The present investigation was undertaken to demonstrate the fungicidal mechanism, mainly the inhibition to the respiratory enzymes of Piricularia oryzae, of 1-(3, 4-dichlorophenyl)-2-acetylhydrazine (I) as an example of β-acylphenylhydrazine derivatives. Compound I inhibited only the increase of oxygen uptake by intact mycelium in the following substrates; glutamate, fumarate, succinate and glucose. In contrast to these of intact mycelium, the respiration of spores was not affected by I. The locus of inhibition of I in the electron transfer system is the site of the SC-factor, and this site coincides with the inhibition site of phenylhydrazines described by Ozawa and Asami. The inhibition patterns to the succinate oxidase system, especially succinate-cytochrome c reductase, and to glutamate decarboxylase support the assumption that compound I shows marked inhibition by the hydrazine decomposed in the mycelium of P. oryzae. Compound I also inhibited moderately the rate of oxidation of NADH2 in transketolase system of the hexose monophosphate shunt.
Quantitative analyses of total phenol, o-diphenol, chlorogenic acid, caffeic acid, tyrosine and Klarson lignin in potato-tuber tissues neighbouring cells infected by an incompatible race of Phytophthora infestans were done. Distribution of phenolic compounds as a function of distance from the infected cut surface showed that the tissue zone where the metabolism of phenolic compounds is markedly accelerated by infection by an incompatible race was about 10∼15 cells in thickness. During the first 24hrs, after inoculation, the content of total phenol and other phenol (total phenol minus o-diphenol and tyrosine) increased more rapidly in this zone than in the corresponding uninfected control. However, later (48hrs, after inoculation), the higher the concentration of inoculum, the lower the content of total phenolie, o-diphenol, chlorogenic and caffeic acid in this zone as compared with the uninfected one. Neverthless, the incorporation rate of radioactivity of D-glucose-14C (U.L.) into chlorogenic and caffeic acid was not reduced by the inoculation with dense inoculum, although total radioactivity of the disk fed with D-glucose-14C (U.L.) was distinctly lower in the tissue underlying the infected zone than in the corresponding control. These results indicate that there is an increase in the turnover rate of phenolic compounds in tissue adjacent to the infection regardless of density of inoculum. An increase in the infection of the so called “Klarson lignin fraction”, which may involve browning substances, suggests that there is an intimate relation between the phenol metabolism in this tissue zone and repairing in the infection.
One Erwinia and two Pseudomonas were isolated from healthy rice Kernels which had been newly croped in Akita Prefecture. The one was identified as Erwinia herbicola (Düggeli) Dye (1964) and the other two were belonging to the fluorescent group named by Iizuka and Komagata. Erwinia one showed agreement with the description of Pseudomonas trifolii Huss isolated by Iizuka and Komagata except for the maximum growth temparature, flagellation and acidity in starch-peptone medium. One of the isolates in the fluorescent group of Pseudomonas showed agreement with the description of Pseudomonas straminea which was newly isolated by Iizuka and Komagata except for the three polar flagella and the assimilation of 2 keto-gluconate. Another white one was believed to be Pseudomonas schuylkilliensis which showed agreement with the description of Iizuka and Komagata except for the feeble hydrogen sulfide production and assimilation of salicylic acid. Rice seeds did not show no symptom on the surface of them when they were inoculated.
The nature of TMV mixed with RNase was investigated. No differences were found in the electrophoretic behavior or anion exchange chromatograms of TMV treated with RNase compared with that of TMV alone. However, the cationic character of the virus was changed by adding the enzyme. The amount of TMV-RNase complex adsorbed to tobacco cell debris was not enhanced by the addition of phosphate in the medium, unlike the phosphate effect with TMV alone. TMV mixed with RNase was very stable in the alkaline solution (pH 10), but a small portion of the TMV-RNase complex was degraded. Moreover, RNase did not stabilize the TMV protein polymer (rod) when the pH of the solution was raised from 5.9 to 7.6. It was then discussed that the reduction of the virus infection by RNase might be caused by the changes in the adsorbing ability to the host and the structural stability of the virus.
A virus isolated from a mosaic plant of amaryllis (Hippeastrum hybridum) in Shizuoka is identified as Hippeastrum mosaic virus (HMV), because of its similarity to the description of HMV by Brants et al. (1965) in host range, physical properties, morphology, and lack of aphid transmission. Namely, the virus is readily transmitted by juice inoculation, but not by Myzus persicae. By mechanical inoculation, it produced systemic mosaic in amaryllis and Lilium formosanum, and local lesions in Chenopodium amaranticolor and Gomphrena globosa. New Zealand spinach, Raphanus sativus, bean (Chajiro and Otebo), cowpea (Kurodane), broadbean, Nicotiana glutinosa, N. tabacum (Bright Yellow), petunia and tomato were not susceptible. In electron microscopy using dip method, long flexuous thread-like particles were observed. Majority of the particles were 600∼800mμ in length, but there was no distinct mode. The virus in vitro withstood heating at 65°C for 10 minutes, dilution to 1:100 and 1 day's aging at 20°C. Twenty-three samples of mosaic plants of amaryllis obtained from Shizuoka, Fujinomiya and Tachikawa were tested for HMV and CMV on a series of differential hosts. All samples proved to include HMV, while about one third of them included also CMV simultaneously. The samples from which only HMV were isolated, showed mosaic symptoms having rather coarse dark green areas irregular in size and shape. CMV, isolated from amaryllis, revealed some differences from the ordinary strain of CMV in host range and in symptoms on tomato, potato, N. glutinosa, G. globosa, petunia, etc.
Chenopodium sap used mainly in this study was the supernatant from centrifuged (9, 000×g, 20min.) leaf homogenate with three fold (by weight) of 0.2M phosphate buffer solution (pH 6.0), avoiding cold acetone soluble parts, being dialyzed against water, and was designated ChIS. To test the inhibitory effect of Chenopodium sap on the infection of non-persistent virus, daikon mosaic virus (DMV) (the Japanese strain of turnip mosaic virus) and its local lesion host, White Burley tobacco were mainly used. Experiments were carried out by abrasion with virus diseased plant sap and also with Chenopodium sap on the half leaves of the lesion host. Inhibitory effect was shown with the percentages obtained by the comparison of the lesion number on treated half leaves with that on the opposite half leaves (controls).1) No decline of inhibitory effect on virus infection of Chenopodium sap heated at 60°C for 10 minutes was observed; while the effect lost when the sap was heated at 100°C for 10 minutes (Table 1, 2, and 3). 2) Inhibitory effect fell remarkably when rubbed with DMV on its original plant, Chenopodium album (Table 4). 3) No inhibitory effect on DMV infection was obtained when DMV was inoculated on upper side of a tobacco leaf soon after its under side was rubbed with ChIS (Table 5). 4) Efficacy of inhibition of ChIS on DMV infection was observed with a 1 to 100 dilution (Fig. 1). Further dilution of the sap resulted remarkable fall of inhibitory effect. 5) Efficacy of inhibition of ChIS on DMV infection was observed even when rubbed 48 hours before the virus inoculation (Table 6). 6) As to after application of ChIS, efficacy of the inhibiting sap on DMV infection was observed up to 30 minutes after the virus inoculation. 7) No inhibitory effect of ChIS on DMV infection was observed when tobacco leaves were syringed or rubbed with ChIS before inoculation of the virus with Myzus persicae (Table 7). 8) No inhibitory effect of ChIS on DMV infection was again observed when tobacco leaves were rubbed with ChIS after inoculation of the virus with M. persicae by brief feeding period of 15 to 20 seconds (Table 8 and 9). To explain the mechanism of the inhibiting action against virus infection of the inhibitor such as Chenopodium sap, the idea that the inhibitor acts on the host cytoplasm and inhibits the formation of virus-receptor complex is now widely accepted. In addition to this a working hypothesis, “Hypersensitive reaction of plant cytoplasm against incompatible inhibitor” is suggested. When such an incompatible inhibitor is introduced and brought in contact with cytoplasm, the latter lose its capability to adsorb virus particles as the results of disorganization due to its hypersensitivity. In the cases of short feeding period of 15 to 20 seconds of M. persicae, the stylet is hardly capable to penetrate through the epidermal layer after it is inserted in between two epidermal cells, and the stylet borne virus as DMV is likely to be transmitted to the plasmodesmata in the intercellular region when stylet tip reaches there. In contrast to this, when DMV is inoculated by abrasion, cuticular layer of the leaf epidermis is scratched off, and the virus is transmitted easily to the disclosed ectodesmata. This histological difference of infectible sites in an epidermal cell-the one plasmodesmata for aphid inoculation, and the other ectodesmata for rubbing inoculations-is the decisive factor why Chenopodium sap is able to inhibit the infection of the virus only when the latter is inoculated by abrasion (Fig. 3).
As has presiously been reported, when mulberry trees which had been infected with Kikuchi mild strain in previous year were cut from the basal part of the trunk in winter or were allowed to remain unpruned throughout the growing season, most of these trees developed normal shoots and leaves in summer. And it has been considered that the recovery from the disease by the cutting back of affected mulberry trees in winter or no cutting back throughout the year is attributable to the retardation of movement of the pathogen from the roots. Results in line with these findings, this experiment was carried out at Kikuchi city in order to establish the economical protection counterplan from damage of the disease. From one of the results of this experiment, it was discovered that the cutting back of mulberry trees from 1 meter above the stump in June (cutting back 1m above) is efficient to protect from the damage as well as the cutting back of trees in winter or no cutting back throughout the year and moreover increase a leaf yield at the same time. Using two methods of the cutting back in winter and of the cutting back 1m above, these experiments were carried out. Experiment A were performed the cutting back in winter in 1964, the cutting back 1m above in 1965, and the cutting back in winter in 1966. Experiment B were the cutting back 1m above in 1964, the cutting back in winter in 1965, and the cutting back 1m above in 1966. Experiment C were the cutting back in winter in 1964, the cutting back 1m above in 1965, and the cutting back 1m above in 1966. Experiment D were cut back, leaving the stubs in June from 1964 to 1966 as to control. The results of A, B, C were excellent in respectively compared with D. Therefore, it seemed to the author that the best economical protection counterplan is the three-year rotation of the method of experiment C is carried out in the field.
Repeated electron microscopic examinations of thin sections of young leaves and shoots from mulberry tree infected naturally or artificially with dwarf disease, have failed to show any such uniform particles, spherical or elongated, as have been described for plant viruses in the past. Presence of specific, pleomorphic bodies, however, have been demonstrated consistently in the siebe tubes and occasionally also in the phloem-parenchyma cells. These bodies are spherical to irregularly ellipsoidal in shape, and 80 to 800mμ in diameter. They possess a two-layered limiting membrane of about 8mμ in thickness, instead of cell wall. The smaller bodies, 100∼250mμ in diameter, are nearly round, and generally filled with ribosome-like granules of about 13mμ in diameter. Sometimes net-strands similar to those found in the nuclear regions of other bacteria were located in the less electron dense area. The larger bodies are occupied by a large central vacuole surrounded with ribosome-like granules at the periphery. Frequently, structures similar to nuclear net-strands are observed inside the vacuolated area. The gross morphology and fine structure of these bodies seem to be similar to the descriptions of either the cells of Mycoplasma species (Pleuropneumonia-like organisms) or agents of Psittacosis-Lymphogranuloma-Trachoma group as given by Domermuth et al. (1964), and others, though any agents of such groups have as yet not been reported from plants, so far as we know. Most of the smaller bodies may correspond to the “elementary bodies”. Occasionally, budding-like protrusion or constriction of the larger bodies, suggestive of small body formation, were observed. The diversity in the size of the bodies found simultaneously in the phloem may represent their developmental stages. Moreover, therapeutic effectiveness of tetracyclines to mulberry dwarf (Ishiie et al., 1967), and disappearance of the specific bodies in the phloem of the plants recovered by tetracycline treatment, may provide an evidence in favor of Mycoplasma hypothesis. In view of the constant association of the organism in considerable amounts in the phloem of dwarfed plants, the consistent absence of those in healthy plants, the failure to demonstrate particles of any true virus nature, and the apparent sensitivity to tetracycline, it is suggested that the Mycoplasma-like organism described above may be the causal agent of mulberry dwarf disease, although further experiments are necessary for an undisputed proof of etiology and taxonomy. Presence of similar Mycoplasma-like organisms in the phloem tissues has been confirmed in the preparations from witches' broom potato, witches' broom paulownia, and petunia infected with aster yellows. Negative results were obtained in those from check plants. An attempt to isolate and culture the organisms in question on artificial media is now in progress. These results suggest that reexaminations of the causal agent would be desirable for the yellows and witches' broom group which are transmitted by leafhoppers, or by grafting, and in which it is difficult to detect “typical virus particles”.
Doi et al. (1967) have demonstrated the consistent association of Mycoplasma- or PLT group-like organisms in the phloem tissue of dwarf diseased mulberry plant. If these organisms proved to be sensitive to tetracyclines, still more evidence for an etiologic relation will be added. Accordingly, effects of chlortetracycline hydrochloride (Aureomycin) and tetracycline hydrochloride (Achromycin), of the tetracycline group, and also Kanamycin were investigated upon the symptom development of dwarf disease in mulberry seedlings. Solutions of the antibiotics were applied to infected seedlings by three methods, singly or in combination: repeated foliage spraying, soil drenching at intervals of 2&3 days, and root dipping prior to planting. Generally, Achromycin and Aureomycin at a concentration of 100ppm showed remarkable effects in suppressing symptom development, whereas Kanamycin did not. Therapeutic effectiveness obtained by root dipping was superior to that of foliage spraying. There was no noticeable effect in the case of soil drenching. The plants recovered by treatment with these antibiotics, however, tended to develop symptoms again some period after the application was discontinued. The length of the period until re-appearance of symptoms seemed to vary with the original disease severity, the size of the seedlings, and environmental conditions. These results may indicate that mulberry dwarf disease is caused by an agent whose multiplication is inhibited by an antibiotic such as chlortetracycline and tetracycline.