In order to know whether the inhibitory action of a phytoalexin (PA) obtained from a certain host-parasite combination is specific to the parasite species used or not, three kinds of PAs were assayed on several phytopathogenic fungi including congenial and non-congenial to the host plants. The following three PAs were used: (1) garden pea pods versus Ascochyta pisi (pea-PA), (2) rape pods versus Glomerella cingulata (rape-PA), and (3) broad bean leaves versus Glomerella cingulata (broad bean-PA). These pods were inoculated, on the inner epidermis, with spore suspensions, incubated for 24 hours at 20°C, then the spore suspensions were collected, and the resulting PAs were extracted with petroleum ether, the latter being evaporated under reduced pressure at 20°C, then dissolved in ethanol. Broad bean leaves were inoculated, after the epidermis of lower surface were peeled off, on this surface with spore suspension of Glomerella cingulata. The PA was prepared by the same procedure as above. Series of PA agar media were made by adding varying amounts of PA ethanol solution to malt extract agar so as to prepare the media containing the PA at the concentrations of 8, 4, 2, 1, 1/2, 1/4, and 1/8 times of that of original PA-solution in the case of pea-PA, and 4, 2, 1, 1/2 and 1/4 times in the cases of rape- and broad bean-PAs, assuming that PAs were extracted throughly from the original PA-solutions with petroleum ether. Each of these media, 0.7ml was poured into a watch glass, 30-32mm in diameter, and was inoculated with a disc of agar, 0.3mm in diameter, on which the fungus to be tested had been grown. After being incubated for 2-7 days at 20°C the diameter of the colony was measured. The results were presented in Figures 1, 2, and 3. These results show that the pea-PA strongly inhibits the growth of Piricularia oryzae which can not infect the pea plant, inhibits to a medium extent the growth of Glomerella cingulata, which is a facultative parasite to the same plant, while inhibits very weakly that of Ascochyta pisi which is really a parasite to the plant. The detoxication of pea-PA by A. pisi and also by Fusarium oxysporum, both being pathogenic to the plant, was certified by in-vitro tests. The disappearance of the opaqueness around the colonies of the fungi on the PA agar media indicated the existance of PA-decomposing ability of both fungi. Ethanol extraction of the transparent zone thus appeared on the agar media around the colony showed a very low inhibitory action on the conidia germination of Glomerella cingulata, and also a remarkable decrease in its optical density at 309mμ by spectrophotometer (Table 5).
In the previous paper, the author concluded that the detoxication of PMA by the rice leaf juice was due to the thiol group present in it. This paper deals with the stoichiometrical reaction of PMA with the known thiol compounds and thiol groups contained in the rice plant. The thiol group was determined by the amperometric titration method, by which SH in the range of 0.03-0.6μM was determined and 96per cent was recovered from glutathione (GSH). The SH contents of egg-albumin, yeast-extract, peptone, γ-globulin were 14.4, 1.23, 0.25, 0.1μM per gram respectively. The detoxication effect varied in this order. In the rice juice, from which protein had been removed, and in the yeast-extract, SH diminished more rapidly than in GSH solution. The SH in the rice juice decreased more slowly during the storage at 5°C than in GSH solution. The SH content was highest in the upper three leaves, about 300μM per 100g fresh weight, decreasing toward the lower leaves. The content was about 1/4 in the leaf still folded and below 1/10 in the leaf sheath, the stem and the root. The SH contents of these tissues varied in parallel with the protein contents. As the stage advanced from the vegetative growth to the reproductive, the SH content in the leaf decreased slowly but the SH became hardly extractable into the juice because of hardening of the tissues. The juice-SH reacted stoichiometrically with PMA at one to one mole ratio. When both PMA and the juice were added to the spore suspension of Cochliobolus miyabeanus, the spore germination was not inhibited until PMA exceeded the equal mole to the juice-SH. The elongation of germ tube was not inhibited until PMA exceeded the half mole of the juice-SH. When PMA dust is applied to the rice plant for the control of blast disease, the amount of PMA deposited on the rice leaves may be at most 1/100-1/1, 000, by mole, to that of SH present in leaves. The author concludes that the effectiveness of PMA for the blast disease is mainly due to the PMA present on the leaf surface or penetrated into the cell membrane, but not due to the PMA penetrated and diffused in the cell sap, because the latter PMA will easily be detoxified by the excessive amount of SH.
Peroxidase and phenol oxidase activity, O2-uptake, total phenols and o-diphenols content were determined for tubers of twenty two white potato varieties. The degree of resistance of the tuber tissues to Phytophthora infestans was evaluated for each variety by estimating spore production on the cut surface of tuber. In the case of resistance of tuber tissues to an incompatible race of P. infestans, the varieties exhibiting higher levels of peroxidase and phenol oxidase activity, and higher concentrations of o-diphenols were always highly resistant, but those having lower concentrations of o-diphenols were not necessarily susceptible. In the cases of tubers which were resistant to the compatible race (that is, field resistance of susceptible varieties), on the contrary, these correlations were not apparent. Apparent correlations were found between phenol oxidase activity and peroxidase activity, phenol oxidase activity and oxygen uptake, peroxidase activity and o-diphenols content, oxygen uptake and total phenol content, total phenol content and increase in o-diphenols content, and o-diphenols content and increase in o-diphenols content.
The authors have carried out taxonomic investigations of the causal fungus of Japanese pear scab for several years. The results are summarized in this paper as follows. 1. The overwintered spots of Japanese pear scab on the twigs are somewhat sunken and show distinct cracks surrounding the lesions, while those of European pears are more or less swollen, producing the stroma under the periderm in the spots. 2. The stroma of Japanese pear fungus develop well and conidiophores abundantly grow in cluster, causing dark sooty appearance of the spots. Conidia are 7∼23×5.0∼7.5μ in size, smaller than those of European pear fungus. 3. The perithecia of Japanese pear fungus are depressed conic globoid in shape and 50.0∼150.0μ in height, while those of European pear fungus are globoid and 77.5∼200.0μ in height, much taller than the former. 4. The ascospores of Japanese pear fungus are unequally 2-celled and measure 10.0∼15.0× 3.8∼6.3μ. They are smaller than those of the European pear fungus, especialy the lower cells are much shorter than those of the latter. 5. According to the inoculation experiments, Japanese pear fungus showed distinct pathogenicity to the leading varieties of Japanese pear, but was unable to attack the European pear varieties such as Flemish Beauty and Bartlett. On the other hand the European pear fungus infected only Flemish Beauty but no Japanese varieties. 6. Based on the data reported in the present as well as the previous papers (23) by the authors, the causal fungus of Japanese pear scab is considered to be a distinct species from European pear scab fungus, Venturia pirina Aderhold. The authors propose the name Venturia nashicola to the present fungus.“Nashi”represents pear in Japanese. The diagnosis of the fungus is as follows. Venturia nashicola n. sp. Perithecia gregarious or scattered under the epidermis of the affected leaves, depressed conic globoid, the wall dark colored, membranous, hard, and consist of two or three layers of brownish cells, 50∼150μ in height and 53∼138μ in diameter; ostiole very long. Asci hyaline, clavate or long ovate, 35∼60×5∼10μ; eight-spored. Ascospores hyaline or light brown, two celled, shoesole-shaped, 10∼15×3.8∼6.3μ. Conidia dark brown, long oval or fusiform, 7.5∼22.5×5.0∼7.5μ, and 14.4×6.0μ on the average. Conidiophores clustered, erect, and simple, 10∼20μ in length. Conidia borne singly on the warty tips of conidiophores. Habitat: On the leaves, twigs, and fruits of Pirus serotina Rehder and Pirus ussuriensis var. sinensis Kikuchi, producing scabby spots. Locality: Throughout Japan
1. Diseased mulberry trees growing in 6-inch pots were placed in constant temperature chambers at 30°, 25°, 20°, 15°, 10°, and 3°C, in December, before defoliation. The trees kept at 30°, 25°, and 20°C developed symptoms on all newly growing shoots, whereas those kept at 15°C produced normal leaves at the beginning, but showed symptoms on later growing apical leaves. 2. Diseased trees were placed in chambers at 30°, 25°, and 15°C, in April, after defoliation. All the trees developed normal leaves at the beginning, but gradually showed symptoms on later growing leaves, irrespective of temperature. 3. Cuttings obtained from diseased trees which had been kept at 10° or 3°C from December to May or June, when grown in a green-house, mostly showed recovery from the disease, while root stocks of the same plants, when placed at 30°C, all developed diseased shoots. 4. It is considered that, during winter, the virus in the affected shoots either becomes inactivated or otherwise decreases at temperatures below 15°C, but not at temperatures above 20°C, whereas in the roots the virus does not decrease even at low temperatures. When diseased trees sprout in April, apparently normal leaves are produced at the beginning, and symptoms appear gradually afterwards. These normal leaves are produced possibly because the virus in the shoots has been inactivated during the winter, and the symptoms are incited by virus moved from the roots, multiplying in the newly growing shoots.
1. Twenty-seven species of non-leguminous herbaceous plants known to be susceptible to virus diseases were selected from twelve families including Cruciferae, Chenopodiaceae, Cucurbitaceae, Solanaceae, Compositae, Amarantaceae, and others. They were inoculated with the sap from the young shoots of Satsuma orange affected by Satsuma dwarf. Among them only sesame (Sesamum indicum L.) proved to be susceptible to this disease, showing local lesions on the inoculated leaves, and also vein clearing, vein necrosis, curling and malformation on the upper leaves. 2. Cross inoculations between sesame and Blackeye cowpea or between sesame and Chashiroingen, a variety of kidney bean were positive. Therefore the virus in the infected sesame plants is apparently the Satsuma dwarf virus which has been transmitted by sap-inoculation to leguminous plants as previously reported by the authors. 3. Sesame proved to be susceptible to Satsuma dwarf virus, but nonsusceptible to tristeza and vein enation viruses which are commonly carried in Satsuma orange plants. 4. Among three varieties of sesame tested, White sesame (Shiro-goma) was the most susceptible. Brown sesame (Cha-goma) was somewhat less susceptible whereas Black sesame (Kurogoma) was infected systemically showing no local lesions. 5. No significant difference in susceptibility to the present virus was shown at different growing stages of individual sesame plant. However, plants inoculated at the first or second true leaf stage developed the most distinct symptoms and such young plants proved to be the most suitable indicator for Satsuma dwarf virus. 6. Sesame plants are much more heat-tolerant than most of the Leguminous plants, and the former is useful as an indicator for the present virus even in the midsummer season. 7. When sesame plants were exposed in a higher temperature than 34°C immediately after inoculation, they usually showed no symptoms, but sometimes systemic infection appeared ten days after inoculation. On the other hand, when the inoculated sesame plants were kept more than eight hours at 25°C, they showed striking symptoms even if they were later exposed to temperatures higher than 36°C. 8. When sesame seedlings were inoculated with the sap from shoots of affected Satsuma orange, adjusted to different pH by McIlvaine buffer, infection was obtained with inocula at pH 5-8, with the optimum at 7-8.
1. Using three wheat varieties, Norin No. 50 (susceptible), Norin No. 61 (moderately resistant), and Yuyake-komugi (resistant), the mode of disease appearence of flag smut (Urocystis tritici Koern.) was examined, when soil temperature was regulated at 5, 10, 15, 20, and 25°C, respectively, during 24 days after the seeds were inoculated with the fungal spores and sown in sterilized soil. In Norin No. 50, 98 to 99per cent of the plants were diseased at 10 to 20°C. In most cases, main culms showed symptoms, and 96 to 98per cent of tillers of the diseased plants were also smutted. At 25°C and 5°C, percentages of diseased plants were lower than at 10 to 20°C (Table 1). In Norin No. 61, many plants were diseased, except for 5°C, and the disease appearence was characterized by very low percentage of smutted main culms in contrast to disease development on tillers in almost all diseased plants. In Yuyake-komugi, no plants were diseased. The mode of disease appearence in these varieties as mentioned above, seemed to be attributable to intrinsic resistance to flag smut of the varieties, which is not much influenced by soil temperature. 2. 14 days after the seeds of each variety were inoculated with the spores and sown in the soil regulated at 15°C, some seedlings were picked up from the soil and the distribution of hyphae within their tissues was examined under a microscope. Remaining seedlings were transplanted to sterilized soil in pots, and disease development was observed as the plants became mature. Hyphal penetration was found to occur through epidermal cell wall of coleoptile, and the hyphae advanced in the direction shown in Fig. 1. In Yuyake-komugi, the penetration was checked by callus formed at the point of hyphal entry. The callus was characterized by a thickening, both inside and outside, of the epidermal cell wall and lustrous yellow coloring. In rare cases, callus was pierced by penetrating hyphae, but the hyphae died within the epidermal cell, presumably due to disorganization of cytoplasm of this cell, and could not penetrate into neighbouring cells. In Norin No. 50, most penetrating hyphae could invade epidermal cells of coleoptile without producing callus, but in some cases, callus was noted around the penetrating hyphae on the inside of the epidermal cell wall. Further development of the hyphae was checked. In Norin No. 61, the mode of penetration of the fungus was not much different from that in the susceptible variety, although callus formation was rather frequent. 3. Percentage of diseased plants observed at maturity stage was 99per cent in Norin No. 50, 63per cent in Norin No. 61, and 0per cent in Yuyake-komugi (Table 2). According to microscopic observations, percentage of seedlings which had coleoptile tissues occupied by the invaded hyphae was 100per cent in Norin No. 50, 50per cent in Norin No. 61, and 0per cent in Yuyake-komugi. These facts seemed to indicate that callus formed at the point of penetration would have an important role in the resistance of the host to the fungal attack. On the other hand, symptoms appeared on main culms in 96per cent of the tested plants in Norin No. 50, 9per cent in Norin No. 61, and 0per cent in Yuyake-komugi. Percentage of plants in which the hyphae reached the epicotyle tissues or near the meristematic regions, developing through the coleoptile tissues, was 90per cent, 25per cent, and 0per cent, in respective varieties. These facts showed that the hypha of flag smut fungus could reach the growing points of main culms when hyphal development in the coleoptile tissues was rapid, and that the hyphal development in the tissues was more rapid in Norin No. 50 than in Norin No. 61.
Comparisons were made among three pathogenic soil fungi. Pythium aphanidermatum, Rhizoctonia solani (an isolate from broad bean seedling) and Sclerotium rolfsii, concerning their vertical distributions in soil. Mycelial density at each depth in soil was measured by contact slide method, and the pathogenicities of the fungi inoculated at different soil depths were determined by the use of indicator plants. R. solani maintained a high mycelial density even at the depth of 10-15cm, P. aphanidermatum at 5-10cm, while S. rolfsii only at the surface layer. When inocula were buried at different depths in soil, the pathogenicity of each fungus was the highest at 5-10cm with R. solani, at 2.5-10cm with P. aphanidermatum, while at the only surface with S. rolfsii. There was better mycelial growth toward the upper and lower layers with both R. solani and P. apanidermatum than that with S. rolfsii.
The bacterial leaf spot of alfalfa caused by Xanthomonas phaseoli f. sp. alfalfae was observed for the first time in Japan at the farm of National Institute of Animal Industry, in September 1962. Leaf spots are at first minute, round, watersoaked. Mature spots 2-3mm in diameter are pale yellow with a dark brown margin surrounded by a yellowish halo. The spots tend to coalesce along midrib and near the tip or the margin of the leaflets. Severely infected leaves often shed. The causal bacteria infected Phaseolus vulgaris by inoculation producing symptom similar to that produced by Xanthomonas phaseoli on the same host. The morphological, cultural and phsiological characters of the bacteria causing the disease were investigated, and hardly distinguishable from those described by Riker et al. (1935) and Sabet (1959). Therefore the causal bacteria were quite identical in all its characteristics with Xanthomonas phaseoli f. sp. alfalfae (Riker, Jones et Davis) Sabet.