The effect of polyoxin on spore germination of A. kikuchiana Tanaka was investigated using transmission and scanning electron microscopes. The entire spore was covered by two-layered cell wall, having a rough surface. During the initial stages of germination, the new cell wall layer being continuous with the spore inner wall layer was formed as the cell wall of germ tube. When the spore was treated with polyoxin, the entire germ tube swelled forming the bulbous structure and the outer layer occurred around the germ tube soon after the germ tube emerged. If polyoxin was absent, such an outer layer did not appear during the incubation period.
Longevity and density of saprophytic survival of Xanthomonas citri (Hasse) Dowson was examined with the leaf infiltration technique on various weeds, dead plant leaves or straws, soils and roots of Natsudaidai (Citrus natsudaidai). The infestation of the materials with X. citri was made by dipping the plant materials into bacterial suspensions with the concentration of approximately 108/ml, or by pouring them in soil samples. 1. Infested with bacteria in early spring, the bacterium survived for 1 to 3 months on weeds, dead rice straw and in soils. With the infestation in autum, however, it survived for 7 months on either intact parts or dead leaves of Zoysia japonica and Vetiveria zizanioides, for 5 months in soils which were kept air drying (Fig. 1).2. The patterns of the seasonal population changes differed depending on the kinds of materials. The population of X. citri maintained for several months the level of about 102 to 103 bacterial cells per gram samples after showing quick decrease at the beginning on intact Zoysia japonica and dead rice straw (Fig. 2, 3). In wet soils kept outdoor, the population quickly decreased in almost linear ways, whereas the rather low level of the population was maintained for the period of 8 months in soils which were left under greenhouse and kept drying (Fig. 4). The survival population on the root surface of C. natsudaidai was characterized by no marked decrease at the beginning and by maintaining the high level for 10 months (Fig. 7). 3. Seasonal population changes of X. citri surviving saprophytically on various materials were compared among three phage-types A, B and C. As shown in Fig. 5-8, the population of phage-type C on Zoysia japonica and rice straw became 10 to 100 times higher than those of the other two types in late autumn through winter season, whereas such clear differences were not recognized in soils. 4. The possibility was discussed from the above evidences as to the existence of two different ecotypes, i.e., the parasitic ecotype, phage-type A and B, and the saprophytic one, phage-type C. Although the former can saprophytically survive under natural conditions, its major habitat would be the diseased tissues of the citrus trees and it perpetuates causing repeated infections on the host plants. On the other hand, the saprophytic ecotype may survive mainly on non-host vegetations without causing severe infections on citrus plants under natural conditions.
The rice stripe virus, a circulative and propagative virus transmitted by Laodelphax striatellus, has been considered to be a spherical particle of about 30nm in diameter. However, branched filamentous particles of, about 400nm in total length and about 8nm in width were found in rice leaves infected with the virus by the direct negative staining method. These particles are quite different in morphology from any other virus particles so far reported, but have never been found in healthy rice leaves. These particles were purified from the virus-infected rice leaves by 10% sucrose cushion centrifugation, chloroform treatment followed by Triton X-100 treatment, sucrose density gradient centrifugation, and finally by D2O sucrose equilibrium centrifugation. The purified preparation containing a large number of the branched filamentous particles but no spherical particles showed to retain infectivity in inoculation tests by microinjections to L. striatellus, and also showed to be a nucleoprotein by UV absorption spectrum. From these results it is concluded that the rice stripe virus is a branched filamentous particle. When observed at high magnifications under the electron microscope, the particle is a slender filament, 3nm in width, of circular structure which usually takes the form of a super-coiled helix. The pitch of the secondary helix is 6nm. No uniformity is found in the number of branches, and sometimes some parts of the particle show loop-like pattern due to partial loosening of the super-coiled helix.
In the course of research studies to detect the antiviral substances for tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV), it was found that some salts of alginic acid have a high inhibitory activity against TMV infection.The methods used are the inoculation of mixture of chemical and virus, and the inoculation of virus after application of chemicals on the leaf surface. Na-, K-, NH3- and triethanolamin-salt of alginic acid showed the same degree of inhibitory activity, 80-100 percent inhibition, in the tests using 0.1-1 percent concentration of chemicals and TMV-Nicotiana glutinosa as the test combination. Equally high inhibitory activity was observed also in the combination of TMV-French bean and TMV-Xanthi nc tobacco, by the test using Na alginate. In TMV-Chenopodium amaranticolor, however, the activity was lower than those in the other combinations mentioned above. The mixture of Na alginate and casein (AC) showed higher inhibitory activity and retained the activity longer than Na alginate alone after application to N. glutinosa leaves. AC inhibited about 30 percent of the transmission of CMV to tobacco plant by green peach aphid in the green house experiment. Mechanism of the inhibitory activity of alginic acid is not clear yet. However, following data were obtained; 1) When Na alginate was applied to the undersurface of leaf and TMV was inoculated on the opposite surface, no effect was observed. Accordingly, there was no sign of translocation of effective substances into the leaf tissues. 2) When mixed solution of TMV and Na alginate was centrifuged at low speed, most infectivity was found in the precipitation. This fact shows that AC has the effect to aggregate TMV particles. 3) No inhibitory activity was found when AC was applied after inoculation, except with in 5 minutes after inoculation. 4) Infection of Bright Yellow tobacco which can be infected systemically with TMV was also inhibited by AC. This fact indicates the effect of AC is not the prevention of local lesion formation but the inhibition of infection itself. The results of further tests including practical application in the fields shall be reported eleswhere.
In order to obtain more conclusive results on tissue degradation by pectolytic enzyme, the present investigation was conducted to confirm the properties of pectin-like polysaccharides liberated from cell wall-materials by the action of endo-PTE. Three hundred mg of radish fiber was incubated with the purified enzyme (2.0 unit) at 30C for 90 minutes in 0.033M tris-HCl buffer, pH8.0. The maceration mixture was fractionated into 6 subfractions of cell wall-materials according to the method of McColloch22). In contrast to non-macerated radish fiber, dil. HCl-soluble carbohydrate subfraction (F-III) was remarkably decreased with a visible increase of water-soluble subfraction (F-I) during the enzymatic maceration. It is concluded, therefore, most of water-soluble carbohydrates in macerating mixture were mainly liberated from cell wall-materials extractable with dil. HCl solution, corresponding to so-called protopectin. Both F-I and F-III subfractions were heterogeneous polysaccharide and separated further into 6 fractions by DEAE-cellulose column chromatography. When each fraction of F-I which originated from macerating mixture was hydrolyzed with 1N H2SO4 at 100C for 20 hours, neutral sugars such as galactose, arabinose, rhamnose and xylose were also detected on paper chromatograms, in addition to galacturonic acid. Major components of F-I subfraction, which eluted from the column with 0.2, 0.3 and 0.4M of acetate buffer (pH6.0), were obviously increased with the enzyme action as compared with these components of non-macerated control. On the other hand, 3 major components of F-III subfraction, which eluted with 0.3 and 0.4M acetate duffer anb 0.1N NaOH, were remarkably decreased during the enzymatic maceration. These visible changes of major components in F-I and F-III were mainly due to the fluctuations of uronide substances, and no visible change was observed in neutral carbohydrate contents. In F-I subfraction prepared from macerating mixture, both 0.2M and 0.3M fractions abounding with uronide components also showed relatively higher contents of unsaturated compounds and methoxyl groups. From the reasons mentioned above, it is supposed that both fractions were released from water-insoluble cell wall-polysaccharides by trans-eliminative reaction of the enzyme used.
Colletotrichum graminicola (Ces.) G.W. Wils. isolated from Zea mays L. formed two different types of conidia on PDA. One of them is ellipse-shaped conidia, 6-21×3.0-6.0μm (12×3.8μm on average), which are formed at the tips of small side protuberance of young hyphae. These conidia did not change into sickle-shaped ones after the culture was incubated for a long time. Another type is sickle-shaped conidia, 24-30×3.3-5.7μm (27.2×4.7μm on average), produced in sporodochia in old culture. The ellipseshaped conidia were more virulent to the leaves of Zea mays than the sickle-shaped ones. The ellipse-shaped conidia seem to correspond to the “free conidia” described by Barnett in 1960 (Illustrated genera of Imperfect fungi: p. 194).
Aggressiveness of a mutant for virulence of rice blast fungus was investigated using isogenic lines of rice which were selected from japonica×indica crosses for incorporating a gene for blast resistance Pi-zt. The mutant fungus strain acquired a specific virulence to Pi-zt, which is resistant to a blast fungus strain Ken 53-33. The mutant blast strain was found to be weaker in aggressiveness on the resistant isolines than on the susceptible ones. Aggressiveness of three mutant strains isolated from different fungus strains was compared with that of the original strains on five susceptible rice varieties. One of the mutants, Ken 53-33-zt+, was less aggressive than the original strain, but the other two mutants were as much aggressive as their parental strains. These results were discussed in reference to the gene-for-gene theory for hostpathogen interaction.
Phytoalexin activity was detected in an obligate parasitic host-parasite combination, barley and Erysiphe graminis. There are two phases of phytoalexin production in the pathogenesis. In the first phase, phytoalexin activity was found 12hr after inoculation and was more prominent in incompatible cultivar-race interactions than in compatible ones. Both resistance and the ability to accumulate phytoalexin activity in leaves of the incompatible cultivar during the first phase were lost by heat treatment at 50C for 5min. In the second phase, phytoalexin activity was detected around the fungal colonies formed on leaves of the compatible hosts. The antifungal activity of the second phase phytoalexin was almost the same against several races. The role of phytoalexin in the pathogenesis in powdery mildew of barley are discussed.
Isolate No.30 of bean yellow mosaic virus (BYMV) was purified from broad bean leaves by extraction with 0.1M Tris-HCl buffer, pH7.0, containing 0.05M EDTA and 1% 2-mercaptoethanol; repeated clarification with carbon tetrachloride and precipitation with 4% polyethylene glycol (#6, 000) followed by differential centrifugation and sucrose density-gradient centrifugation. ISCO fractionator scanning patterns indicated a high degree of purity but serological results revealed the presence of some host contaminates. The yield of virus was about 2mg/100g of broad bean leaves. The titer of the antiserum against isolate No.30 was 1/2048 by ring precipitin tests. Although the intact virus particles could not diffuse in agar gel plates, a clear precipitin line was observed when 0.5% SDS (or LIS) was added to the agar or virus suspension. Isolate No.30 was compared serologically with a necrotic strain and a chlorotic spot strain of BYMV in agar gel diffusion tests. Spur reactions were observed between isolate No.30 and these strains.
A mosaic disease was found in potato cultivars of Irish Cobbler, Benimaru, Tarumae, and Eniwa at the Potato Foundation Stock Seed Farm, Obihiro, Hokkaido, in 1967 and succeeding two or three years. The diseased plants showed pale-green small stipples along leaf veins, followed by general mottling, on leaves at their flowering stage, and later showed necrotic spots on lower leaves. In Irish Cobbler blonzing of upper surface of leaves was also observed at the later stage of growth. Potato virus X was not detected in the diseased plants by the serological method, but the virus which would be potato virus S was isolated from diseased plants of Irish Cobbler. When inoculated with the virus by sap inoculation, similar symptoms to potato virus infected plants developed on virus-free plants of Irish Cobbler. Positive reaction was obtained between the virus and the antiserum against potato virus S. By sap inoculation, the virus caused veinclearing and mosaic symptoms on Nicotiana debneyi, chlorotic small local lesions and systemic veinclearing on Chenopodium murale. Chenopodium amaranticolor C. quinoa, Solanum villosum, Cyamopsis tetragonoloba (Texsel), Comphrena globosa, and Tetragonia expansa were also susceptible to the virus. The virus was inactivated in ten minutes between 55 and 60C, and after 3-4 days at room temperature (ca. 20C). Dilution end point was between 1:1, 000 and 1:10, 000. By the direct negative staining method, elongated particles of about 650nm in length were frequently observed in the diseased plants. The virus could not be transmitted by green peach aphid, Myzus persicae (Sulz.). In cross protection tests, potato virus S isolated from apparently healthy plants of Irish Cobbler completely protectedthe this virus infection. Mild mosaic symptoms appeared on virusfree plants of Irish Cobbler by sap inoculation with potato virus S. From these results it is concluded that the mosaic disease examined is caused by a strain of potato virus S, designated PVSM by the present authors. Symptoms caused by the virus on several potato cultivars are described. The symptoms on Irihs Cobbler were very clear at 20C, but not at 25C.
The activity of pectolytic and cellulolytic enzymes excreted by different isolates of Pyricularia oryzae Cav. was examined by using agar plate method. No correlation was found between the pathogenicity or virulence and these enzyme activities. The phenomenon of sugar repression was observed. The striking outcome was the finding of a virulent isolate P-2 which does not excrete the lytic enzymes.
The typical symptom of the disease are yellowing, and sometimes brownish necrotic spots on the middle leaves of rice plants, and stunting. The disease is transmissible by green leafhopper, Nephotettix cincticeps, and occasionally infected doubly with rice dwarf. It occurs in the paddy field of Kyushu and may be a new virus disease in Japan.
The “waika” disease of rice plants has occurred since 1971 in Kyushu, Japan. The characteristic symptoms on diseased rice plants are stunting, discoloration and drooping of leaves. The causal agent which was transmitted by Nephotettix cincticeps Uhler and N. virescens Distant, seemed to be non-persistent in the vectors, and was found to be spherical virus approximately 30nm in diameter.
Small spherical virus particles, 30nm in diameter, were found in DN preparations from rice plants infected with the “waika” disease which had been collected in several areas of Kyushu, Japan. The virus particles could be partially purified by differential centrifugation followed by sucrose density gradient centrifugation. In thin section experiments, the same virus particles were observed in vacuoles and in cytoplasm. Development of cytoplasmic inclusion bodies (viroplasms ?) containing the virus particles was found in cytoplasm of some vascular cells.