Citrinin, which markedly inhibits local lesion formation on Nicotiana glutinosa leaves infected with tobacco mosaic virus (TMV), inhibited TMV multiplication in tobacco and N. glutinosa leaves. TMV multiplication was inhibited by 70% when leaf-discs from inoculated leaves were floated on 100ppm citrinin solution for 48 hours. The inhibition rate increased progressively with the time of treatment, while TMV produced in treated leaves had the same specific infectivity as that produced in untreated leaves. When ¹⁴C-amino acid was infiltrated into infected N. glutinosa leaves and labeled TMV was separated by gel filtration, citrinin was found to inhibit the incorporation of ¹⁴C into TMV. Effect of citrinin on RNA synthesis in tobacco and N. glutinosa leaves infected with TMV was investigated by using methylated albumin kieselguhr (MAK) column chromatography and disc electrophoresis in polyacrylamide gel. In the MAK column chromatographic pattern, TMV-RNA overlaps with 28S rRNA peak. Citrinin inhibited the incorporation of ³H-uridine into 28S rRNA fraction of both uninfected and infected leaves, whereas the incorporation into sRNA, DNA-like RNA, and 18S rRNA was inhibited only to a negligible extent. Analyses using the polyacrylamide gel electrophoresis showed that citrinin inhibited cell RNA synthesis to some extent even at 25ppm. Citrinin, at a low concentration, inhibits the 28S rRNA but at a high concentration, 100ppm, inhibits both 28S rRNA and TMV-RNA synthesis. Since citrinin at 75ppm completely inhibits local lesion formation on N. glutinosa while inhibits TMV synthesis by about 50%, the effect of citrinin on lesion formation is not directly connected with that on virus synthesis but the effect may be caused via the suppressed function of ribosomes.

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Relations between seed transmission and embryo infection were studied on cowpea and azuki bean plants inoculated with seed-borne viruses. Virus-host combinations tested in the present study were, (1) azuki bean mosaic virus (AzMV)/azuki bean, (2) cowpea aphid-borne mosaic virus (CAMV)/cowpea, (3) cucumber mosaic virus (CMV)/azuki bean, (4) subclover mottle virus (SMV, a strain of broad bean wilt virus)/cowpea, and CAMV/azuki bean. Seed transmission occurs in the former two combinations, but does not occur in the remaining three combinations as described in a previous paper (Tsuchizaki, T., et al., 1970).
Young seedlings of cowpea or azuki bean were inoculated with a respective virus. Ten to fifteen days after flowering immature embryos were collected from the inoculated plants, and were examined individually for the presence of virus. The result showed that virus was recovered from immature embryos only in virus-host combinations that seed transmission occurred. It was also found that some embryos contained virus in high concentration, but others contained virus in very low concentration. When these virus-infected embryos were sown in pots containing sterilized soil and were grown into young seedlings, seed transmission was demonstrated only in seedlings developed from embryos containing virus in high concentration. In the following experiment cowpea or azuki bean plants were inoculated with CAMV in flowering stage. Ten to fifteen days after flowering immature embryos were collected from these plants, and were examined individually for the presence of virus. The result also showed that virus was recevered from immature embryos only in virus-host combinations that seed transmission occurred. But in this experiment virus concentration in infected embryos was always very low. It is evident that gamate infection cannot occur under these experimental conditions. Hence low virus concentration observed on the infected embryos may be a result of infection through direct invasion by virus from mother plants.
In another experiment both mature and immature seeds were obtained from cowpea as well as azuki bean plants. Removing their seed coat, the embryo surface were inoculated with CMV, AzMV, CAMV, or SMV by the carborundum method. Virus infection was observed to occur in every virus-host combinations tested, although the percentage of infected embryos were sometimes low.
Then an experiment was carried out on cowpea or azuki bean plants about 10 days after flowering. Immature embryos developing on mother plants were inoculated with a virus in that condition. Viruses used for the inoculation were CMV, AzMV, and SMV. After maturity these inoculated seeds were collected and were examined for seed transmission. Total 190 inoculated seeds including four virus-host combinations were thus tested. Among them seed transmission was observed only in two azuki bean seeds inoculated with CAMV and one cowpea seed inoculated with SMV. This result suggests that developing immature embryos are resistant to virus infection.
Considering these results, the difficulty or impossibility of embryo infection through direct invasion by virus from mother plants may be explained by the following two facts; (1) virus movement to embryo from mother plant is difficult, and (2) developing immature embryo is resistant to virus infection.

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Ultra-thin sections of apical meristem from flower buds of cowpea and azuki bean plants, infected with cowpea aphid-borne mosaic virus (CAMV) or with azuki bean mosaic virus (AzMV), were examined under electron microscope for the distribution of virus particles and cytoplasmic inclusions. In a cowpea variety “Zairai-Tsurunashi-Kintoki” infected with CAMV, both virus particles and cytoplasmic inclusions were seen in the preparation of meristematic tissues at a distance of 0.1-0.2mm from apical end, but these were found more frequently in plants infected with a strain (CAMV-3) known to be transmitted through seeds at a moderately high percentage (approx. 28%) than in those infected with another strain (CAMV-1) of a lower percentage of seed transmission (approx. 6%). No virus particles and inclusions were detected in the cell layer of 0.1mm from apical end. Similar results were obtained with another cowpea variety infected by CAMV-3 and -1, although in meristem infected by CAMV-1 the virus particles appeared to be located farther than 0.2mm from apical end.
In azuki bean plant, virus particles and inclusions were detected in meristematic tissues at a distance of 0.1-0.2mm from apical end, infected by AzMV that is seed transmissible, but none in the comparable region, down to 0.2-0.3mm, of tissue infected by CAMV that is not transmitted through seed of azuki bean.
Electron microscopic examination of individual pollens for virus particles revealed that 6.8 and 18.6% of pollens from cowpea plants infected with CAMV-1 and CAMV-3, respectively, contained virus particles.
A hypothesis is proposed to explain seed transmission of virus or lack of it in cowpea and azuki bean as follows. In seed transmitted virus-host combinations: virus is distributed in meristematic tissues of flower buds except a layers of 0.1mm from apical end, and can move into male or female gametes, resulting in infection of some embryos with virus in concentration high enough to produce infected seeds. Developing immature embryos may occasionally be infected through direct invasion by virus from mother plant, but virus concentration is not sufficient to cause seed transmission. In non-seedtransmitted virus-host combinations: virus is not distributed in meristematic tissues of 0.2 or 0.3mm from apical end of flower buds, and infection of both gamete and embryo does not occur. In addition, developing immature embyos are not liable to infection through direct invasion by virus from mother plant.

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Foster and Ross (1968) found that when Turkish tobacco leaves inoculated with tobacco mosaic virus (TMV) were 2-5 days later immersed for 40sec. in water at 50°C, local lesions appeared 1-2 days after the heat treatment. We determined the conditions for more effectively inducing local lesions, and also compared the modes of infection and multiplication of TMV between systemic hosts and local lesion hosts. N. tabacum ‘Xanthi’and N. tabacum ‘Samsun’ were used as the systemic hosts, and ‘Xanthi nc’ and ‘Samsun NN’ as the local lesion hosts. When leaves of 2.5 months old plants were used, optimum treatment was for 2min. at 50°C, or for 10sec. at 55°C. Optimum incubation after the heat treatment was for 2-3 days at 25°C, or for 1-2 days at 30°C. When leaves of younger plants were used, 1min, treatment at 50°C was adequate, whereas 2min. at 50°C resulted in a visible injury of the treated leaves. In leaves of plants older than 3 months, treatments even longer than 2min. at 50°C induced no lesions at all. The number of lesions induced by heat treatment on leaves of the systemic hosts inoculated with TMV was of about the same level as on leaves of the local lesion hosts inoculated with TMV of the same concentration. There was also no significant difference in virus dilution-infectivity curves between the systemic hosts and the local lesion hosts. No significant difference was observed in the number of ectodesmata in epidermal cell walls of healthy leaves between both hosts. Local lesions on leaves of the systemic hosts induced by heat treatment 2-4 days after inoculation were much larger in size than those on leaves of the local lesion hosts on the same days after inoculation. TMV seemed to spread from each infective center more rapidly in the leaves of the systemic hosts than in those of the local lesion hosts. It is considered that the numbers of infectible sites are about equal between the local lesion hosts and the systemic hosts, but the rate of TMV multiplication after the establishment of infection is lower in the local lesion hosts than in the systemic hosts.

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Inhibitory effects of four kinds of tea catechins, (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), and (-)-epigallocatechin gellate (EGCg), against TMV and CMV were investigated.
Nicotiana tabacum (Ky-57) was used as the assay host for CMV, and tomato and N. glutinosa for TMV. Each of four tea catechins was added at a concentration of 5mg/ml to the sap expressed from leaves of tobacco or cucumber systemically infected with the virus, and inoculated on the leaves of the assay plants.
When each of four tea catechins was added at a concentration of 0.5% to 1% gelatin solution, (-) EC and (-) EGC did not give precipitation, whereas (-) ECg and (-) EGCg immediately produced precipitates. The same results were obtained when the above described virus inocula were used instead of the gelatin solution.
(-) ECg and (-) EGCg, which easily combine with water-soluble protein, were more inhibitory against infection with CMV than (-) EC and (-) EGC, and no symptom appeared even 30 days after inoculation.
No symptom appeared also in both systemic and local lesion hosts when inoculated with TMV plus (-) ECg or (-) EGCg.
The number of local lesions on inoculated leaves decreased in proportion to the concentration of (-) ECg and (-) EGCg in the inoculum, and no lesion was produced at 0.5% of catechins.

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Many watermelon plants cultivated in the Chiba and Ibaraki prefectures showed symptoms similar to those of mosaic disease in June 1968. In these districts it became evident that apparently healthy fruit that were harvested harboured some form of abnormality. Infected plants were retarded in growth. The young leaves showed mosaic symptoms but in the majority of cases the symptoms were not distinct and often disappeared as the leaves grew older. Fruit stalks of infected plants generally showed necrotic lesions and sometimes the fruit showed mosaic-like dark green elevated areas on the surface. The white peripheral regions of infected fruit which were cut open appeared to be water-soaked and whitish yellow. The inner pulp also turned to water-soaked dirty red and contained crescent-shaped crevices while there was an increase in the yellow fibres.
The above symptoms were induced on fruit as well as leaves of healthy watermelon by juice inoculation with a virus isolated from infected plants showing the abnormalities described. It became apparent that the symptoms in watermelon were most marked when inoculated at the time of fruit set.
The virus was identified with cucumber green mottle mosaic virus (CGMMV) by mechanical inoculation and observation under electron microscope using the dip method. This virus was, however, distinguishable from that isolated from cucumber in the western part of Japan in 1966, on the basis of differential reaction on Datura stramonium, Chenopodium amaranticolor and from serological comparison. The virus isolated from watermelon was designated as watermelon strain and the one isolated from cucumber in 1966 as cucumber strain.
Specimens of fruit exhibiting the fruit pulp symptoms were collected from various localities and virus isolations were made. From most of them CGMMV was isolated, while from certain specimens watermelon mosaic virus (WMV) and cucumber mosaic virus (CMV) also were isolated, and from some no virus was isolated. The fruit abnormality referred to here is certainly caused by CGMMV, but WMV, CMV and some physiological disorder may also cause similer fruit abnormalities. These, however, can be distinguished from the symptoms caused by CGMMV.
Bottlegourd, Lagenaria siceraria var. hispida, is generally employed as a root-stock in the cultivation of watermelon. The presence of CGMMV was detected in the seed coat of bottlegourd seeds which were collected in Chiba and Ibaraki prefectures from left-over stocks of one season, while it was not detected in the seeds of watermelon. Also in the Tochigi prefecture where bottlegourd is cultivated on a large scale the crop showed a high percentage of mosaic symptoms, and from these mosaic plants CGMMV was isolated.

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Primary leaves of two oat varieties were inoculated with Puccinia coronata Corda and the changes in ³²P incorporation into nucleic acid fractions were studied by MAK column chromatography.
In the susceptible variety, Victoria, the inoculated/uninoculated ratio of radioactivity in the rRNA fraction increased slightly at the initiation period of reproductive growth of the rust fungus (3 days after inoculation), then largely during the uredosorus development (4 to 5 days). The ratio in the sRNA fraction increased only at the beginning of sporulation (5 days). The change in the ratio for the TB-RNA as well as rapidly labeled RNA fractions showed a similar trend to that for the rRNA fraction.
In the resistant variety, Shokan No.1, the increases in the ratio were noted to be of similar extent among the sRNA, rRNA, and TB-RNA fractions: being slight at the prehaustorial stage of infection (20hr after inoculation), and thereafter being large until 48hr. The rapidly labeled RNA was also increased by infection.
The base analysis of labeled RNA's indicated that the increased synthesis of rRNA may be originated from the rust fungus in the susceptible leaves, and from the host in the resistant leaves.

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The effect of temperature and of some other factors on multiplication, plaque formation, and inactivation of Bdellovibrio bacteriovorus isolated from paddy fields of Japan were investigated. The mode of multiplication of the organism on Xanthomonas oryzae was also studied by means of one-step growth experiment.
The optimum temperature for multiplication of B. bacteriovorus isolate Bd-N6801 in X. oryzae cells in liquid culture was 30 to 32°C. The plaque formation was much influenced by incubation temperature, kind and concentration of indicator bacterium, and concentration of agar in the medium. The highest plaque forming efficiency was obtained by using a highly concentrated suspension (3.5×10⁹ cells per ml) of X. oryzae in a medium containing 0.6% of agar. Plaque forming efficiency was almost independent of temperature in the range of from 22 to 34°C. Plaques did not appear when agar concentration in the medium was above 1.0%.
All isolates of B. bacteriovorus tested in this experiment were partially inactivated at 40°C, and completely inactivated at 45 to 50°C, in 10min in sterilized distilled water.
Bd-N6801 produced in average 8 to 9 progenies per cell of X. oryzae, with a latent period of 3 to 3.5hr following the termination of 30min attachment period.

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Two types of callus were recognized in cultured tissues of potatoes, i.e., fragile callus on variety Norin No, 1 and Irish Cobbler, and compact callus on Interspecific hybrid 96-56.
Nine and 12 hours after the inoculation with zoospores of Phytophthora infestans (Race 0 and Race 1), browning and active protoplasmic streaming were observed in the callus tissue cells of Irish Cobbler and Norin No.1. When zoospores of Phytophthora infestans Race 0 and Race 1 (non-compatible races) were inoculated on interspecific hybrids 96-56 and SH-469, a high grade of browning in the suscept cells was recognized 6 and 9 hours after inoculation. Resistant hybrid showed dark browning in the infected cells.
Browning was not observed in free cells from potato callus tissues of variety Irish Cobbler obtained by shaken culture, even 72 hours after inoculation with zoospores of Phytophthora infestans.
Both types of hypersensitive flecks and susceptible lesions were recognized on the callus in Interspecific hybrid 96-56 after treating with the DNA fraction obtained from variety Norin No.1.

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When the roots of young tobacco seedlings raised in soil or in culture solution were inoculated with extracted juice of tobacco leaves infected with cucumber mosaic virus ordinary strain (CMV-O), 25-100 percent of seedlings showed systemic symptoms within 7-40 days after inoculation, while with CMV yellow strain (CMV-Y) about 5-20 percent of inoculated plants showed systemic symptoms. The rate of plants which showed systemic symptoms was lower than in the case of inoculation on leaves. The period required for the appearance of systemic symptoms was also longer. The difference in the rate of appearance of systemic symptoms between CMV-O and CMV-Y might be due partly to seasonal variation in susceptibility of tobacco roots and partly to the difference in the virus strains. Multiplication of CMV in the inoculated roots showed a similar pattern as in the roots of plants inoculated on leaves.
Tobacco seedlings were raised in the pots (diameter 12cm) with soil, two plants per pot, and the leaves of one of the plants were inoculated with CMV-O. The other plant was covered with polyethylene bag to avoid infection by leaf contact. From more than 50 percent of root samples from uninoculated and appearently healthy tobacco plants, CMV was recovered within 31 days after inoculation on the donor host. In an experiment using tobacco plants grown in wooden-frame, and inoculated with CMV-Y, a similar result was obtained. In a test of field grown tobacco plants, growing in the vicinity of CMV infected plants, CMV was recovered from the roots of 5 plants out of 7 plants tested, even though the tested plants were showing no symptoms and no virus was detected from the leaves. The transmission of the virus may have been caused by root contact, because transmission occured more frequently in abundantly rooted plants than in poorly rooted ones, and no virus was detected from root-samples collected from root parts on the other side of neighbouring infected plant.
Contact transmission has been thought to be negligible in the case of comparatively labile viruses such as CMV. From the results reported here, however, transmission by root contact may not be neglected in natural occurrences of CMV in tobaccoo plants.

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