Search

The red pigmented strain F-1-1 of Serratia marcescens shows a strong antagonism against Phytophthora capsici, the pathogen of damping-off of cucumber seedlings, and its antagonistic activity is due to the production of antibiotic red pigments. In the present study, purification and identification of the red pigments produced by the strain F-1-1 were performed. The water-insoluble red pigments were extracted from cultures on agar plates with ethanol and then chloroform, and purified by silica gel column chromatography. An active substance was isolated using the antifungal activity assay and was identified as prodigiosin, a known compound based on the spectral data. Purified prodigiosin inhibited the germination of cystospores and the growth of the fungal hyphae of P. capsici. It also showed antimicrobial activity against some plant-pathogenic fungi including Cochliobolus miyabeanus, Pythium spinosum, P. ultimum and against plant pathogenic bacteria including, Clavibacter michiganensis subsp. michiganensis and Erwinia carotovora subsp. carotovora. These results suggest that S. marcescens F-1-1 could be used as a biocontrol agent against many plant diseases other than cucumber damping-off.

Two kinds of biphenyl phytoalexins accumulated in leaves of Photinia glabra Maxim (Rosaceae) in response to inoculation with spores of Entomosporium mespili, a pathogen of P. glabra, or to treatment with 10-4M HgCl2. The phytoalexins were identified as 2'-methoxyaucuparin and 4'-methoxyaucuparin. The contents of 2'-methoxyaucuparin and 4'-methoxyaucuparin were 3.578 and 1.233μg/g of leaf (fresh weight), respectively, 4 days after the inoculation with spores of E. mespili, whereas their contents in the uninoculated leaf were minute. The two phytoalexins exhibited toxicity to Pestalotia photiniae, a pathogenic fungus to P. glabra, and to Botrytis cinerea and Cochliobolus miyabeanus, the non-pathogens. The toxicity of 4'-methoxyaucuparin to P. photiniae and Botrytis cinerea was higher than that of 2'-methoxyaucuparin.

We produced a polyclonal antibody which reacts with antigens of mycelia of the rice blast fungus. Using this antibody, we developed an ELISA method which can measure small amounts of mycelia of Magnaporthe grisea (Hebert) Barr in a rice leaf. More than 0.1μg mycelia of the rice blast fungus in 100mg of fresh leaf can be measured by this method. The antibody, acquired using lyophilized mycelia of the fungus, reacts as well with fresh mycelia, conidia, and even those of different races as it does with lyophilized ones. But it does not react with mycelia of several fungal pathogens of the rice plant. Thanatephorus cucumeris (Frank) Donk, Rhizoctonia sp., Cochliobolus miyabeanus (Ito et Kuribayashi) Drechsler ex Dastur, Sphaerulina oryzina Hara and Nigrospora oryzae (Berkeley et Broome) Petch do not react with the antibody at 20μg dry weight mycelia/ml of PBS (pH 7.5) containing 0.05% Tween 80. But at about 2mg/ml, the antibody slightly reacts with mycelia of Sphaerulina oryzina and Nigrospora oryzae. This method should be useful in understanding the interaction between blast fungus and rice plant.

The segregation and distribution patterns of the mating types of Cochliobolus miyabeanus were determined. The segregation ratio of the two types A, B, and C, D was about 1:1 in monoascosporic isolates and 35:49 in monoconidial isolates from field. In field, both mating types were distributed almost equally and moreover co-existence (sympatric distribution) of these types in the same localities or on the same rice plant was demonstrated. Over 60% of both monoconidial and monoascosporic isolates of the fungus were hermaphroditic. The remainder showed a loss of female character(s) in mating tests. Crosses between isolates of H. oryzae from Japan and U.S.A. were attempted. Fertile ascocarps were produced with the correct combinations. The identity of this fungus from both countries was confirmed by the formation of the perfect state.

The optimum culture conditions for pseudothecia formation by Cochliobolus miyabeanus have been determined: Sachs medium with rice straw, 20-24C (development was slower at 20C), pH4.65-8.10 (in the case of Czapek's agar-no carbon source medium with rice straw: pH3.7-5.9), Sachs medium with gramineous crop or weed plants added showed fairy good pseudothecia fromation as well as with rice straw. As a natural medium for perfect state formation, rice straw decoction agar (50g, 10g, or 1g/l) or potato decoction agar (150g, 30g, or 3g/l) was adequate when a rice straw was placed on the agar surface. Use of rice seed coats on Sachs medium gave scanty pseudothecia formation.

The sexual reproductive pattern of Cochliobolus miyabeanus was determined. This fungus is heterothallic and basically hermaphroditic. Two mating types were recognized. Hewever, some long term stock cultures such as KU-13 and IFO-6631 Were unable to form pseudothecia-forming ability. These cultures were fertile when crosses were undertaken with protothecium-forming isolates of an opposite mating group. The phenomenon has not been reported previously in Cochliobolus spp.

Annular depressions and perforations in fungal cell walls were observed on hyphae and sclerotia of Rhizoctonia solani and on conidia of Cochliobolus miyabeanus following burial of these respective fungus propagules for 4 to 8 weeks in vegetable field soils obtained from various locations in Shikoku, Japan. The annular depressions and perforations were 1.0 to 5.5μm in diameter with 1 to 7 perforations per hyphal cell or conidium. Vampyrellid amoebae were isolated from soils and were estimated to number 2.0 to 33.0 per gram of soil. Trophozoites of the amoebae extended 500μm and more with extremely fine filopodia. Plasmotomy was common in large trophozoites before encystment. Digestive cysts were pale yellow, orbicular or elliptic and averaged 33.5×40.1μm in diameter. During excystment, trophozoites emerged through 10 to 20 holes 2.0 to 10.0μm in diameter in the cyst walls. Trophozoites engulfed hyphae in 5 to 20min after attaching to the fungal cell and emptied the contents of the cell within 40 to 90min for hyphae of R. solani and 60 to 120min for conidia of C. miyabeanus. Morphology of the trophozoites and cysts, the mode of encystment and excystment, and the feeding behaviour of the amoebae all match the descriptions for Arachnula impatiens Cienkowski.

Cochliobolus miyabeanus, perfect state of Helminthosporium oryzae, has been produced in culture by certain crosses. Media and methods for formation of the perfect state are described. In routine experiments, the authors employed Sachs agar medium plus rice straw at the constant temperature of 24C for 25-30 days. Morphological data have concurred with the original data of Ito and Kuribayashi (1927, 1931), with the one exception of the length of the ostiolar beak.

In the previous paper, the author concluded that the detoxication of PMA by the rice leaf juice was due to the thiol group present in it. This paper deals with the stoichiometrical reaction of PMA with the known thiol compounds and thiol groups contained in the rice plant.The thiol group was determined by the amperometric titration method, by which SH in the range of 0.03-0.6μM was determined and 96per cent was recovered from glutathione (GSH).The SH contents of egg-albumin, yeast-extract, peptone, γ-globulin were 14.4, 1.23, 0.25, 0.1μM per gram respectively. The detoxication effect varied in this order.In the rice juice, from which protein had been removed, and in the yeast-extract, SH diminished more rapidly than in GSH solution. The SH in the rice juice decreased more slowly during the storage at 5°C than in GSH solution.The SH content was highest in the upper three leaves, about 300μM per 100g fresh weight, decreasing toward the lower leaves. The content was about 1/4 in the leaf still folded and below 1/10 in the leaf sheath, the stem and the root. The SH contents of these tissues varied in parallel with the protein contents. As the stage advanced from the vegetative growth to the reproductive, the SH content in the leaf decreased slowly but the SH became hardly extractable into the juice because of hardening of the tissues.The juice-SH reacted stoichiometrically with PMA at one to one mole ratio. When both PMA and the juice were added to the spore suspension of Cochliobolus miyabeanus, the spore germination was not inhibited until PMA exceeded the equal mole to the juice-SH. The elongation of germ tube was not inhibited until PMA exceeded the half mole of the juice-SH.When PMA dust is applied to the rice plant for the control of blast disease, the amount of PMA deposited on the rice leaves may be at most 1/100-1/1, 000, by mole, to that of SH present in leaves.The author concludes that the effectiveness of PMA for the blast disease is mainly due to the PMA present on the leaf surface or penetrated into the cell membrane, but not due to the PMA penetrated and diffused in the cell sap, because the latter PMA will easily be detoxified by the excessive amount of SH.

1. The present paper deals with the result of the investigations on the relation between the different kinds of culture media and the activity of some carbohydrases, i.e., α-amylase, β-amylase, invertase, and maltase, produced by Cochliobolus miyabeanus. The estimation was made using both intra- and extra-cellular preparations.2. As to α-amylase and β-amylase, extra-cellular activity is greater than intra-cellular activity. On the contrary, greater activity of intra- than extra-cellular invertase was detected.3. All the enzymes tested seemed to be the adaptive enzymes because their production markedly increased when the corresponding carbohydrates were added to the media.4. All of the four carbohydrases were produced more abundantly in the maltose medium than in the other media used.5. The activity of these enzymes is not always parallel with the growth amount of mycelial mats and the change in pH-value of the culture media.

The resistance of the leaf-sheath cells of rice plants against Cochliobolus miyabeanus was markedly decreased by adding reducing agents, such as ascorbate or glutathione, to the inoculum.Phytoalexin-like antifungal substance was produced as a result of interaction between the cells of rice plants and the germinating spores of C. miyabeanus. However, since ascorbate and glutathione did not either decrease the activity of phytoalexin, or did not inhibit the formation of such a substance, the active principle in resistance which was inactivated by adding reducing agents would not be referred to the activity of phytoalexin.The invaded cells of leaf-sheath and their neighbouring cells were oxidatively stained with various rH indicators at the begining of hyphal penetration. It is noticeable that the parts of mycelia which were in contact with the membranes of inner epidermal cells of the leaf-sheath were also oxidatively stained in deep colour. The invaded cells of the resistant variety, Kameji, were stained with methylene blue in green colour at the begining of invasion, while in the susceptible variety, Asahi, the colour reaction was nearly blue.Judging from these results, it was concluded that the resistance of rice plant against hyphal penetration of C. miyabeanus is, to some extent, attributable to the fungal oxidation products, perhaps quinones, which are derived from the host cells or membranes.

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.

In this paper microflora, especially of fungi, on the surface of apparently healthy leaves of rice plants is discussed. The second leaf from the top of variety Nakate Shin-Senbon was used for isolation, and many fungi, such as Candida, Rhodotorula, Penicillium, Alternaria, Aspergillus, and bacteria were isolated. Among them Candida sp. was most frequently isolated. The presence of Candida or other fungi in the conidia suspension of Cochliobolus miyabeanus did not disturb the conidia germination of the latter. However, if the mixed inoculation of Cochliobolus with Candida was performed to rice plants, number of the brown leaf spots decreased, representing about 50 per cent of the control. No microorganism, isolated from leaves of rice plants, inhibited the mycelial growth of C. miyabeanus on agar media.

When conidia of Pyricularia oryzae were compressed at 0, 500, 1, 000, 1, 500, and 1, 800kg/cm2 for 10 minutes, the germination was reduced from 97% to 16%. The infectivity of these conidia to rice leaves was correspondingly reduced. At 0-1, 000kg/cm2 the number of conidia with appressoria exceeded those with ordinary germ tubes, whereas at 1, 500 and 1, 800kg/cm2 conidia with appressoria were less than those with germ tubes. With increasing pressures (0-1, 800kg/cm2) the number of susceptible type lesions decreased from 7.5 to 0.5/10cm of rice leaf, whereas the number of resistant type lesions remained approximately constant at 1/10cm.When conidia of Cochliobolus miyabeanus were exposed to increasing pressures as above, the germination and infection were reduced, but not as much as with P. oryzae. Conidia exposed to pressures above 1, 500kg/cm2 formed smaller lesions on rice leaves than those exposed to lower pressures.

The fine structures of conidia of Cochliobolus miyabeanus were investigated by electron microscopic observations of ultrathin sections of conidia fixed in a solution of 2per cent permanganate buffered with veronal acetate to pH 7.4 and the results were shown in Figs. 1∼7.Fig. 1. General view of a conidium. The conidium was cut parallely to the long axis of cell.Fig. 2. Higher magnification of a small area (cf. rectangle in Fig. 1). Note the different orientation of cellulose microfibrils in outer and inner layers.Fig. 3. Various profiles of mitochondria and endoplasmic reticulum.Fig. 4. General view of nuclear area.Fig. 5. Portion of protoplasmic junction. The junction was cut parallely to the long axis of the cell.Fig. 6. Portion of protoplasmic junction. The junction was cut perpendicularly to the long axis of cell.Fig. 7. Diagrammatic view of conidium of Cochliobolus miyabeanus reconstructed from the data obtained.C: cytoplasm N: nucleusCW: cell wall NM: nuclear membraneER: endoplasmic reticulum OL: outer layerIL: inner layer PJ: protoplasmic junctionM: mitochondria PM: protoplasmic membrane

The fungicidal activity of phenylmercuric acetate (PMA) was assayed on the conidia of Cochliobolus miyabeanus by the use of slide-germination test method. The ED 50 of PMA was 0.04ppm, when PMA alone was dissolved in water. But the addition of 1ml of rice leaf juice per 2.5ml of the test solution raised the ED 50 of PMA up to about 20ppm. The juice was thus prepared by grinding 10g of leaves with water at 5°C, filling up to 150ml and filtrating.The rate of the detoxication of PMA by the juice increased parallel to the amount of the added juice. The effect of the juice decreased by the oxidation during extraction and storage at 5°C. The dialysis of the juice revealed that the effective substance was present mainly in the protein fraction and partly in the low molecular fraction. PMA was detoxified by the addition of thiol compounds, such as cysteine, glutathione (reduced), egg-albumin, yeast-extract and sodium thiosulfate, as well as the rice leaf juice. Amino acids, vitamins, carbohydrates and inorganic salts, all of which did not contain the thiol-group, did not detoxify PMA.From these results, the author concluded that the detoxication of PMA by the rice leaf juice was due to the formation of the mercaptide of phenylmercuric group with the thiol-group mainly of protein and partly of low molecular substances such as glutathione (reduced) present in the juice.

Dehydrogenase activity in the rice plant tissues inoculated with Cochliobolus miyabeanus (Helminthosporium leaf-spot fungus) and in the causal fungus was investigated using blue tetrazolium (BT). This reaction was positive in the sections of healthy leaf tissues after 2 hours' incubation at 30∼32°C and the rate of the staining became constant after 8 hours' incubation. The reaction was negative when the sections were pretreated with formalin (10%, 1hr.) or treated at 50∼60°C, 10min. In leaf tissues, the parenchymatous cells were most strongly stained and the sites of the enzyme activity seemed to lie on grana-like region in chloroplasts.The leaf spot caused by the present fungus became visible 20 hours after inoculation. The dehydrogenase activity in this early stage of infection was demonstrated in cells near browned epidermis. With the enlargement of spotted area, the tissue around the lesion stained with BT; but the reaction became constant 48 hours after inoculation, although the enlargement of the lesion was still proceeding. In the typical leaf spot produced 5∼7 days after inoculation, localization and intensity of formazan deposition could not be distinguished from those in healthy leaf tissue.The reaction was weak in leaf-sheath cells even when the pathogen penetrated them. On the other hand, the penetrating hyphae, especially the appressoria and the germ tubes, showed strong, reaction. When the spores germinated on slide-glass, the reaction was detected on the terminal cells of the spores and on the germ tubes as well as on the appressoria. This positive reaction on spores of the present fungus seemed to localize on mitochondria of the fungus.

1) In the presFent paper, the writers described the results of investigations on the starch accumulation in the area surrounding the spots on the leaves of rice plants affected by Cochliobolus miyabeanus.2) After the inoculation, the starch disappeared in the area around the spots within 20 hours and then it began to accumulate in the area surrounding the zone where starch disappeared. The starch accumulation increased with the development of spotted area. After several days, the value of starch accumulation index, the ratio of area of starch accumulation to the spotted area, became constant, while the concentration of starch continued to increase. It seems that the effect of infection gives rise to some abnormal physiological changes in the host immediately after the invasion of parasite.3) The area of starch accumulation was small in the early morning, but increased rapidly with the increase of insolation and remained constant until the sunset, while the concentration increased markedly in the evening. Under rainy or cloudy weather, starch decreased or disappeared around the spot. By keeping plants in a dark place for 24 hours, the starch disappeared. However, in some samples collected on the 10 th day after the inoculation, starch was still detectable.4) In plants cultured with nutrient solution containing excess amount of manganese or decreased amount of nitrogen and also in Akiochi plants (rice plants physiologically declined in autumn), the starch accumulation was promoted. Generally, it seems that in the surrounding zone of spots revealed on plants grown under abnormal nutritional condition, starch accumulates greatly.5) Judging from the results of our experiments, it may be suggested that the accumulated starch around the spotted area is assimilatory, and that the mechanism of this abnormal starch accumulation is partially based on the increased activity of starch assimilating enzymes, e. g. phosphorylase etc., in the surrounding area of spot.

In the present paper fine structures of chloroplasts in cells of rice plants and their ultrastructural changes due to the infection of Cochliobolus miyabeanus were described.In a longisection of healthy leaves of rice plants, chloroplasts were enveloped with a double membrane, and the lamellar system in chloroplasts showed a distinct compartmented grana stacks.Fifty hours after the inoculation of this fungus, chloroplasts in cells swelled about 1.5-2 times, and they were oblong to spherical in shape, having partially disintegrated double membrane of the envelope. The outermost sac of grana stacks of chloroplasts was firstly swollen into vesicle-like structure, with no matrix detectable in the interior. In chloroplasts adjacent to hyphae the disintegration of lamellar system may first take place in the grana stacks near the envelope. It gradually spread to every stack of grana and finally they degenerated entirely, forming abundant small vesicles, 0.1-0.5μ in diameter.

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.

A new antifungal antibiotic was found from the culture filtrate of a bacterium, which had been isolated from the spot of Helminthosporium blight of rice plant. The bacterium is Bacillus funicularis Cluyver et Van Niel, and the antibiotic produced by the bacterium inhibits the germination of the conidia of Cochliobolus miyabeanus. This substance is named “Funicularin” temporalily. Bouillon medium is one of the most suitable media for the production of the antibiotic and the optimum conditions for the production are 4 days culture at 25-30°C. in the medium with the initial pH 6-7. The method of extraction and purification of this antibiotic is shown in Fig. 1. When B. funicularis was successively cultured in the medium containig Funicularin, the production of the substance markedly increases. The susceptibility of rice seedlings to Helminthosporium blight appears to be lowered by the immersion of roots in the culture filtrate, the index of the disease spot enlargement being considerably smaller than that of the nontreated.

1) The present paper deals with the results of experiments through the paper-electrophoresis on the proteins contained in mycelia of several phytopathogenic fungi.2) Under the current of 0.25mA/cm for 3 hours using veronal-K2HPO4-Na Borate buffer solution, three components of proteins (I, II, III) were recognized in mycelia of Piricularia oryzae, Cochliobolus miyabeanus, Gibberella fujikuroi, Phytophthora infestans, Phytophthora macrospora and Guignardia camelliae, but in mycelia of Corticium sasakii and Shiraia bambusicola four components of proteins (I, II, II', III) were recognized.3) There were no qualitative difference between the two paper-electrograms of mycelia of different races of Piricularia oryzae, Cochliobolus miyabeanus and Gibberella fujikuroi.4) The proteins detected by the paper-electrophoresis in mycelia of Piricularia oryzae decreased with the growing stages, and with in the extent of the writer's experiments, no electrophoretic proteins were recognized six weeks after the inoculation.5) Proteins, especially I and II components, contained in mycelia of Piricularia oryzae decreased, if the mycelia were contacted with 10-4 molar solution of copper sulfate or mercuric chloride for 20 hours. The decrease of the components were probably caused by the degeneration of the proteins contacted with heavy metalic ions, but in the case of adding the metalic solutions to the juice macerated from mycelia of P. oryzae just before starting electrophoresis, the proteins did not decrease even at the concentration of 10-3 molar.

1. The conidia of Cochliobolus miyabeanus and the chlamydospores of Ustilaginoidea virens, and the conidia of Piricularia oryzae lost germinability by compression of 2, 500kg/cm2 and 2, 000kg/cm2, respectively, for 10 minutes. The germ-tubes of C. miyabeanus affected by hydrostatic pressure were slender and shorter than those of the normal. Conidia of C. miyabeanus compressed at 2, 000kg/cm2 for 10 minutes, and those of P. oryzae pressured at 1, 000kg/cm2 for 20-30 minutes, germinated bearing a 3-5-branched germ-tube.2. The germination index of these fungi decreased with the length of time of compression at 1, 000, 1, 500 and 2, 000kg/cm2, except of that of C. miyabeanus which compressed at 1, 000kg/cm2. Tolerance of conidia to the pressure seemed to be largest in C. miyabeanus, though tolerance is variable with spore individuals. The conidia of P. oryzae, however, were found to be lowest in pressure tolerance among the fungi employed, and variation in tolerance due to spore individual was also restricted.3. Elongation of the germ-tubes of the conidia of C. miyabeanus and P. oryzae was restricted in parallel with increase of pressure.

When conidia of Cochliobolus miyabeanus were held for 5 hours at 28°C under increasing hydrostatic pressures (100-500kg/cm2), the length of germ tubes decreased with increasing pressure, and their forms changed from filamentous to elliptical to spherical. In control germ tubes the nuclei were scattered in a cell, whereas at 200kg/cm2 the nuclei were clustered. No germination occurred at 600kg/cm2 and above.When conidia which were obtained by compression at 100-700kg/cm2 as above were kept under suitable condition for germination on slide, ordinary germ tubes elongated vigorously from abnormal germ tubes and from ungerminated conidia. Percentage of abnormal branching in these germ tubes increased with increasing pressure.