An investigation was made to estimate the damage of Cercospora leaf spot caused by Cercospora beticola Sacc. in sugar beet, Beta vulgaris var. rapa Dumot. The disease in field grown sugar beet plants was controlled by spraying with copper fungicide and therefore this chemical was used for establishing different degrees of damage. When the seeds were sown in the Tohoku district in late April, the damage was most severe at forty leaves stage of the plant. At the stage, generally, the top growth of the plants was most vigorous. When the infection was severe, the plants showed severe defoliation and reduction of the vigorous leaves. As the result, reduction of root growth yield and sugar content were corresponded. When the infection occurred after forty leaver stage of the plants, the damage was not so severe.
Two causal viruses were isolated from crotalaria (Crotalaria spectabilis) showing yellow ringspot and mosaic symptoms. The first virus was identified with bean yellow mosaic virus. The virus was transmitted mechanically and also by Myzus persicae. The hosts of this virus were crotalaria, broad bean, bean, soybean and Chenopodium amaranticolor. In electron microscopy using dip method, long flexuous thread-like particles were observed, and the length of them was 700-800mμ. Juice from diseased broad bean leaves reacted positively with antiserum of bean yellow mosaic virus (Komuro and Tochihara, 1964) in a slide flocculation test. The second virus was identified with tobacco ringspot virus. The virus was transmitted mechanically and also by Myzus persicae. The host range of this virus was comparatively wide, namely, aster, zinnia, Nicotiana tabacum (Bright Yellow, Xanthi), N. glutinosa, petunia, okra, broad bean, crotalaria, bean, soybean, turnip, New Zealand spinach, Gomphrena globosa, C. amaranticolor, beet and others. Characteristic ring-like pattern symptoms were observed on N. tabacum (Bright Yellow, Xanthi), N. glutinosa, and Datura stramonium, and these symptoms had a tendency to be masked under high temperature conditions. This virus isolate showed some differences from the hitherto reported tabacco ringspot viruses in noninfectivity to cucurbitaceous plants and in the symptoms on soybean. In electron microscopy using direct negative stain methond, spherical particles of about 26mμ in diameter was observed. The virus in vitro withstood heating at 65°C for 10 minutes, dilution to 5, 000 and 7 day's aging at room temperature. Kahn et al. (1963) reported that bean yellow mosaic virus was isolated from crotalaria, but the disease of crotalaria by tobacco ringspot virus has not yet been reported. This is also the first report in recognition of tobacco ringspot virus in Japan.
1. Rice plants in various stages (seedling, tillering and heading stages), were inoculated with conidia of Helminthosporium oryzae (Cochliobolus miyabeanus) and incubated for 8, 16 and 24 hrs. at 28°C. The preparation of protein was carried out by a modified method of Danielsson as shown in Fig. 1, and after the preparation, protein was fractionated by employing paper-electrophoresis and column chromatography. 2. The paper electrophoretic analysis showed that protein from rice plants had 5 fractions determined by densitometric scanning. However, there was no characteristic difference between healthy and diseased leaves. 3. By the step-wise elution from DEAE-cellulose column with 0.01-1.0M tris buffer solutions (pH 7.2), 6 fractions were obtained. Among them, 3 fractions were changed quantitatively with the infection. Although the different patterns of protein fraction were given with the growing stages and culture conditions of the rice plant, the protein fractions eluted with 0.5M and 1.0M tris buffer were always found to be changed quantitatively after the inoculation. The protein from 0.5M tris buffer elution increased in the leaves of susceptible variety, Asahi, 24 hrs. after inoculation. In the heading stage of rice plants, the increment of protein was noticeable 16 hrs. after inoculation.
In the present paper, some biochemical characteristics of the protein fractions of which increased in leaves inoculated with Helminthosporium oryzae are discussed. 1. Neither spore germination nor the elongation of germ tubes was inhibited by the protein fraction increased in the inoculated leaves. 2. Catalase activity was increased markedly in the protein fraction eluted with 1.0M tris buffer (pH 7.2), while no β-amylase activity was detected in the protein fraction. Unexpectedly the protein fraction inhibited β-amylase activity. The β-amylase inhibition by the proteins was discussed in relation to the mechanism of starch accumulation which was found usually in the surrounding area of diseased spot. 3. The amino acid contents of protein fraction eluted with 0.5M tris buffer (pH 7.2) were analysed by using amino acid auto-analyser. The protein fraction obtained from diseased leaves contained more aspartic acid and less glutamic acid than that from healthy leaves.
The occurrence of yellow dwarf disease of pea and broadbean was noticed in Wakayama, Aichi, and Okayama Prefectures since 1963. In general, the outbreak was sporadic, but was observed to be epidemic in occasional fields. The virus causes yellowing, leaf-curling or rolling, and dwarfing in various leguminous plants, such as pea, broadbean, bean, azuki-bean, soybean, milk-vetch, crimson clover, and subterranean clover, and also in Datura stramonium. In tobacco (Samsun and White Burley), N. glutinosa, N. rustica, and spinach leaf-curling and stunting are observed as well, although the virus is not yet recovered. The causal virus is not transmitted by plant juice. It is transmitted by Aphis craccivora, but not by Myzus persicae, Acrythosiphon pisum, and Megoura viciae japonica. In the dip-preparation from diseased plants, no elongated virus particles are detected under the electron microscope. No inclusion body is demonstrated in epidermal cells of infected pea and broadbean. Aphis craccivora could acquire the virus in as short time as 5 minutes. Efficiency of transmission, however, increased with more time than 4 hours on the source plants. The aphids which had received sufficient acquiring feed, transmitted the virus to about 7 per-cent of plants if fed for inoculation for 5 minutes on test plants, but infection increased to above 30 percent when aphids fed for 4hr. or more. Latent period invector aphid was found to be at least 36 hours at alternating temperatures of 24°C (day) and 18°C (night). As a result of daily serial transfers, individual aphids seemed to transmit the virus as long as they lived, although not every plant in a series was infected. In one case, a single aphid was able to infect all the test plants during a period of successive 15 days. Thus, the virus can be considered a persistent virus. By reference to the literature, the virus in the present work is apparently distinct in symptoms and agents of transmission from such persistent aphid-borne viruses as pea ena-tion mosaic virus, pea leaf roll virus, groundnut rosette virus and subterranean clover stunt virus, but identical with milk-vetch dwarf virus (MDV) which was described in Japan by Matsuura in 1953 and by Hino et al. in 1967.
Comparisons were made on the fine structure of hyphae of Helminthosporium oryzae Ito et Kurib. and Pyricularia oryzae Cavara by electron microscopy. The fine structure of the cell wall, number of nuclei, mitochondrial volume and the arrangement of cristae were found to be highly differentiated in both hyphae of H. oryzae and P. oryzae. The results obtained are as follows: (1) Hyphae of Helminthosporium oryzae The cell wall of hyphae was consisted of three layers (outer, middle and inner layers), as seen in conidia, and the hyphae of this fungus were recognized to be multinucleate. The cytoplasm contained well-defined mitochondria (0.76×0.51μ), endoplasmic reticula (ER), vacuoles and unidentified spherical bodies. (2) Hyphae of Pyricularia oryzae Two layers (outer and inner layers) were detectable in the cell wall structures of hyphae. Each cell seemed to possess a single nucleus surrounded by a double membrane with nuclear pores. The nucleus contained a comparatively dense body identified as chromatin substance. The cytoplasm also contained coiled membrane fragments, twisted-ER, variable surfaced ER, large mitochondria (1.28×0.62μ) and lipid granules (1.50μ in diameter).
Mosaic plants of annual bluegrass, Poa annua L., and soil sample are collected from a barley field in Tottori. X-bodies were found in the epidermal strips of diseased leaves. Electron microscopic examination of leaf-dip preparation revealed rod-like particles measuring 110-160×25mμ. Inoculation with the sap of mosaic annual bluegrass plants effected infection of not only annual bluegrass but also wheat, as determined by mosaic symptoms, and presence of X-bodies and rod particles. Back inoculation from infected wheat to annual blugrass was successful. There was no evidence of virus transmission through seed. Soil transmission of the virus to annual bluegrass and wheat, however, was proved by sowing the seed in the infested soil or by immersing the germinated seeds for 15 days in water mixed with infested soil. Virus was partially purified by differential centrifugation from sap clarified with chloroform and dihydrostreptomycin-sulfate. This virus preparation reacted well with a soil -borne wheat mosaic virus antiserum in complement fixation tests. Resting spores of Polymyxa graminis were found in the roots of naturally infected annual bluegrass. From these results, the virus from Poa annua was identified as soil-borne wheat mosaic virus. Annual bluegrass, a new host, may play some role in the spread of soil-borne mosaic disease of cereals in the field.
A growth-depressing substance to young roots of rice seedlings was assumed to be existed in the culture medium which was inoculated with Xanthomonas oryzae. In order to isolate the active principle in pure crystal state and to identify chemically, the following procedures were adopted: The Wakimoto's potato semi-synthetic culture medium was prepared, inoculated with the causal bacterium and kept at 28°C in shaking conditions. Seven days after, the culture solution was extracted with ethylether and then re-fractionated to acidic part. After steam-distillation, the residue was submitted to column-chromatography over silica gel, to elute the biologically active zone by chroloform and to fractionate by fraction collecter. Finally, about 50mg of the crystal was obtained in pure state from 10l of the inoculated culture medium by the above-mentioned procedures. The melting point lies at 76.5°C and was not depressed by admixture with authentic phenyl-acetic acid. The substance was identical in infrared spectrum with authentic phenylacetic acid. Judging from these characters, the authors concluded that the growth-depressing substance to young roots of rice seedlings which was produced by X. oryzae in culture was identical with phenylacetic acid.
The behaviors of conidia of Helminthosporium oryzae Ito et Kuribayashi on coleoptiles of two rice varieties, resistant (Kameji) and susceptible (Shiga Asahi No. 27) were compared by using light microscope. Conidia germinated on the coleoptiles of both varieties to almost the same percentage, whereas the length of germ tubes was shorter and the percentage of appressorium formation was smaller on the coleoptile of resistant variety than those on the coleoptile of susceptible one. On the other hand, the fine structure of a germ tube, appressorium, invading hypha of the causal fungus and the host cells (Shiga Asahi No. 27) were observed under the electron microscope. Mature germ tubes and appressoria formed on both varieties gained a two-layered cell-wall, 0.2μ in thickness. The outer layer was thin and electron-dense, and the inner layer thick and electron-transeparent when stained with potasium perma nganate and osmium tetroxide. The cell wall of immature germ tubes, however, possessed only a mucilaginous inner layer. This substance was further detectable around germ tubes and appressoria, and appears to serve for the attachment of these to the epidermal cell wall. When an appressorium firmly attaches the epidermal cell wall, the pectic layer is resolved and the cuticle layer is often ruptured just around the appressorium by being pulled up with the mucilage. The epidermal cell walls of the resistant variety was thicker than those of the susceptible one by about 27 per cet.
An unidentified ninhydrinpositive substance (NPS) was detected in the culture filtrate of several plant pathogenic fungi by thin layer chromatography when cultured on the modified Czapek's medium containing authentic amino acid (s). The chemical property was different from that of alanine, glycine, threonine, glutamic acid, glutamine, glucosamine, galactosamine, and anthranilic acid in thin layer chromatographic behaviors. At an early stage of the mycelial growth, NPS appeared in each combination between amino acids used in the modified Czapek's medium and plant pathogenic fungi employed. Consequently, the authors concluded that plant pathogenic fungi have an ability through a common metabolic pathway to produce NPS in the culture medium.
When tobacco, Nicotiana tabacum variety Xanthi (scion) grafted on Nicotiana glutinosa (stock) was inoculated with TMV on its t6bacco leaf several weeks after grafting, systemic necrosis of lateral shoot of N. glutinosa was observed 9 to 16 days after inoculation, in 13 out of 18 tests. Similarly, when N. glutinosa (scion) grafted on tobacco (stock) was inoculated with TMV on its tobacco leaf, systemic necrosis was observed on the apical portion of N. glutinosa 15 to 24 days after inoculation, in 4 out of 11 tests. From these necrotized portions TMV was recovered in high concentration. When any of the leaves of N. glutinosa grafted with tobacco were inoculated with TMV, necrotic lesions appeared only on the inoculated leaves. However, no systemic infection was observed in the tobacco part. When doubly cleft-grafted plant-N. glutinosa (scion), tobacco (middle part), and N. glutinosa (stock)-was inoculated with TMV on its tobacco leaf, systemic necrosis began to appear on the lateral shoots of the stock N. glutinosa 10 to 14 days after inoculation. Contrary to this, when doubly cleft-grafted plant-tobacco (scion), N. glutinosa (middle part), and tobacco (stock)-was inoculated with TMV on the leaves of either of the two tobacco shoots, mosaic symptoms appeared on the young leaves of the opposite tobacco shoot 15 to 24 days after inoculation. This fact indicates that TMV is able to move from one tobacco shoot to another without hindrance from the middle part, N. glutinosa. Immediately and 5 days after the appearance of systemic necrosis due to TMV infection, the affected lateral shoots of N. glutinosa were fixed with FAA and serial sections from these materials were examined for the distribution of the necrotized portions caused by the TMV which had been moved. The first appearanance of necrosis was observed within young phloem tissues next to the apical meristem. Then the necrotic portion extended gradually to the surrounding tissues. No necrotic portions were found at the growing point and its peripheral meristematic cells. And no necrosis was observed in the mature phloem at least for 2 weeks after inoculation. It is concluded that TMV moved through enucleate mature sieve tubes of N. glutinosa plant into the young phloem and multiplies there to form necrotic lesion, and that TMV is incapable to multiply within the meristematic tissues.
Cytological evidences offered by several authors6, 16) on the leaf cell of Nicotiana glutinosa inoculated with tobacco mosaic virus (TMV) revealed that the degeneration process of the host protoplasm, that began by disorganization of chloroplast and nucleus, followed by accumulation of brown pigment within the degenerated protoplasm, proceeded in parallel with TMV reproduction. The necrotic lesion of N. glutinosa by TMV infection usually increased in size especially in case of young lesion (Fig. 1). Occasionally, TMV escaped from the necrotic lesion of N. glutinosa, reached the stem-tip passing through inner tissue, without leaving any outer symptom of infection on the stem, and caused stem-tip necrosis (Fig. 2). Then the necrotic lesion spreaded systemically down to the whole plant tissue. The necrotic lesion of N. glutinosa by TMV infection, that increases in size and even traslocates, is entirely different in its nature from the necrotic lesion formed within the peripheral zone of a hypersensitive wheat variety against the invasion of an incompatible race of the obligate parasite, Puccinia graminis. On the contrary, the necrotic lesion of N. glutinosa may be compared with the diseased spot of a moderately susceptible bean plant affected with Colletotrichum lindemuthianum.