In the present paper, the inhibitory activities of some antibiotics and fungicides, such as Antipiriculin-A (Antimycin-A, AP), Blastmycin (BM), and phenyl mercuric acetate (PMA), against different growing stages of P. oryzae were studied. The results obtained are as follows: (1) Inhibition to spore germination. Under an adequate condition of temperature and moisture, 20∼30per cent spores of the fungus germinated within 6 hours and 80∼90per cent germinated 24 hours after incubation. All chemicals prevented comparatively the germination in the early stage of incubation, whereas these activity became feeble 24 hours after incubation. Among the chemicals tested, PMA showed to be the most effective and the crystalline antibiotics, AP and BM, did not show so much effect, probably because of their lower solubility in water. However, one of the solubilized antibiotics (e. g. AP-emulsion) showed satisfactorily the same inhibitory effect as did PMA. (2) Inhibition to growing mycelium. When the chemicals was added into flasks, in which mycelia of the fungus had been inoculated, a remarkable inhibition to mycelial growth was observed. However, the addition of chemicals at the stationary growth phase could not obtain the inhibitory activity except the addition of AP-emulsion. AP-emulsion not only prevented the mycelial growth, but also lysed the mycelial content. (3) Inhibition to sporulation. Spore suspension of the fungus was inoculated in Petri dish containing agar medium and was incubated at 27°C. When the vegetative mycelia were grown, the chemicals were added by spraying them on mycelia. No chemicals showed the inhibitory effect against the sporulation of the fungus. (4) The clinical concentration of growth inhibition. Using the agar dilution method, PMA was found to prevent the growth of the fungus at 0.05mcg/ml and AP-emulsion at 0.1mcg/ml. It is supposed, therefore, that the satisfatory effect against the rice blast fungus may be obtained by AP-emulsion spray in the field test.
In the present paper, the inhibitory effects of Antimycin-A (Antipiriculin, AP), Blastmycin (BM), and Phenyl mercuric acetate (PMA) on the fundamental metabolic systems of P. oryzae are reported. The enzyme solution of P. oryzae was able to oxidize some organic acids found in TCA cycle, and this oxidation, especially that of succinate or lactate, was remarkably inhibited by AP, BM, and PMA. The endogenous respiration of the enzyme solution, however, was not inhibited by these inhibitors, but was inhibited by a high concentration of PMA. As to the terminal respiratory systems in P. oryzae, the following series of hydrogen doner and acceptor systems were presented, that is, succinic dehydrogenase, succinoxidase system, and cytochrome oxidase system. Both AP and BM showed definitely a specific inhibition to the succinoxidase system, whereas PMA inhibited all these systems. The oxidative phosphorylation and glycolysis in P. oryzae were also found not to be affected by AP and BM, although PMA seemed to inhibit the glycolysis slightly.
This paper describes the nitrogen metabolism of P. oryzae and its inhibition by Antimycin-A (AP), Blastmycin (BM), and Phenyl mercuric acetate (PMA). According to the rate of growth of P. oryzae, the various nitrogen sources tested were divided into three groups. The most suitable group for fungal growth contains, for instance, glutamic acid, aspartic acid, serine, leucine, NH4Cl and so on. The amino acids belonging to the most suitable group were oxidized by P. oryzae in a short period of incubation, and the most of these oxidation was completely inhibited by AP, BM, and PMA. Two types of transamination were found in P. oryzae. One of these reaction was that of between α-ketoglutaric acid and alanine, and another was between α-ketoglutaric acid and aspartic acid. In the case of cell level, the former reaction was completely inhibited by AP, BM, and PMA at 18 hours after incubation, while the latter was moderately inhibited even by PMA. Using the extracted enzyme solution, however, no inhibitory effects of both AP and BM on these two reactions were confirmed, although a strong inhibition was detected by PMA.
The effect of light on maturation of apothecia of Sclerotinia trifoliorum Eriks. was tested by means of planting the sclerotia in moistened sand under the different light conditions in the glass house (15∼20°C). The maturity of apothecia was observed by dividing into the following three grades: i. e., the mature apothecia of which discs expand normaly, and discharge numerous ascospores; the intermediate apothecia having fleshy and long stipes, and bearing relatively less amount of ascospores; and the immature apothecia which have slender, longer and peaked apical ends and bear no ascospore. The treatment of sclerotia under continuos darkening for 10 days did not affect the maturation of apothecia if they were transplaced under the natural light condition, but the dark treatment for more than 15 days disturbed their maturation in proportion to the length of treatment. Namely, by the continuous 25-days-darkness, 70 per cent of the immature apothecia was resulted despite the keeping of natural light for 15 days after the same darkness mentioned above, but in the continuous darkness for 40 days all the apothecia lasted immature. The apothecia matured also under the contiunous fluorescent light (White 1, 800lux), and 3-days-alternation of darkness and light, but 15-days-alternation of darkness and light affected slightly to maturation. On the contrary, most of apothecia were immature by 15-days-alternation of light and darkness. And by the other different treatments of change from darkness to light or from light to darkness, mature, intermediate and immature apothecia appeared at the different percentages (v. fig. 2). Under the light conditions filtered through the cellophanes of different colours, i. e., colourless, yellow, blue, green, and violet, apothecia matured generally, except red cellophane under which many of them lasted immature and failed to produce spores. As regard to the intensities of illumination, apothecia lasted immature under the illumination less than 100lux, but 50per cent of them were intermediate under 200lux, and 78per cent matured under 600lux, in comparison to full maturation under the conditions of 1, 000∼1, 400lux.
The growth stimulating effect of the contents of Satsuma orange fruit rind to Penicillium digitatum (green mold) and P. italicum (Blue mold) was studied. Extract from the orange rind by 80∼99.5% methanol or 80% acetone showed a promotive effect for the mycelial growth of both fungi. The growth stimulative substance was readily adsorbed by active carbon; and on the agar media containing the rind extract treated with carbon, the mycelial growth of P. digitatum was decreased even when treated with a small amount of carbon (0.5g per 80cc). In the case of P. italicum more vigorous growth was obtained when cultured on the rind extract agar treated with small amount of carbon than on the untreated media. On the media, however, containing the rind extract treated with much carbon (1 to 2g per 80cc) under weakly acidic, the growth of P. italicum was also inhibited as in the case of P. digitatum. From these experimental results it was concluded that the extract from the orange rind had two factors, the one stimulative and the other inhibitory to the growth of P. italicum, and the latter did not play its role in case of P. digitatum. It was indicated that the vigorous growth of P. digitatum on the media of orange rind tissue and also on orange fruit would depend upon this stimulative effect of the rind contents.
1. This paper deals with the host range, properties, purification, and electron microscopy of the radish P virus. In a preliminary survey of the viruses of Japanese radish mosaic disease complex in the suburbs of Tokyo, made jointly with Mr. Yasuo Komuro, it was found, that besides the cucumber mosaic virus, three other viruses, which we have termed P, Q, and R, are involved. These viruses occur either singly, or as combinations of two, three, and four. While the virus Q is apparently distinct, both the viruses P and R are considered to belong, in the conventional taxonomy, to the turnip mosaic virus group. There are, however, some indications that these two viruses are rather related remotely with each other. Most of the experiments reported here, unless otherwise stated, were made using a virus isolate P0, which was isolated in May 1956, from a mosaic radish plant in Matsudo, Chiba Prefecture, and was maintained in a greenhouse, by successive juice inoculations on turnip plants. 2. Host range. (Mosaic, or mottle): Brassica rapa var. glabra, B. rapa var. pervidis, B. juncea (two subvarieties), B. nigra, B. campestris, Raphanus sativus var. acanthiformis (five subvarieties), Matthiola incana, Spinacia oleracea (subvariety Ujo), Chrysanthemum coronarium, and Petunia hybrida. (Symptomless carriers): B. oleracea var. capitata (subvariety Yoshin), and B. napus. (Necrotic local lesions on inoculated leaves): Gomphrena globosa, and Chenopodium album. (Not susceptible): N. tabacum (subvariety Bright yellow), N. glutinosa, Vigna sesquipedalis, Vicia faba, Cucumis sativus, Beta vulgaris var. flavescens, Datura stramonium, Lycopersicum esculentum, Zinnia elegans, Brassica oleracea var. capitata (subvariety Hachigatsudori), B. oleracea var. botrytis, and Spinacia oleracea (subvariety Nihon). N. glutinosa was found to be infected by the P virus, when inoculated together with cucumber mosaic virus. 3. Properties. Dilution end-point in extracted leaf juice was 1:2000∼1:5000. Thermal inactivation point was between 55 and 60°C, in 10 minutes treatment. Longevity of the virus in crude juice was between 4 and 7 days at 25∼26°C, but this period became much longer, when a little KCN was added to the leaf juice. In a frozen-dried preparation of leaf juice, the virus still retained infectivity after 23 months. 4. Aphid transmission. The P virus is readily transmitted by the aphid, Myzus persicae, except for the particular virus isolate P0, which has been maintained in a greenhouse for more than two years by successive juice inoculation. Although this isolate P0 was found not transmissible by the aphid, a sub-isolate P'0, which was derived from the same source as P0 but which had been kept in a frozen-dried juice preparation, was found to be transmissible by the aphid. 5. Purification. Attempts were made to obtain a purified virus preparation, using diseased turnip (subvariety Kanamachikokabu) or petunia leaves. The final procedure adopted was as follows: (freezing and thawing of leaf tissues or juice) → combined procedures of repeated salting-out by ammonium sulfate and differential centrifuging → (treatment with chloroform). The final product was almost colorless, and the yield was about 10∼15mg per 100ml leaf juice. This purified virus preparation diluted to (2∼3)×107 (dry weight basis) was still capable of infecting 20 per cent of inoculated turnip plants. 6. Electron microscopy. Examination of the purified virus preparation revealed the presence of sinuous rod-shaped particles, of about 12∼13mμ in width, with the most frequent length of 650∼700mμ. The preparation was found to be almost free of other particulate matter.
1. This paper deals with serological experiments using the radish P virus. The virus used as immunizing antigen was the isolate P0, which was obtained from a mosaic radish plant in Matsudo, Chiba Prefecture, in May 1956, and was maintained in a greenhouse by successive juice inoculation on turnip plants. 2. By a combined procedure of salting-out with ammonium sulfate and differential centrifuging, a partially purified virus preparation was obtained from infected turnip leaf juice. Antiserum was prepared by injecting rabbits with this virus preparation. The rabbits were given 7∼8 intravenous injections (equivalent to total leaf juice volume of 150ml), at 2∼7 days intervals. The titer of the antiserum obtained was 1:10, 000∼1:40, 000, in precipitation reaction tests. 3. The antiserum reacted specifically with leaf juice of Brassica rapa var. glabra, Raphanus sativus, Brassica pekinensis, Petunia hybrida, Chrysanthemum coronarium and Spinacia oleracea, which were infected with the radish P virus; also with leaf juice of mosaic radish plants collected in the suburbs of Tokyo, which were judged, by host range tests, to be infected with the radish P virus. The antiserum did not react with leaf juice of healthy plants, plants infected with radish Q virus, radish R virus, cucumber mosaic virus, or several other viruses. In absorption tests, the antiserum completely absorbed the radish P virus, but not other viruses. 4. Virus dilution end-point was: ×300 in agglutination reaction on slide glasses, and ×2, 000 in precipitation reaction, using turnip leaf juice; 0.002∼0.005mg/ml (virus dry weight basis), using purified virus preparations. 5. Out of 231 mosaic radish plants collected in the suburbs of Tokyo, 106 reacted with the antiserum. In host range tests of the viruses involved, radish P virus, radish Q virus, radish R virus, and cucumber mosaic virus were detected. As judged from the combined results of these serological tests and host range tests, 31 out of 76 plants tested were found to be infected with a single virus, the rest being infected with two, three, or four viruses.
(1) The activity in vitro of a new antifungal antibiotic named Blasticidin S and phenyl mercuric aceate against rice blast (Piricularia oryzae) was studied. The germination of spores was completely prevented at a concentration of 1μg/ml of both substances, when tested by the drop culture method. The inhibition effect of Blasticidin S against the formation of spores was also almost the same as that of phenyl mercuric acetate. But Blasticidin S was more effective to the inhibition of mycelial growth than was phenyl mercuric acetate. The concentration at which the growth of mycelium was almost completely inhibited, when grown in shaken liquid media, was less than 1μg/ml for Blasticidin S, and more than 10μg/ml for phenyl mercuric acetate. (2) The stability of Blasticidin S on leaves of rice plants growing in the greenhouse was studied by the cup method. Blasticidin S could be detected at a concentration of 0.1μg/ml by the cup method assay using a spore suspension of Cladosporium fulvum as the test organism. The activity of Blasticidin S on leaves decreased gradually, and about one fifth of the original activity was found after four days. (3) When the first internode of the rice seedling was coated with paste containing Blasticidin S, the antibiotic was found to be translocated to the upper stems and leaves. The paste was composed of 2 per cent antibiotic, 38 per cent petrolatum liquidum, 50 per cent petrolatum album, and 10 per cent glycerin. The leaves and stems above the treated stem were found to contain about 30μg/g of the antibiotic one day after the treatment. (4) In the greenhouse Blasticidin S was found to be effective as a therapeutic fungicide against rice blast. When plants were sprayed with a solution containing 5μg/ml of Blasticidin S one or two days after inoculated with the spore suspension of P. oryzae, the number of leaf-spots developed on the plants was less than on those treated with 20μg/ml of phenyl mercuric acetate. However, when sprayed four days before inoculation, the protective effect of Blasticidin S was inferior to that of phenyl mercuric acetate.
Correlation between laboratory and field effectiveness of various fungicides against Piricularia oryzae, which causes rice blast, was studied. The fungicides tested were seven chemicals, copper sulfate, phenyl mercuric acetate, Zineb, Urbazid, Dichlone, Nirit and s-Triazine, and two antibiotics, Antimycin A and Blasticidin S. Among them phenyl mercuric acetate, Antimycin A and Blasticidin S had been known to be more effective as protectant fungicides against rice blast in the field than others. In the laboratory we observed the inhibition effects of these fungicides on spore germination, mycelial growth by shaking culture, and spore formation of P. oryzae. And also the minimum inhibitory concentration by the agar dilution method and the size of inhibition zone on agar plate by the cup method were measured. The spore germination was perfectly inhibited by all the fungicides tested, and the minimum inhibitory cencentrations of all the substances tested except copper sulfate were almost the same. The result measured by spore germination or agar dilution method seemed not to agreed with the field results. The mycelial growth and the spore formation were markedly inhibited by phenyl mercuric acetate, Blasticidin S and Antimycin A, but littele or less by others. Satisfactory correlation between the laboratory results measured by mycelial growth or spore formation method and the field results was obtained. Therefore the inhibition effect on mycelial growth and spore formation seemed to play an important role in determining a compound as a practical fungicide against rice blast in the field than that on spore germination. The sizes of inhibition zone were larger in the case of the three fungicides having field effects than in the case of others.