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.     

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 usedfor inoculation.     

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.     

Partial Purification,of Extracellular Cellulase from Pyricularia oryzae and Comparison of the Elution Patterns among Various Strains

In order to explain the difference in extracellular cellulase activities (C₁ and C_x enzyme activities) among various strains of P. oryzae, the elution patterns from the column were compared among various strains, following each step of the partial purification.

The crude enzymes, prepared by ammonium sulfate fractionation (0.2~0.8 sat.) from the culture filtrates, which were obtained from various strains of P. oryzae cultured on rice plant powder as the carbon source, were fractionated by DEAE-Sephadex A–50 chromatography into two components; the passing-through fraction (I) and the fraction (II) adsorbed and eluted from the column with 0.5 M NaCl The percentage of the enzyme activity (C_x enzyme activity) in fraction I to that of the crude extract was found to vary chracteristically according to the strain, and the variation was in a good correlation to that of the extracellular cellulase activities.

Fractions I and II were then separated by Sephadex G–100 into two (peaks a and b) and at least five (peaks c, d, e, f and g) components, respectively. The activities in peaks a, b and g were found to vary according to the strain, while those of peaks c and e were common among various strains.

The cell wall fraction prepared from C–3 strain, which was previously shown to be low in enzyme activity and thus out of the correlation between the degree of pathogenicity and extracellular cellulase activity, was found to exhibit higher cellulase activities (C₁ and C_x enzyme activities) than those of other strains examined. Thus, the low extracellular cellulase activity in the case of C–3 strain was suggested to be due to the abnormality in the mechanism of enzyme excretion.     

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.     

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.     

茶多酚对稻瘟病菌的抑制作用及抑菌机理

用不同浓度茶多酚对稻瘟病菌进行抑菌和抑菌机理研究。结果表明,不同浓度的茶多酚对稻瘟病菌菌丝生长和分生孢子萌发具有很强的抑制作用。随着茶多酚浓度的增加,其抑制作用增强,其中5.00 mg/mL和10.00 mg/mL抑制效果最好,其抑制率高达100%,且分生孢子畸形,细胞破裂,原生质外溢。其作用机理主要是破坏菌体的细胞膜结构,抑制CAT、POD酶活,使其丧失细胞膜的屏障和酶系的保护功能,最终导致菌体生长受到抑制或死亡。     

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.     

稻瘟病菌效应蛋白的分泌特征及其在水稻细胞中的转运

许多病原菌能够通过分泌不同的效应蛋白以调控植物的防御以及胞内进程,从而助其有效入侵植物组织。稻瘟病菌的效应蛋白根据其不同的定位特点,被分为细胞质效应蛋白与质外体效应蛋白两类。在侵染过程中,细胞内的侵染菌丝被源于宿主植物的交界面菌丝膜(extrainvasive hyphal membrane, EIHM)包围,由EIHM与侵染菌丝细胞壁形成的质外体隔间是效应蛋白分泌的"必经通道"。此外,在稻瘟病菌侵染过程中会形成一个高度局部化的结构—活体营养表面复合体(biotrophic interfacial complex, BIC),它能够聚集由侵染菌丝分泌的细胞质效应蛋白。该文综述了稻瘟病菌效应蛋白的功能及其迁移过程,介绍了BIC的两个重要的形成阶段,阐明了不同效应蛋白的定位特点以及胞间转运的动态过程,揭示了效应蛋白分泌、转运至水稻细胞质以及在水稻细胞之间移动的分子机制。     

茶树轮斑病的发生及病原菌分生孢子萌发特性

对豫南茶园茶树轮斑病的发生与病原菌分生孢子萌发特性进行了调查和研究。茶树轮斑病在豫南茶区发生较普遍,一般夏秋发病较重,冬春发病较轻;轮斑病的病叶率和病情指数与温度、湿度、光照等生态因子关系密切;病原菌孢子在离体25℃条件下,4 h开始萌发。在茶树叶面上,病原菌分生孢子萌发明显比非叶面条件下好,病原菌菌丝生长较快,说明此病原菌与茶树叶片有高度的亲和力和较强的适应茶树叶面微环境的能力;茶树轮斑病病原菌分生孢子在pH5~7范围内萌发及芽管伸长较好,pH过小或过大均不利于病原菌的分生孢子萌发和菌丝生长。     

Cellulase and ({beta}-Glucosidase Components in Culture Filtrates from Botryodiplodia theobromae Pat.

Cellulase and ß-glucosidase components in culturefiltrates from Botryodiplodia theobromaePat. were separatedby polyacrylamide-gel electrophoresis and gel-filtration onSephadex. A correlation between the electrophoretic mobilitiesand gel-filtration behaviour of the components was established.Four cellulase components (Ca, Cb, Cc, and Cd) were recognized with approximate molecular weights of 162 000 (Ca), 15 000(Cb), 10 000 (Cc), and 4500 (Cd). Four ß-glucosidasecomponents (Ba, Bb, Bc, and Bd) were also recognized with molecularweights of 112 000 (Ba), 56 000 (Bb), 27 000 (Be), and 13 300(Bd). The cellulase components appeared to be composed of aggregatesof similar subunits. Similarly, the ß-glucosidasecomponents appeared to be aggregates of similar subunits. Similarly,the ß-glucosidase components appeared to be aggregratesof a subunit that differed from the cellulase subunit. The activitiesof the cellulase components differed on different cellulosicsubstrates. Cotton flock was readily solubilized by a mixtureof cellulase components Ca, Cb, and Cc, an effect which wasenhanced in the presence of component Cd. Any appreciable solubilizationof native cotton fibres required component Ca or, more effectively,Cd.     

Purification of Peptidases of Aspergillus oryzae and Some Properties of the Purified Peptidases

The purification of Aspergillus oryzae peptidases was attempted by the fractional precipitation with acetone, ammonium sulphate, and by starch zone electrophoresis. We, thus, achieved a great success in the separation of dipeptidase free from aminopolypeptidase and proteinase as well as in the separation of aminopolypeptidase free from dipeptidase and proteinase.

The specific activity (C₀) of the former (leucylglycine hydrolysis) was 7000 and that of the latter (leucylglycylglycine hydrolysis) 22000.

The leucylglycine dipeptidase was remarkably activated by Zn⁺⁺, and Co⁺⁺. Some other enzyme properties were also found and are discussed.