The virus which transmitted mechanically from Satsuma dwarf tree to herbaceous plants was used for retransmission. Inoculation was made by injection. The retransmission occurred from infected kidney bean to the four of the five seedlings of Satsuma mandarin and three of the eight of Natsudaidai. When the RNA specimen of this virus prepared from sap of infected kidney bean by the treatment of phenol was used for retransmission, four of the thirty seedlings of rough lemon were infected. In the seedlings retransmitted with this virus, the flecking and line pattern were produced on the new growth of Satsuma mandarin made following infection, chlorotic ring or spot on rough lemon and flecking and mottle on Poncirus trifoliata. The warping of young leaves and downward boat or spoon shape of mature leaves which are typical symptoms of Satsuma dwarf appeared on the shoots of Satsuma mandarin grown secondary after cutting. On the basis of these evidence, it was confirmed that this mechanically transmissible virus is the causal virus of Satsuma dwarf. In Satsuma seedlings inoculated with Satsuma dwarf virus, characteristic flecking and line pattern developed on the several shoots which sprouted on the same side of stem as inoculated points and from these shoots the causal virus was detected. All of the shoots grown secondary after cutting of first growth showed symptoms and positive reaction on detecting test of causal virus.
The present paper describes the fine structure of hyphal cells of Helminthosporium oryzae which invaded the coleoptile of two rice varieties, Kameji and Shiga Asahi 27, resistant and susceptible to the brown leaf spot respectively. Leaf tissues were fixed in KMnO4 (2% in distilled water) and postfixed in a 1% solution of phosphate buffered OsO4. 1. In the susceptible variety the hyphae of the fungus remarkably increased its diameter after penetrating the cuticle of epidermal cell walls, and developed through the middle layer of radial walls of epidermal cells and then, in most cases, intruded into epidermal cells. 2. The cell wall of young immature hyphae which invaded the parenchymatous cells of the resistant and susceptible varieties consisted of only a mucilaginous inner layer, similar to that of immature germ tubes. However, two-layered cell walls were observed in the matured hyphae, electron-dense outer layer (about 0.12μ in thickness) and electron-transparent inner layer (about 0.20μ). Vacuoles in old hyphae developed extensively, some reaching 3μ in diameter, and occupied a greater part of the cytoplasm. 3. In the susceptible variety accumulation of electron-dense substances, 0.5μ thickness was recognized around the hyphae in the radial wall of epidermal cells. This might be a response product of the host cell to the exudate of the fungus. 4. In the parenchymatous tissues of the susceptible variety the hyphal tips swelled again after penetrating the cell wall by a slender hypha, about 1μ in diameter. In these newly formed hyphal cells many mitochondria and lipid bodies were recognized 5. In an affected epidermal cell of coleoptiles of the resistant variety, substances which appear to be due to the degeneration of cytoplasm of the host cells were recognized around the hyphae. Less amount of these substances was detectable around the hyphae in the susceptible variety. 6. The hyphae of the causal fungus in coleoptile of the resistant variety were generally slender, about 1μ in diameter. They had large vacuoles in cytoplasm, and finally they showed a vacuous appearance retaining only the outer layer of cell walls. 7. In the resistant variety, Kameji, chloroplasts in parenchymatous cells at first swelled after the invasion of the causal fungus. Thereafter, the intergrana-lamellae expanded and the grana-lamellae changed into many small vesicles, 0.1-0.5μ in diameter. Thus the chloroplasts were finally degenerated. This degenerative process is referred to so-called vacuolar degeneration.
In the previous paper1) it was reported that a ninhydrin-positive substance (NPS) is produced in a modified Czapek's medium, in which NaNO3 was replaced by an amino acid, inoculated with several plant pathogenic fungi. NPS is produced non-biologically in the modified Czapek-tryptophan medium when the medium was adjusted at pH 2.8 or 4.8, whereas it was not produced non-biologically at higher hydrogen-ion concentrations, 7.0, 9.0 and 11.0. NPS was produced in the same medium even at high hydrogen-ion concentrations, 6.8, 7.2, 8.2 and 9.0, when inoculated with plant pathogenic fungi such as Alternaria kikuchiana, Cladosporium herbarum and Colletotrichum dematium. In order to check the non-biological production of NPS at the acidic micro-environments distributed locally on the mycelial surface in liquid medium, silica gel particles were added, in place of fungal mycelium, to the medium and the production of NPS was tested, however, no NPS was detected. These results indicate that NPS is produced by the fungi through some metabolic process. NPS is produced in the medium within two days, reaching its maximum about 6th day after inoculation, at a wide pH range, from 6.8 to 9.0. In order to identify NPS, its behavior on thin layer chromatography was compared with those of artificially synthesized N-glycosides. The result indicated that NPS contains fructose as sugar moiety.
In warm parts of Japan, in spring and fall, especially in the months of April and May, there can be observed many cases of a plant disease, which causes brown spots on ryegrasses and fescues and blackish-brown stripes on bromegrasses. The damage on Italian ryegrass is severe. The pathogens isolated from the above-mentioned 4 sorts of grasses can be classified into 8 groups, according to some minor bacteriological characteristics. However, since they all can infect the above 4 original hosts, the author can conclude that they belong to the same species. From its bacteriological characteristics, the symptom of disease it causes on oats, and its polyxenic pathogenicity, he identifies the pathogen with Pseudomonas coronafaciens var. atropurpursa (Reddy et Godkin) Stapp. The bacteriological characters of the pathogen are as follows: Cylindrical rods, 0.6-0.8×1.2-4.2μ, motile with 1 or 2 polar flagella, gram-negative. Aerobic, colonies on 5% sucrose agar domed and mucoid. Production of fluorescin and liquefaction of gelatin variable. Milk no change, some coagulated. Litmus reduced, but not nitrates. Indol, hydrogen sulphide, ammonia and acetoin not produced. Methyl red negative. Acid but no gas produced from arabinose, xylose, glucose, fructose, galactose, mannose, sucrose, glycerol, mannitol and sorbitol, but variable from rhamnose, lactose and raffinose; neither acid nor gas from maltose, starch, inulin, dextrin or salicin. Anaerobically neither acid nor gas produced from glucose. Starch, aesculin, arbutin, margarine and Tween 80 not hydrolysed, Cytochrome oxidase and tyrosinase positive, but not oxidase, arginine dihydrolase or lecithinase. Oxidation of gluconate negative. Optimum pH 6.7-7.2. Optimum temperature 20°C. Thermal death point 51° or 52°C. Occures naturally on Lolium spp., Bromus spp., and on Festuca spp. Produces infections, following spray inoculation, in seedlings of Dactylis glomerata, Agropyron intermedium, Hordeum vulgare, Triticum sativum, Secale cereale, Avena sativa and of Arrhenatherum elatius.
Pentachlorobenzyl alcohol (PCBA), a specific rice blast control agent is known to have scarce antifungal activity but prevents the hyphal penetration of blast fungus into host tissue. The present work is aimed to clarify, by using a model "fungus-artificial membrane" system, whether the inhibition of hyphal penetration is due to the direct action of PCBA to the fungus itself or the compound is metabolized within the host tissue to some fungitoxicants. Blast fungus can penetrate through cellophane membrane when a drop of conidia suspension is placed on a cellophane sheet which is floated on a boiling water extract of rice straw and incubated for 48hr at 27°C. PCBA prevents the penetration through cellophane by blast fungus at low concentration, so one of the most important mode of actions of PCBA might be the deprivation of pathogenecity of blast fungus and the possibility of biotransformation to some toxic compounds in rice leaves which actually blocks the penetration of hyphae is most likely ruled out. PCBA has no effect on boring of cellophane membrane by Cochliobolus miyabeanus and Alternaria citri which are uncontrollable pathogens by the compound. PCBA inhibits neither production nor activity of cellulase of blast fungus, therefore these results may suggest that the mechanism of hyphal penetration is rather mechanical than enzymatical.
The present paper describes the results of experiments carried out with the aim of obtaining evidences other than the transovarial virus transmission, of multiplication of the rice stripe virus in its insect vector, the smaller brown planthopper Laodelphax striatellus Fallén, and information about the possible site of virus multiplication and pathological effects of the virus on the insect vector. Serial transfers of the rice stripe virus from insect to insect were made using micro-injection method described by Maramorosch (1952). The virus was successfully carried in 4 serial passages in the smaller brown planthopper. The final dilution of the inoclum used to inject the last group of the series is calculated to be at least 1.25×10-6 of the original inoculum, while the virus derived from viruliferous insects was shown to have a dilution end-point between 10-2 and 10-3. Groups of planthoppers which had been injected with the juice from viruliferous insects, were macerated 2, 5, 10 and 15 days after the injection, and the preparations were tested for infectivity by inoculating to virus free insects. The inocula prepared from the planthoppers 2 and 5 days after injection were found noninfective, in coincidence with the incubation period in the vector. A 1:10 dilution of the macerate from planthoppers 10 days after injection and a 1:50 dilution of those from planthoppers 15 days after injection proved infective. This suggests that the virus may have multiplied in its vector at least fivehold during the period from 5th to 15th day after injection. Blood and various organs from viruliferous planthoppers were tested for the presence of virus by the injection method. Virus was recovered from eggs, male reproductive organs, and blood and fat body, while no virus was recovered from alimentary canal. Male planthoppers following 2 days of acquisition feeding on diseased rice plants were dissected to remove legs, wings, alimentary canal, Malpighian tubules and reproductive organ, and the remaining part was separated into head, thorax, and abdomen. These body parts were incubated for 13 days at 25-27°C in hanging drops of a medium consisting of Takami's salt solution, D-glucose and extract from their respective part. The virus was not recovered from fresh material of these parts, but was recovered from the abdominal part incubated in vitro for 13 days. Female and male reproductive organs excised from planthoppers which had fed for 3 days on diseased plants, were incubated for 12 days in vitro. From both organs virus was recovered after having been incubated for 12 days, but not before incubation. Cytological examinations of blood corpsucles, reproductive cells, salivary glands, alimentary canal, dermis, and fat body of viruliferous planthoppers showed no consistent, conspicuous changes in these tissues. Histochemical tests for hyaluronic acid, RNA, DNA, lipid, and phospholipid, and phosphatase activity showed no distinct difference between viruliferous and virus-free planthoppers. Relative amount of glycogen and of polysaccharides in the fat body and of mycetome, as determined by Bauer-Feulgen stain and by Lillie method, appeared to be somewhat less in viruliferous planthoppers than in virusfree ones.
A tomato strain of tobacco mosaic virus, TMV-L, was treated with nitrous acid in the extract of infected tobacco leaf, and a mutant was isolated by transferring individual local lesions on Nicotiana glutinosa to tomato seedlings. An isolate, designated as LN27, caused no symptoms on tomato, but when the extract of such symptomless leaves was inoculated on N. glutinosa, and extracts of single-lesions were further inoculated on tobacco plants, a portion of the tobacco plants developed mosaic symptoms after varying periods. Some of the plants which remained symptomless contained virus systemically, while others only in inoculated leaves, or in no parts of the plants. The mosaic symptoms developed on these plants were apparently similar to those caused by the parent virus TMV-L. Lesion productivities of the viruses contained in individual lesions of N. glutinosa were different. The viruses which produced many lesions caused mosaic symptoms on tobacco about 5 days after inoculation. Some of the viruses which produced only a few lesions (2-10 lesions per leaf) caused mosaic symptoms 12-41 days after inoculation, while others caused no symptoms at least during this period. When Xanthi nc tobacco was inoculated with the LN27-infected tomato extract, the leaves of the tobacco showed both distinctly large and small lesions. The viruses from the large lesions caused mosaic symptoms on tomato, while the viruses from the small lesions caused no symptoms for two weeks or more after inoculation, developing symptoms afterwards in a half to a third of them. Inoculation from the tomato which remained symptomless for some time and showed symptoms later gave rise to an increased number of large lesions. It was considered that a part of LN27 somehow recovered its virulence to tobacco and tomato during multiplication.
Polyoxins, antifungal antibiotics produced by Streptomyces cacaoi var. asoensis, are consisted of many active fractions such as A, B, D, E, F, G and etc., and have antifungal activities selectively against several plant pathogenic fungi. In this paper, fungicidal activities of Polyoxins against sheath blight of rice Pellicularia sasakii (Shirai) S. Ito, and behaviours in plant tissue were investigated. Among many active fractions, D fraction was the most effective for control of this disease. In vitro, Polyoxin complex was more active than organic arsenic compounds and other fungicides against the fungus. In the pot test in greenhouse, Polyoxin complex showed the same protective effect and the same inhibitory effect on development of lesion as methanearsonic acid-ferric, ammonium salt complex. Treating on rice plants, Polyoxin complex showed about the same stability for ultra violet as those of organic arsenic compounds. By bioassay method, it was recognized that Polyoxin complex penetrated into leaves of kidney bean from back side to surface and sheaths of rice plants from surface to tissue inside. Dipping the cut end of stem of kidney bean and no wounded root of rice plant into the solution of Polyoxin complex, a remarkable amount of Polyoxin complex was found to be absorbed and translocated. Being treated by Polyoxin complex on sheaths of rice plant, no rising of active fraction of Polyoxin was observed in the upper part of no wounded sheaths or diseased sheaths of rice plants, but a remarkable amount of active fraction of Polyoxin was observed in the upper of sheaths which were injured by needle prick method. We could not obtain any definite results of penetration of Polyoxin complex into mycelium of sheath blight of rice plant.
Polyoxin is the antibiotic having extensive activities against various fungi, especially, Alternaria spp. Tests were carried out to evaluate fungicidal activities of Polyoxin against Alternaria mali and Alternaria kikuchiana in vitro and in vivo. Among Polyoxin A, B, D, F, G fractions, B fraction especially inhibited development of mycelium, spore germination and sporulation. G fraction showed the high activity in vitro test, but low activity in vivo test. On the one hand, A and D fractions showed the reverse tendency. All fractions of Polyoxin caused abnormal bulbous phenomenon on germ tube of spore and mycelium at low concentration. Polyoxin complex permeated little into spore. Therefore, Polyoxin did not inhibit spore germination at the usual concentration, but made bulbing germ tube as soon as spore germinated. Antisporulation activity of Polyoxin was very strong and kept for a long time. Results of Field test showed that Polyoxin is more effective than the compared fungicides. Polyoxin penetrated into apple leaf and was absorbed from the cut end of apple stem.
Variability in virulence, and some important physiological characters of 6 Indian isolates of Xanthomonas oryzae (Uyeda and Ishiyama) Dowson, the incitant of bacterial blight of rice and their sensitivity to antibiotics in vitro were investigated. Morphologically, all the isolates were alike. However, with regard to cultural and physiological characters and antibiotic sensitivity, they could be divided into two groups. The first group comprised of 4 isolates, which were slower in growth in culture media with optimum temperature between 25-30°C and thermal death point between 51-52°C, liquefied gelatin slowly, hydrolysed starch partially or not at all, gave alkaline reaction in litmus milk, did not produce hydrogen sulphide and were highly sensitive to all the 5 antibiotics tested, including penicillin. These isolates belonged to the highly virulent group. The other group comprised of 2 isolates, which grew faster on culture media with optimum temperature between 25-35°C and the thermal death point between 52-54°C, liquefied gelatin rapidly, hydrolysed starch completely, gave acid reaction in litmus milk, produced hydrogen sulphide and were insensitive to penicillin and comparatively less sensitive to the other 4 antibiotics. These isolates were comparatively less virulent than the isolates of the first group.
Necrosis mosaic of rice, a new soil-borne virus disease described by Fujii and others in 1967, is characterized by the symptoms of necrotic lesions on basal portions of stems and sheaths, elongated yellowish flecks especially on lower leaves, and a decumbent growth of the whole plant. In dip-preparations from roots, sheaths, and leaves of naturally infected rice plants and rice seedlings grown in greenhouse on infected soil, rod-shaped Particles were almost consistently found. Two peaks of the particle-length distribution (275 and 550mμ) were observed in dip-preparations from infected roots. Diameter of the particles was found to be about 13-14mμ.