Morphological Variation in Spores of Pyricularia oryzae Cavara

Two mutant isolates of Pyricularia oryzae formed abnormal, longer, cylindrical spores with more septa than those of normal, obpyriform spores of wild isolates. These isolates also produced normal spores, although their number was less than that of the abnormal spores. When normal and abnormal spores were single-spore-isolated from the lesions caused by the mutant isolates and inoculated to an agar medium, each colony produced both types of spores, regardless of the type of single spores used for inoculation.     

Occurrence and Some Properties of Cellulase in the Filtrates of Conidiospores and Mycelia of Pyricularia oryzae Cavara

Occurrence of cellulase activity was demonstrated in the filtrates of germinating conidiospores and growing mycelia of P. oryzae. Activity and some properties of cellulase in the filtrate of mycelia grown on rice plant powder as carbon source were compared among various strains.

Cellulase activity (C₁ and C_x enzymes; cellulose and carboxymethylcellulose as substrates, respectively) in the filtrate of germinating conidiospores was detected in the pathogenic T–l (Ken 53–33) strain as well as nonpathogenic 0 (THU 3 × 1) strain of P. oryzae. The activity was higher in the former than the latter strains. Cellulase activity (C_x enzyme) in the filtrate of growing mycelia was detected in the four strains used, T–l (Ken 53–33), C–3 (N 87), N–1 (H373), and 0 (THU 3 × 1). Cellulase activity (C_x enzyme) in the filtrate of mycelia was optimal at pH 5.0 and 40°C, and stable up to 40°C. Their properties did not differ significantly except for the pH-activity curve at alkaline side among various strains; but cellulase activity (C₁ enzyme) was found to be correlated with their pathogenicity except for the case of C–3 strain.     

Studies on cellulases of a phytopathogenic fungus, Pyricularia oryzae Cavara. IV. Kinetic studies on beta-glucosidases

Kinetic studies of the two beta-glucosidase isozymes, GB-1 and GB-2, which were from the culture filtrate of a phytopathogenic fungus Pyricularia oryzae, revaled that the latter isozyme was an allosteric protein with two substrate binding sites. The homotropic effects of o- and m-nitrophenyl-beta-D-glucosides on GB-2 showed positive cooperativity, whereas that of cell cellooligosaccharide showed negative cooperatively. The affinity of GB-2 for cellooligosaccharide tended to increase with decreasing chain length, in contrast to that of GB-1. Glucono-delta-lactone and glucose acted as competitive inhibitors of GB-1 and GB-2. As regards the control of the level of glucose formed by the cellulose system, it appears that the rate of formation of glucose by beta-glucosidase is reduced by the presence of the substrate, cellooligosaccharide, as well as by the product, glucose.     

The Role of Toxin(s) Produced by Germinating Spores of Pyricularia oryzae in Pathogenesis

Pyricularia oryzae produced toxin(s) during spore germination which induced susceptibility to infection by non-pathogenic Alternaria alternata of rice leaves. The induced susceptibility was independent of the compatibility between the races of blast fungi used for obtaining the toxin(s) and the rice cultivars used for bioassay. Susceptibility was also induced in other susceptible species (e.g. barley, Italian ryegrass, perennial ryegrass and wheat), results suggesting that the toxin(s) are host-selective and determine the host specificity at plant species level.     

The Role of Toxin(s) Produced by Germinating Spores of Pyricularia oryzae in Pathogenesis

Pyricularia oryzae produced toxin(s) during spore germination which induced susceptibility to infection by non-pathogenic Alternaria alternata of rice leaves. The induced susceptibility was independent of the compatibility between the races of blast fungi used for obtaining the toxin(s) and the rice cultivars used for bioassay. Susceptibility was also induced in other susceptible species (e.g. barley, Italian ryegrass, perennial ryegrass and wheat), results suggesting that the toxin(s), are host-selective and determine the host specificity at plant species level.     

Studies on Pyricularia oryzae Cav. in Sierra Leone: Morphological and Physiological Variability of Some Isolates

The morphological and physiological variability of six isolatesof Pyricularia oryzae, the causal organism of rice blast disease,were investigated. The rate of growth, colony characters, andtime of sporulation were found to vary with the different isolatesthough not by appreciable amounts. Each of the isolates showedconsistently better growth on Takahashi's B medium than on Czapek-Dox'smedium although the growth trend was the same in both media.The colony characters developed by each isolate are not dependenton the medium on which it is growing—a pointer to thefact that such characters may be genetically controlled. Germinationwas faster in distilled water than in 2 per cent agar. All theisolates produced appresoria in vivo and in vitro; those producedin vivo were, however, considerably larger than those producedin vitro. On the basis of appresorial types, the isolates werefound to fall into two physiological races—smooth-walledand rough-walled. Each isolate produced consistently only oneappresorial type in vitro from the apical or basal cell of theconidium. The utilization of carbon and nitrogen compounds variedfrom one isolate to the other, carbon compounds being generallybetter utilized than nitrogen compounds. Pyricularia oryzaecan metabolize a wide range of carbon compounds. However, mannose,sucrose, glucose, fructose, and maltose proved to be most suitablecarbon sources. The variations in the utilization of the variouscarbon and nitrogen compounds seem to reflect inherent biochemicaland physiological differences among the isolates.     

Role of Energy in Pathogenic Variability of Pyricularia oryzae

Pyricularia oryzae Cav. reacts differently to different varieties. IB race group attacked Zenith for three consecutive years for both rabi and kharif seasons under artificial inoculation condition. Three different isolates were obtained in IB race which differed in their pathogenicity giving a constant susceptible reaction to Zenith. The difference in energy potential of three isolates of P. oryzae was tested biochemically. Total sugar, protein and protein patterns were studied following modern methods. W isolate contained maximum amount of total sugar (18.3 μg/g), total protein (23.8 μg/g albumin equivalent) and seven distinct protein bands on polyacrylamide disc electrophoretic gel which was directly correlated with maximum infection value. So it was concluded that the aggressiveness of P. oryzae depends on its energy potentiality in terms of total protein and protein patterns.     

Studies on cellulases of a phytopathogenic fungus, Pyricularia oryzae Cavara. III. Multiplicity of beta-glucosidase, and purification and properties of a second component.

To determine the relationship between the induction patterns of three components of beta-glucosidase of Pyricularia oryzae and carbon sources in the growth medium, various culture conditions were examined. Avicel, hydroxyethylcellulose and methyl-beta-D-glucoside as the carbon source induced both beta-glucosidase components, GB-1 and GB-2, whereas cellobiose and gentiobiose induced only one component, GB-1. Thus, these two components were induced independently and hence thought to be isozymes. The GB-2 was purified to homogeneity by ion exchange and gel filtration chromatographies from two different cultures on methyl-beta-D-glucoside and Avicel. The specific activity of GB-2 when salicin was used as substrate was approximately 5.9 mg glucose/min/mg protein. GB-2 was found to be an oligomeric glycoprotein, which consisted of two subunits with molecular weight of approximately 120,000, comprising a relatively large number of acidic amino acids and mannose, as is the case with GB-1. These two isozymes were clearly different in thermostability, GB-2 being more thermolabile than GB-1. However, the same carboxyl group (pKa 4.2--4.8) was found to be strongly implicated in the formation and dissociation of the enzyme-substrate complex for both of the enzymes, from the analysis of kinetic parameters as a function of pH.     

Kitazin P,Inhibitor of Phosphatidylcholine Biosynthesis in Pyricularia oryzae

The effect of organophosphorus fungicide, Kitazin P (IBP, S-benzyl diisopropyl phosphorothiolate), on lipid biosynthesis of Pyricularia oryzae was investigated. Addition of IBP to the mycelial cells suspension of P. oryzae induced a striking decrease in incorporation of methionine-methyl-¹⁴C, S-adenosylmethionine-methyl-¹⁴C, and glycerol-1-¹⁴C into phosphatidylcholine, which is the most abundant phospholipid in P. oryzae, but incorporation of choline-methyl-¹⁴C into phosphatidylcholine and that of methionine-methyl-¹⁴C into simple lipids were not affected. Incorporation of methionine-methyl-¹⁴C into phosphatidylcholine is found to be directly proportional to mycelial cells growth of P. oryzae. Enzymes responsible for the biosynthesis from glycerol to phosphatidylcholine through Greenberg’s pathway, except phospholipid N-mefhyltransferase, were not inhibited by IBP. IBP concentration required for 50% inhibition of phospholipid N-methyltransferase was 40 ppm. IBP had no effect on activities of glycerokinase, glycerophosphate acyltransferase, phosphatidic acid cytidyltransferase, phosphatidylserine synthetase, and phosphatidylserine decarboxylase, respectively. Therefore, the specific inhibition of conversion from phosphatidylethanolamine to phosphatidylcholine by the transmethylation of S-adenosylmethionine might be regarded as one of the modes of action of IBP.     

Induction of Enzymes in Dormant Spores of Aspergillus oryzae

In dormant conidia of Aspergillus oryzae, alpha-amylase, invertase, and glucose dehydrogenase were induced by their respective inducers. Neither germination nor swelling occurred during this period.     

Phenolic Azo Dye Oxidation by Laccase from Pyricularia oryzae

Laccase oxidation of phenolic azo dyes was examined with a commercially available laccase from Pyricularia oryzae as the model. Methyl-, methoxy-, chloro-, and nitro-substituted derivatives of 4-(4(prm1)-sulfophenylazo)-phenol were examined as substrates for this laccase. Only the substituents on the phenolic ring were changed. Among the dyes examined, only 2-methyl-, 2-methoxy-, 2,3-dimethyl-, 2,6-dimethyl-, 2,3-dimethoxy-, and 2,6-dimethoxy-substituted 4-(4(prm1)-sulfophenylazo)-phenol served as substrates. Preliminary kinetic studies suggest that 2,6-dimethoxy-substituted 4-(4(prm1)-sulfophenylazo)-phenol is the best substrate. Laccase oxidized the 2,6-dimethyl derivative of 4-(4(prm1)-sulfophenylazo)-phenol to 4-sulfophenylhydroperoxide (SPH) and 2,6-dimethyl-1,4-benzoquinone. The 2-methyl- and 2-methoxy-substituted dyes were oxidized to SPH and either 2-methyl- or 2-methoxy-benzoquinone. Six products were formed from laccase oxidation of the 2,6-dimethoxy-substituted dye. Three of them were identified as SPH, 4-hydroxybenzenesulfonic acid, and 2,6-dimethoxybenzoquinone. A mechanism for the formation of benzoquinone and SPH from laccase oxidation of phenolic azo dyes is proposed. This study suggests that laccase oxidation can result in the detoxification of azo dyes.     

Novel insights into host specificity of Pyricularia oryzae and Pyricularia grisea in the infection of gramineous plant roots

Pyricularia oryzae and Pyricularia grisea are pathogens that cause blast disease in various monocots. It has been reported that P. oryzae infects the leaves and roots of rice via different mechanisms. However, it is unclear to what extent the tissue types affect the host specificities of P. oryzae and P. grisea. Here, we evaluated the tissue‐specific infection strategies of P. oryzae and P. grisea in various gramineous plants. Generally, mycelial plug inoculation caused root browning but the degree of browning did not simply follow the disease index on leaves. Interestingly, the Triticum and Digitaria pathotypes caused strong root growth inhibition in rice, wheat, and barley. Moreover, the Digitaria pathotype inhibited root branching only in rice. Culture filtrate reproduced these inhibitory effects on root, suggesting that some secreted molecules are responsible for the inhibitions. Observation of root sections revealed that most of the infection hyphae penetrated intercellular spaces and further extended into root cells, regardless of pathotype and host plant. The infection hyphae of Digitaria and Triticum pathotypes tended to localize in the outer layer of rice roots, but not in those of wheat and barley roots. The infection hyphae of the Oryza pathotype were distributed in both the intercellular and intracellular spaces of rice root cells. Pathogenesis‐related genes and reactive oxygen species accumulation were induced after root inoculation with all combinations. These results suggest that resistance reactions were induced in the roots of gramineous plants against the infection with Pyricularia isolates but failed to prevent fungal invasion.