Two acidic proteins involved in toxoflavin production by Pseudomonas glumae, causal agent of rice seedling rot and grain rot, were identified. Non-toxigenic (Tox^-) Tn5 mutants which did not produce a yellow pigment, were avirulent to rice plants. When proteins from wild-type and Tox^- strains were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), two acidic proteins, designated as TRP-1 and TRP-2, were found in the wild-type but not Tox^- strains. The synthesis of TRP-1 and -2 was confined to the late exponential phase of culture growth, corresponding to toxoflavin production during the late exponential to early stationary phases of culture growth. The anti-TRP1 antibody reacted with a single protein band of about 30kDa only on blots of SDS-PAGE separations of those P. glumae and P. gladioli strains able to produce toxoflavin.

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Probenazole, an inducer of systemic acquired resistance against fungi and bacteria, and its metabolite saccharin were tested for their effect on virus infection. When applied to the soil of potted tobacco (Nicotiana tabacum cv. Xanthi nc) plants, both compounds promoted the appearance of local lesions by tobacco mosaic virus and reduced lesion size. Probenazole enhanced the masking of mosaic symptoms in the systemic host, tobacco cv. Samsun. These facts indicate that probenazole and saccharin induced a broad spectrum of disease resistance in plants.

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A membrane fraction was isolated by differential centrifugation from PVX-inoculated tobacco protoplasts to investigate in vitro viral RNA synthesis. This fraction was capable of catalyzing the in vitro synthesis of a genomic-length (6.4kb) and two subgenomic-length RNAs (2.1 and 0.9kb). The different sensitivities of the RNAs to S1 nuclease at high and low salt concentrations suggested that the synthesized genomic-length RNA was mainly the replicative intermediate RNA (RI) and that the two subgenomic-length RNAs were single-stranded. Most of the synthesized RNAs were shown to be of a positive polarity based on ribonuclease protection assay. Dependent on the exogenous template, the fraction treated with micrococcal nuclease synthesized only the genomic-length RNA but not the subgenomic RNAs. Treating the membrane fraction with protease abolished synthesis of the subgenomic-length RNAs, but not that of genomic-length RNA. The synthesizing activity for the subgenomiclength RNAs was released into the soluble fraction after treatment with the detergent Brij 58. These results suggested that genomic and subgenomic RNAs are replicated at different sites in the membrane; genomic RNA synthesis occurs inside the membranes, whereas subgenomic RNA synthesis occurs on their surface.

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Mutants of Burkholderia glumae, the causal agent of rice seedling rot and grain rot diseases, were isolated by transposon (Tn) mutagenesis with Tn4431 harboring a tetracycline (Tc) resistance gene, which was carried on the suicide vector pUCD623. The resulting Tc-resistant colonies were screened for a loss of toxin production as well as for a lack of pathogenicity to rice seedlings. One of the screened mutants, the mutant No.19, was shown to have a single copy of Tn insertion on the genomic DNA by Southern hybridization test. Restriction enzyme fragments containing Tn4431-flanking sequences from the genomic DNA of mutant No.19 were cloned and used as probes for colony hybridization of the genomic DNA cosmid library from a wild strain of B. glumae. Six recombinant clones showing homology to the probes were isolated and conjugated into mutant No.19 by triparental mating. The recombinant cosmid pNP147, one of six clones, was able to complement the mutant No.19 for toxin production and pathogenicity. The toxin, purified from a culture of mutant No.19 with pNP147, was identified as toxoflavin by NMR and Mass spectra analyses. These results suggest that toxoflavin is a critical factor in disease development of B. glumae, since the nontoxigenic mutants were unable to induce seedling rot disease to rice plants.

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When plants interact with pathogens, they protect themselves with various chemical and physical barriers. Some barriers, such as phytoalexin production, are induced by molecules called elicitors that are produced by pathogens. Although bioassays for measuring elicitor activity in suspension-cultured rice cells have been established, a bioassay for measuring elicitor activity in rice plants has been lacking. Phytoalexin accumulation in rice leaves in response to elicitor treatment is highly dependent on environmental conditions, establishing the right combination of conditions has been difficult. We have succeeded in developing a new bioassay by cultivating rice plants under conditions of low temperature (22°C), high light intensity (30, 000lux) and high humidity (80%). When the fifth leaves of rice plants cultivated under these conditions were fully expanded, the treatment of the fourth leaves with mycelial extracts of Magnaporthe grisea or Phytophthora infestans induced the accumulation of the rice phytoalexins, momilactones and phytocassanes.

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Single-strand DNA conformation polymorphism (SSCP) analysis using internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA) amplified by polymerase chain reaction (PCR) was performed to detect DNA polymorphism of nine species of Melampsora rust fungi on willows. The rDNA ITS 1 region (approximately 300-320bp in size), was amplified from 49 collections. The SSCP patterns in the amplified ITS 1 region of five species, M. capraearum, M. epiphylla, M. larici-urbaniana, M. microsora and M. yezoensis were species-specific. On the other hand, no distinction was found in the SSCP patterns between M. chelidonii-pierotii and M. coleosporioides, and between M. epitea and M. humilis. In improved PCR-SSCP analysis using restriction fragments of the amplified ITS 1 and ITS 2 regions, no difference was detected between M. chelidonii-pierotii and M. coleosporioides, and between M. epitea and M. humilis. These results suggest the respective two species have a close genetic and taxonomical relationship. We revealed the PCR-SSCP analysis may be beneficial as a tool for distinguishing species of Melampsora because the analysis is simple and sensitive.

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Three nucleic acids were found in 5% polyacrylamide gel electrophoresed with extracts from several cultivars of Japanese pear (Pyrus spp.) that had no symptoms. They were cultivar-specific, and were detected from bark, leaf, petals and flowers plus petals. They were not digested by DNase I and were resistant to RNase A in 2×SSC buffer, but were susceptible to RNase A in 0.1×SSC buffer. These nucleic acids were absorbed by CF-11 cellulose in 15% ethanol. These results indicate that the three nucleic acids were double-stranded (ds) RNA. Molecular weights were estimated at about 1.08, 0.97, 0.90×10⁶ daltons according to electrophoresis with dsRNAs of the rice dwarf virus as a marker. Several cultivars that have these dsRNAs had genetic relationships. The results of investigating the transmission of dsRNAs to the next generation by crossing showed that dsRNAs were detected in 31/40 when only the seed parent had dsRNAs, in 12/12 when only the pollen parent had dsRNAs, and in 5/5 when both parents had dsRNAs. These dsRNAs were transmitted high frequently to the next generation through both ovule and pollen, but transmission of dsRNAs by grafting to other cultivars without dsRNAs did not occur. These results suggest that the dsRNAs from Japanese pear are similar to dsRNAs derived from the phytocryptovirus group.

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[in Japanese]

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[in Japanese]

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[in Japanese]

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[in Japanese]

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Host specificity of the powdery mildew fungus Blumeria graminis (Erysiphe graminis) isolated from Asperella longe-aristata was clarified by inoculating 16 graminaceous plant species. The fungus was virulent on Triticum spp. in addition to the original host plant species. Isolates of B. graminis f. sp. tritici were virulent on both A. longe-aristata and Triticum spp., however, other formae speciales of Blumeria graminis in Japan were avirulent on A. longe-aristata. Cross-compatibility tests between formae speciales of the fungus showed that an isolate of B. graminis from A. longe-aristata was fertile when crossed with f. sp. tritici of the fungus. These findings show that B. graminis isolated from A. longe-aristata should be classified as f. sp. tritici.

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The optimum temperature and relative humidity (RH) for frogeye leaf spot development on sweet pepper were 20-25°C and above 95% RH, respectively. Latent periods after inoculation were closely related to temperature and RH conditions. For lesion appearance in a plastic house, about 22 hours of the optimum condition, or about 150 hours at 15°C and above 95% RH. The accumulated hours would be useful for predicting the onset of this disease in winter.

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The effect of fluazinam WP (active ingredient 39.5%, trade name: Frowncide SC) on white root rot (causal agent, Rosellinia necatrix) of grapevine was examined in the vineyard. Fluazinam soil-drench at either 790ppm or 395ppm showed high efficacy against white root rot of grapevine. Treatment with fluazinam at 790ppm after harvest time (October-December) was more efficient than treatment before sprouting time (March). The treatment protected grapevine roots from the disease for at least half a year even in highly infested soil, and was not phytotoxic against grapevine.

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