The present paper deals with a study on tobacco mosaic virus (TMV) contained in manufactured tobacco. The following kinds of commercial tobacco which had been manufactured in Japan in 1954, were tested in order to see to what extent they might carry the TMV: 1) six kinds of cigarettes and their scraps, 2) one kind of cigars, 3) two kinds of pipe obaccos, 4) three kinds of fine cut tobaccos and their scraps. The samples were collected from 21 factories all over the country, and were tested by rubbing inoculation of tobacco seedlings with extracted juice with carborundum. Of 126, 126, 3, 6, 42 and 42 samples tested, 123 (97%) of cigarettes, 124 (98%) of their scraps, 3 (100%) of cigars, 3 (50%) of pipe tobaccos, 36 (86%) of fine cuts, and 41 (98%) of their scraps, respectively, caused mosaic on inoculated tobacco plants. None of samples of Momoyama, pipe tobacco, caused mosaic. However, the yellow component separated from the sample caused mosaic, though the blackened one did not. According to the tests of local lesion method on Nicotiana glutinosa, there were no consistent differences in TMV concentration between cigarette and fine cut, and between each of them and its scrap. The TMV concentrations examined in 3 cigarette samples were about 5×10-4-10-4 of that in fresh mosaic leaf grown in a greenhouse, per same weight. These figures are rather high, regarding the danger of infection of tobacco plant in the field. Electron micrographs of three preparations of TMV purified by ultracentrifuge from distilled water extracts of cigarette, from tobacco plants inoculated with the extracts, and from plants inoculated with the ordinary strain of TMV (a strain stocked in our laboratory as an ordinary one), show that the length distribution of particles is generally similar, but there was some indication that the former two preparations contained more short particles than the last one.
The present state of knowledge on the chemotherapy for plant virus diseases has recently been considerably broadened. The discovery of purines and purine analogs has made it possible, with greater success, to provide a reliable information on this subject. Furthermore, R.A. Gray has reported a new antibiotic, noformicin, which inhibits both the production of local lesions and systemic infections caused by southern bean mosaic virus and by tobacco mosaic virus in intact plants. In the following are presented the results of the studies, although the preliminary nature, obtained in an effort to extend a fundamental investigation on this approach, especially on the effect of the new antibiotics upon the tobacco mosaic virus (TMV) multiplication in tobacco leaf tissues. The mature leaves which had been inoculated one day ago by rubbing with a gauze pad soaked in TMV-infected tobacco juice, were removed from tobacco plants (Nicotiana tabacum, Turkish) and split along the midrib. One-half of each detached leaf was floated on the solution containing the antibiotics in Petri dishes under fluorescent lamp in an incubator at 20-25°C. The opposite leaf-halves which had been inoculated with the same virus were kept on water and served as controls. After 3-4 days incubation, the leaf tissues were removed and ground in a mortar for virus analysis. The procedure shown in figure 1 was undertaken for precipitating virus protein, the relative concentration of which in each samples was determined by a colorimetric procedure using the Folin reagent. Readings were obtained with an electrophotometer. Phytotoxicity of the antibiotics: Injury symptoms were noted for each antibiotics over a wide range of concentrations. Moreover, the rate of respiration of leaf disk floated on the antibiotic solution was measured by using Warburg's manometer and was compared with the control disk placed on water. Antibiotic translocation: Translocation in the detached tobacco leaves of the antibiotic solution was ascertained. Leaf tisssues floated on the solution were crushed with mortar and pestle to secure an extract for antibiotic assay. These extracts were assayed by the cup-method using the test organisms for each antibiotics. Inhibition zones were measured after 18 hours incubation at 37°C. The antibiotics used in these studies were supplied from the Research Laboratory, Takeda Pharmaceutical Industries, Ltd. and most of them were the crude substances. Some of the results obtained are summarized in tables 1 and 2. As seen in the tables, 24 samples out of 62 samples tested were found to exhibit 20 per cent or more virus-inhibition. Most effective inhibition to virus multiplication was 58 per cent shown by the sample No. 62, whereas 8-azaguanine (10-3M) and noformicin (200 ppm, crude), the latter was kindly supplied by Dr. R.A.Gray, showed 32 and 39 per cent virus-inhibition, respectively, under our experimental conditions. The successive experiments, applying the antibiotic solution by leaf-spraying method and so on, are now under progress.
In the author's former report 5-8, it was shown that the content of epidermal midrib cell of resistant potato varieties degenerates according to the following scheme, when infected by P. infestans. To ascertain whether such a chain of cell physiological phenomena in degeneration process is in a causative linkage or not, following experiments were carried out. Petioles of Hokkai No. 10 (inter-specific hybrid variety) were cut in halves longitudinally, and washed with running water, and then the cut surfaces were inoculated with zoospore suspension of P. infestans (race O). The variety used is highly resistant to race O. About 2.5hrs. after inoculation, 2, 4-dinitrophenol (DNP) 5×10-4M. solution or H2O (as control) was vacuum-infiltrated into the inoculated tissue, which was left in the solution for 30min. Then the tissues were kept in petri dishes at 19-20°C. DNP is known to be an inhibitor of oxidative phosphorylation, and poisonons for protoplasmic streaming. At the time when the tissues were vacuum-infiltrated, a large number of appressoria were beginning to extend the infection hyphae into the host cell. The vitality of the infected cells was judged by means of vital staining with neutral red or by recognizing the existence of protoplasmic streaming. The results obtained are graphed in fig. 1. The death of the invaded cell was remarkably delayed by the post-infectional DNP treatment. Accordingly it will be presumed that acceleration of cytoplasmic movements induced by infection of P. infestans is closely associated with the death of that invaded cell. In addition, it will be reasonable to suppose that the energy is required for the quick occurrence of the death of the hyper-sensitive cell invaded by P. infestans. Other possible explanations of the delay of the cell-death by post-infectional DNP treatment, however, should not be overlooked. It is possible that the effect of DNP upon the intra-cellular hyphae gives rise to the delay of the cell-death. In fact, the velocity of development of intra-cellular hyphae in a cell treated with DNP was inhibited by about 30% as compared with that in untreated one (Table 1). The vigorous hyphal growth in later stages in the living cells treated with DNP, however, seems to indicate that the effect of DNP upon the hyphal development cannot be the cause of delay of the cell-death. It is possible, however, that the treatment would affect the production of toxic substance by intra-cellular hyphae of P. infestans, had it been really produced, thus inducing the delay of cell-death. This possibility should be ascertained in future expemiments.
The pathogenic races of Phytophthora infestans isolated in Japan have been classified according to the international system of nomenclature, using a series of differential hosts of Dr. W. Black. Results obtained are as follows; Domestic International group 0 (H1): race 0 group I (H3): race 1 group II (H2): race 4 group III (H2, 3): race 1, 4 group IV (BH4): race 2 ( ) Strains representing each group, and used in the former reports. As to genes for resistance, Takase's genes Ra and Rb have been found to be identical with international genes R1 and R4 respectively in their specific reactions to races of the fungus.
1. The mycelial strand of Helicobasidium mompa TANAKA penetrates the cork layer of sweet potato by breaking through middle lamella of it. The pectic material in contact with the mycelia is gelatinized and has a very low pH value. Following the penetration of the cork layer, the mycelia rapidly macerate the starchy parenchyma and liquefy it. The liquid thus produced is strongly acidic. 2. The optimum pH value for the protopectinase activity of the fungus lies between 2.5 and 3, 0. Above 5.0 the enzyme is almost inactive. 3. From expressed juice of Halicobasidiumrotted sweet potatoes crystalls of itaconic acid were isolated. 4. Itaconic acid was also obtained from culture filtrate of the fungus, yielding 0.5 to 1.0g. of crude crystals from 500ml. of 30 day's culture filtrate. In young culture, for example up to 30 days old, oxalic acid was not detected. The above results suggest that itaconic acid produced by the fungus plays an important role in lowering the pH of the infected tissue, and enhancing the protopectinase activity and intensifing the pathogenic action of the fungus.
An antibiotic substance has been purely isolated from the culture filtrates of Gloeosporium olivarum Alm. which was grown for 12 days in the presence of 0.03% 2-methyl-4 chlorophenoxyacetic acid (MCP). The yield was 0.181mg/ml of the original filtrate. This compound of white needle-shaped crystals, m. p. 55°C, is thermostable, nearly insoluble in cold water, but soluble in hot water and many organic solvents. It contains C, H, O, Cl and the hydroxyl group characteristic to a primary or secondary alcohol, but does not contain S, P and N·The one-dimensional paper-partition chromatogram of the antibiotic developed by ascending technique with acetone-methanol-water mixtures (30:5:65v/v) showed a single inhibiting zone at Rf 0.73. When the antibiotic was incorporated into agar media at the dilution of 1:5, 000, the growth of G. olivarum and Cochliobolus miyabeanus was almost completely inhibited, the dilution end-point being about at I:40, 000 and 1:30, 000 against the former fungus and Corticium centrifugum respectively. Conidial germination of G. olivarum was entirely inhibited at 1:3, 000. In the other experiment by paper disk method, it also exerted a toxicity against other 5 species of phytopathogenic fungi and 5 species of bacteria. The inhibitory activity of MCP in vitro on G. olivarum is considered to be attributable to this antibiotic produced by addition of MCP rather than to its direct effect on the pathogen.
The Corticium foot-rot disease caused by Corticium gramineum Ikata et MATSUURA, occurring particulary on the basal part of wheat stem, is characterized by the dark-brown lesion and later by the decay of stem lesion and infection of young leaves in severe case. The effects of soil types, organic manure, chemical fertilizers such as ammonium sulphate, calcium superphosphate, potassium chloride and lime on the development of the disease were studied by the artificial inoculation during two seasons at Akashi, Pref. Hyogo. The results of these investigations showed that the least injury occurred on the wheat plants grown in the sandy soil in which added organic manure, chemical fertilizers together with lime. The sandy soil seemed to be more unfavorable to the development of the disease than the loam and clay soil. There were marked seasonal changes in the microbial populations with the difference of the soil types and the fertilizers. The severity of the disease generally increased with the number of fungi in soil, while decreased with that of bacteria.
Cochliobolus miyabeanus was inoculated on the normal and "Akiochi" (autumn-decline) rice plant and the changes of nitrogen compounds, carbohydrates, reducing ascorbic acid and respiration of the plants accompanied by the infection, were examined. In both plants, soluble and insoluble nitrogens decreased by the infection, though the "Akiochi" plant showed less insoluble nitrogen from the outset. Reducing sugar, soluble cardohydrate also decreased while insoluble carbohydrate increased. Reducing ascorbic acid suddenly decreased in both plants, but the "Akiochi" plant had little amount of the acid before the infection. Qo2 increased and R·Q decreased in both plants. The decrease of R·Q was much greater in the "Akiochi" plant than in the normal one. The examination of the existence of hyphae in the spots showed that the fungus lived in every part of them. From the perthophytic action of this fungus and these results, the writer thought that the defence layer was formed and the fungus was shut out by the layer and the spread of spot was ceased.