Oxolinic acid (5-ethyl-5, 8-dihydro-8-oxo [1, 3] dioxolo [4, 5-g] quinoline-7-carboxylic acid, Starnere^®) is a synthetic bactericide, developed from quinoline derivatives. Oxolinic acid completely inhibited the growth of gram negative bacteria including Pseudomonas glumae, P. plantarii, P. avenae, Erwinia carotovora subsp. carotovora and E. carotovora subsp. atroseptica at the concentration of 0.4μg/ml or below in vitro. Oxolinic acid was effective against various isolates of P. glumae from rice grains collected from various districts of Japan. Antibacterial activities of oxolinic acid on P. glumae varied depending on the growth phase of the bacterium. Activity of oxolinic acid was bactericidal for the bacterium at the lag phase, but was bacteristatic for the bacterium at the early logarithmic phase and decreased after the middle logarithmic phase. Oxolinic acid sometimes induced abnormal elongation of P. glumae cells at the early logarithmic phase.

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An antagonistic rhizoplane bacterium (Serratia Marcescens) and a chitin-degrading microbe (Streptomyces anulatus) were used for establishing a binary microbe system for biological control of Fusarium wilt disease of tomato. For effectively tracing root-colonization, the rhizoplane bacterium was genetically marked with the lux genes and the tetracycline resistance gene through bacterial conjugation. Successful colonization by the bacterium on roots from bacterized seeds was visualized by bioluminescence of the transconjugant into which the lux gene operon had been integrated. Antifungal activity of the transconjugant was stable and effectire even when the bacterium was colonized on tomato roots. Multiplication of the transconjugant in soil was supported by the chitin-degrading microbe which had been previously added in chitin-supplemented soil. The chitin-degrading microbe produced extracellular endochitinases and hydrolyzed chitin which was added to soil. Chitohydrolytic products supported the multiplication and root-colonization of the rhizoplane bacterium in soil. The binary microbe system comprising of chitin-degrading microbe and gene-marked transconjugant was effective in controlling of tomato wilt disease caused by Fusarium oxysporum f. sp. lycopersici.

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Data on host-pathogen relationship of rice (Oryza sativa) and blast (Pyricularia oryzae) obtained in Niigata Prefecture, Japan were analyzed by virulence analysis, and analysis of distribution pattern of virulence gene numbers. The percent growing areas (frequencies) of rice varieties with various genotypes for blast resistance were obtained from statistics data, and frequencies of blast-fungus genotypes for virulence were calculated from the data from 1976 to 1986 in six areas in Niigata Prefecture. The results of virulence analysis showed that nonrandom association was found between two virulence genes, Av-a⁺ and Av-i⁺. Nonrandom associations of virulence genes were also detected by analysis of the distribution pattern of virulence gene numbers. The deviation of distribution pattern from that expected from random association of virulence genes was mostly explained by specific interaction of virulence genes, Av-a⁺ and Av-i⁺.

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Data on host-pathogen relationship of rice (Oryza sativa) and blast (Pyricularia oryzae) obtained in Niigata Prefecture, Japan were analyzed by application of a simulation model for following the change of pathogen genotype frequencies. The proportion of growing areas of rice varieties with various genotypes for blast resistance were obtained from statistics data and frequencies of blast-fungus genotypes for virulence were calculated from the data from 1976 to 1986 in six areas in Niigata Prefecture. The lowering of fitness values for each of avirulence genes, Av-a, Av-i, Av-k and Av-z, accompanying the mutation to virulence was estimated by simulating the change in the frequencies of each genotype of blast fungus on the frequencies of genotypes of rice plant. Lower fitness values of virulence genes, Av-a⁺ and Av-i⁺, were obtained in all areas in Niigata Prefecture. This explains the recent predominance of a pathogen genotype, Av-a Av-i Av-k Av-z (main race 001) in Niigata Prefecture, though predominance of the genotype, Av-a⁺ Av-i Av-k Av-z (race number 003) was previously obtained in the Kanto District with similar gene frequencies for host resistance to Niigata Prefecture. Reverse tendencies between two genes were observed: fitness values obtained were slightly higher for the Av-a⁺ gene and significantly lower for Av-i⁺ gene in mountainous areas in comparison with plain areas.

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Alternaria tenuissima affecting pigeon pea produces a host-specific toxin in spore germination fluids with selective toxicity to susceptible pigeon pea cultivars/lines. The primary site of action by the toxin in host cells was examined by ultrastructural observation and morphometry. The first toxin-induced change in ultrastructure of susceptible leaves was the disappearance of mitochondrial matrices and disruption of cristae within 5 hr after toxin exposure. The number of affected mitochondria in susceptible leaf tissues increased with time, until 100% were affected at 39 hr. Obvious effects on other organelles were not evident at 20 hr in the toxin-treated susceptible leaves. No ultrastructural changes were observed in cells of resistant Tanzania line at 20 hr after toxin treatment. The data indicate that the initial site of toxin action may be located on the mitochondria.

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An isolate of Alternaria alternata from citrus was found to be pathogenic to both Japanese pear (cv. Nijisseiki) and Emperor mandarin. The fungus produced a toxin that is highly toxic to the pear and less toxic to the original host, Emperor mandarin. The toxin was isolated and was designated ACT-toxin IIb. ACT-toxin IIb caused vein necrosis and loss of electrolytes from leaves of cv. Nijisseiki at concentrations more than 1×10^-7 M. The toxin caused vein necrosis at 4×10^-5 M, and increased losses of electrolytes from leaves of the citrus Emperor mandarin at 10^-5 M. The toxin did not affect leaves of Japanese pear cv. Chojuro and rough lemon at 10^-4 M. Electron microscopy showed that ACT-toxin IIb caused invaginations of the plasma membrane in leaf cells of susceptible Japanese pear cv. Nijisseiki, but did not show such effect on resistant pear cv. Chojuro. These results indicate that the mode of action of ACT-toxin IIb is similar to that of ACT-toxin Ib and AK-toxin (from the Japanese pear pathotype of A. alternata). ACT-toxin IIb also induced susceptibility in Japanese pear cv. Nijisseiki to a non-pathogenic isolate of A. alternata. The finding that the pathotype of A. alternata from citrus also has host-selective pathogenicity to certain Japanese pear indicates the danger of growing such susceptible pears near citrus-growing areas, and a potential problem from the introduction of the citrus pathogen into areas of Japanese pear cultivation.

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[in Japanese]

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[in Japanese]

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[in Japanese]

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Relationship between population dynamics of Pseudomonas glumae on rice seedlings and the efficacy of oxolinic acid (5-ethyl-5, 8-dihydro-8-oxo[1, 3]dioxolo [4, 5-g]quinoline-7-carboxylic acid, Starner^®) as a seed disinfectant in the control of bacterial seedling rot of rice caused by P. glumae was studied. When rice seeds were cultivated at 30°C in hastening of germination and emergence of seedlings, the bacterium grew remarkably on plumules during hastening of germination and caused rice plants bacterial seedling rot of rice. The disease severity was correlated with the bacterial growth on plumules during hastening of germination. When seeds were treated with oxolinic acid before and after seed soaking, an increase in the bacterial populations on plumules was inhibited. And the chemical was highly efficient in the control of the disease. On the other hand, when oxolinic acid was treated after hastening of germination, the bacterial populations on plumules increased as much as on untreated controls. And the chemical did not control the disease.

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Seed treatment with oxolinic acid (5-ethyl-5, 8-dihydro-8-oxo[1, 3]dioxolo[4, 5-g]quinoline-7-carboxylic acid Starner^®) showed high efficacy against bacterial seedling rot of rice caused by inoculation of Pseudomonas glumae at the density of 10⁴-10⁸cfu/ml at any stage of a seedling growth except for inoculation of 10⁵cfu/ml after seed soaking. Secondary infection in the nursery box was observed as the diseased seedling patch from the inoculated seeds outward. When seeds treated with oxolinic acid were sown around inoculated seeds, secondary infection of the disease was inhibited and the bacterial populations on healthy seedlings 21 days after sowing were less than 10³cfu/g fresh weight which was the detectable limit.

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Infection of the disease and bacterial movement in the tissue of new canes were examined using 3-year-old grafted potted vines of kiwifruit (cv. Hayward) and 3-year-old potted seedlings grown from seeds of “Hayward”. Pseudomonas syringae pv. actinidiae was used as inoculum. Lesions in the field developed on the leaf surface and leaf margin of “Hayward” after wound and spray-inoculation. Since a lot of water droplets were observed on the leaf margin during high humidity, it was thought that the pathogen invaded through hydathode, in addition to the stomata and wounded parts. Lesions were formed when the bacterial suspensions at more than 10²CFU/ml were inoculated with or without wounding onto the leaves. In the case of spray-inoculation at 10²CFU/ml, however, the lesions appeared 20 days after the inoculation compared to 5 to 8 days for lesion formation using the suspensions at more than 10³CFU/ml. Therefore, this may be due to the pathogen surviving on lower surface of the leaves, and the infection occurring after growing of the pathogen during high humidity. The water-soaked lesion without a yellow halo appeared as a mosaic 4 days after inoculation and these lesions expanded rapidly when rain continued after inoculation. It indicates that the latent period is remarkably influenced by relative humidity. When the lesions reached the midrib, the bacterium was isolated from their sections before the appearance of disease symptom. The bacterial population in the sections decreased as the distance apart from border of lesions grew. When they reached the base of leaf blades, the bacterium was isolated similarly from each section of the petioles. Also, when the tender stem was affected severely with the disease, the bacterium was isolated from each section of the petioles and midribs in the process of disease development. These results indicate that the bacterium moves in the tissue from the lesion on the leaves through the midrib and petiole to the stem. Also, the bacterium moves in the tissue from the diseased stem through the petiole to midrib. This action was remarkable for the seedlings in comparison with the grafted vines of “Hayward”.

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Susceptibility of the new leaves of kiwifruit (cv. Hayward) to bacterial canker increased remarkably when the leaf blades reached ca. 2cm in length. However, it decreased as the leaves matured. The disease development increased rapidly when the mean temperature per 10 days was 15±3°C (April to May). Then, it began to be less serious when the temperature approached 20°C (late May to early June). When the temperature was over 20°C and approached 25°C (June to early July), the disease occurred only under cool and rainy conditions. Over 25°C (after mid.-July) the symptom was not observed. Exudation of bacterial ooze occurred actively on the affected tender canes from April to May. The canes began to wither in early May when the disease developed severely. In case of the disease development remaining in the limited part on the stem, a small amount of oozing was observed from the respectable minority of affected parts until late June (21.8°C). In early July (23.0°C), the number of canes which showed oozing from affected parts decreased remarkably. Moreover, the wound-healing tissue bagan to develop on the affected parts. The wound-healing tissue developed vigorously in mid. to late July (25°C), and oozing stopped. From these results, the suitable mean temperature per 10 days for infection on new canes was concluded to be in the range of 10 to 20°C as optimum at 15±3°C. However, exudation of bacterial ooze occurred again for 20% of the diseased canes in next early spring. It suggested that pathogen over-winters in diseased tissue. Also, the water-soaked spots were produced on the leaves after spray-inoculation at high pressures, and the pathogen was reisolated from those spots. Bacterial canker of kiwifruit is severe in areas where strong wind blow. This phenomenon may be an important factor causing the infection.

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The seasonal changes in bacterial population of Pseudomonas syringae pv. actinidiae in the infected leaves and of bacterial exudation from the lesions were investigated from May to December for kiwifruit (cv. Hayward) in the field. The following relations were observed between mean temperature per 10 days before isolation of the bacteria and the population in the lesion. The population was highest in early May (13 to 17°C), and it began to decrease from 10^6-7CFU/ml when the temperature was over 18°C (mid. to late May). It decreased rapidly from 10^4-5 to 10^2-3CFU/ml when the temperature rose to the range from 20 to 24°C (early June to early July). It decreased more after the rise in temperature and became 10^0-1CFU/ml in late August (25 to 26°C). The bacterium was not isolated in some lesions. However, the bacterial population began to recover when the temperature dropped gradually to the range from 22 to 20°C (late September to early October) in fall. It increased rapidly, when the temperature dropped further to the range from 17 to 15°C (late October to early November), and approached the population at 10^4-7CFU/ml in June. After that, a high population was maintained though the temperature dropped gradually from 13 to 10°C (late November to mid.-December). Bacterial exudation from lesions was observed immediately after floating them in 10ml of sterile distilled water in early May. The amount of exuded bacteria was 10^6-7CFU/ml. It decreased below 10^4-5CFU/ml in early June and took more time for exudation. The bacterial exudation was not observed during July to September. However, it occurred again in early October when the bacterial population in lesions began to recover. Both the percentage of lesions in which bacterial exudation was observed and the amount of exuded bacteria increased rapidly during early November to mid.-December. However, the amount of exuded bacteria did not become over 10^2-3CFU/ml. The above results indicate that the most suitable mean temperature per 10 days ranged from 10 to 20°C for the growth of pathogen in the leaf tissue. It suggests that the bacteria may attack canes in spring and in mid.-fall to early winter because the optimum temperature for growth occurs.

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Rice plants fixed in glutalaldehyde were cut into 30μm thickness with Automatic MT-2 (Nihon-Ika). The cross sections were immersed in the fluorescent antibody labelled with FITC against Pseudomonas glumae and observed under a fluorescent microscope. Specific fluorescences derived from the bacteria were found in and on an upper and lower epidermis, in a parenchyma and in a sclerenchyma of palea and lemma of grain. They were found in an upper and lower epidermis and a vascular bundle sheath of a flag leaf blade, and around a vascular bundle and a lysigenous aerenchyma of a flag leaf sheath. These results suggest that this immunofluorescent antibody technique is useful for detecting P. glumae on rice plants.

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Viral wilt in cucumber plants grafted on squash rootstocks occurred in summer and autumn in Kagawa prefecture, whereas no viral wilt was observed in winter and spring. Mosaic disease occurred at a high percentage in summer and autumn. Both cucumber mosaic virus (CMV) and zucchini yellow mosaic virus (ZYMV) were detected in the grafted cucumber plants showing viral wilt in the fields at a high frequency. The inoculation with the samples containing CMV and ZYMV caused severe wilt on the grafted cucumber plants.

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