 1967): the former produces characteristic ringspot, while the latter top necrosis and veinal necrosis. Many nematodes belonging to genus Trichodorus were collected from soils around roots of the infected aster in fields with Seinhorst elutrator and sieving techniques. The species was identified to be Trichodorus minor Colbran, 1956 (≈T. christiei Allen, 1957). Experimental transmission of the virus by T. minor was successfully made, although a comparatively low infection rate (20 out of 225 seedlings tested) was secured. Several plant species were grown in infected fields, and two months later virus isolation test from the plant roots were carried out. The virus was recovered from aster and spinach, but not from pea, cabbage, corn, etc.
Citrinin, which was isolated from cultures of Penicillium sp. AK-019, inhibited local lesion formation on detached leaves of Nicotiana glutinosa infected with tobacco mosaic virus (TMV). Peroxidase and polyphenoloxidase activities in infected leaves decreased with the prolonged times of treatments with 100ppm citrinin. Five hour's treatment with citrinin inhibited peroxidase activity, but did not so much polyphenoloxidase. Both oxidase activities increased simultaneously with lesion appearance. Citrinin reduced most of the oxidase activities at early infection stages, reaching a maximum 48 hours after inoculation, then the rate of inhibition decreased. Both oxidase activities in infected leaves were recovered by the addition of 250ppm Fecitrate or 100ppm CuSO4 with citrinin. Inhibition by citrinin of local lesions formation and the oxidases activities is discussed.
1) A virus isolated from mosaic carrot plants (Daucus carota) from Nagano Prefecture was identified as celery mosaic virus. The symptoms on carrot were mainly mosaic, but a few plants showed fern leaf symptoms. The virus was transmitted by juice inoculation, and also by Myzus persicae and Semiaphis heraclei in a non-persistent manner. By mechanical inoculation, 8 species in 3 families became infected out of 22 species in 7 families tested. The infected plants were carrot, celery, coriander, parsley, Nicotiana clevelandii, Chenopodium amaranticoler, and C. quinoa. The virus in vitro withstood heating at 40°C for 10 minutes, dilution to 640, and 6 hours at 20°C. In electronmicroscopy using dip method, long flexuous particles of about 750-800mμ in length were observed. This is first record of celery mosaic virus in Japan. 2) Viruses isolated from mosaic carrot plants from Chiba and Hiratsuka were identified as cucumber mosaic virus. The host range and symptoms indicated that it belonged to ordinary strain of the same virus. In agar gel diffusion tests, the virus reacted specifically with an antiserum to cucumber mosaic virus. The virus was readily transmitted from carrot to tobacco, C. amaranticolor, etc. by juice inoculation and from carrot to carrot by Myzus persicae, although some isolates were difficult to infect carrot by juice inoculation.
In the present paper fine structures of chloroplasts in cells of rice plants and their ultrastructural changes due to the infection of Cochliobolus miyabeanus were described. In a longisection of healthy leaves of rice plants, chloroplasts were enveloped with a double membrane, and the lamellar system in chloroplasts showed a distinct compartmented grana stacks. Fifty hours after the inoculation of this fungus, chloroplasts in cells swelled about 1.5-2 times, and they were oblong to spherical in shape, having partially disintegrated double membrane of the envelope. The outermost sac of grana stacks of chloroplasts was firstly swollen into vesicle-like structure, with no matrix detectable in the interior. In chloroplasts adjacent to hyphae the disintegration of lamellar system may first take place in the grana stacks near the envelope. It gradually spread to every stack of grana and finally they degenerated entirely, forming abundant small vesicles, 0.1-0.5μ in diameter.
Six isolates of ectoparasitic bacteria having different host ranges were collected from paddy field soil and irrigation water in Japan, by using Xanthomonas oryzae, the pathogen of bacterial leaf blight of rice, as indicator. The ectoparasitic bacteria produced plaques which resembled in appearance to those due to bacteriophages, 2 to 4 days after plating on the bacterial lawn. The plaques enlarged gradually showing somewhat irregular circumference, and finally covered the entire surface of the plates. The size of the plaques showed much variation. The average size seemed to differ according to incubation conditions, but not to isolates. In any of the six isolates, a single plaque usually comprised two different type of cells. The Vibrio form cells were 0.75 to 1.87μ in length and 0.28 to 0.34μ in thickness, while spherical cells were 0.28 to 0.34μ in diameter. Each cell possessed a single thick polar flagellum. The ectoparasitic bacteria attacked 5 to 30 species of Gram-negative bacteria out of 64 tested, but none of the tested Gram-positive or Gram-variable bacteria. All 45 tested isolates of X. oryzae were attacked by any of the six isolates. The ectoparasitic bacteria were identified from their characteristics as Bdellovibrio bacteriovorus Stolp and Starr.
Thirteen isolates of Xanthomonas oryzae and six phage isolates collected from the various localities of West Malaysia were employed to elucidate some of their properties in comparison with Japanese isolates. The phages are almost completely inactivated at 50-55°C. The phage particles of all the isolates are tadpole-shaped. They are composed of a spherical head 63mμ in diameter, and a tail 156-176mμ in length and 15mμ in width. The Malaysian phage isolates show differences in the host range among them. From serological observations it appears that the Malaysian phages belong to the OP1 group. The latent period for Malaysian phage isolates is 40-45min. On average 13-45 phage particles, depending on the phage isolates, are finally liberated from a single bacterial cell. The bacterial isolates tested are less virulent to rice varieties in comparison to Japanese isolates, and therefore, are placed in the intermediate or less virulent group.