Effects of Dazomet on Phenolase Activity in Sclerotia of Sclerotinia sclerotiorum

Developing sclerotia of the fungus Sclerotinia sclerotiorumexude a clear liquid which contains the enzyme o-diphenol oxidase.The activity of this enzyme, which is also present in the sclerotialtissue, is inhibited by Dazomet (a soil fumigant), sodium azide,and DIECA. These inhibitions can be prevented in the presenceof sufficient quantities of Cu²⁺. The activity of mushroom o-diphenoloxidase is affected by Dazomet and Cu²⁺ in a similar manner. Unpigmented, exposed surfaces of cut sclerotia darken withina few days due to the synthesis of a melanin-like pigment. Theformation of this pigment is prevented by Dazomet. This effectof Dazomet is compared with its action on the darkening of cutsurfaces of potato tubers which also possess appreciable amountsof o-diphenol oxidase.     

Electrophoretic Karyotypes of Sclerotinia sclerotiorum

Electrophoretic karyotypes (EKs) of 83 isolates were variable within agricultural and natural populations of Sclerotinia sclerotiorum, as well as among S. sclerotiorum, Sclerotinia minor, and Sclerotinia trifoliorum. Variation in EKs was not observed within six mitotic or three meiotic lineages of isolates. EKs of 8 to 10 chromosome-sized DNAs were observed. Homologous and heterologous probes hybridized to four linkage groups.     

Differential Responses of Host and Non-host Substrata on Germination of Ascospores of Sclerotinia sclerotiorum

Effect of host.(Cicer arietinum L.) and some non-host (Allium cepa L., A. Sativum L., Ocimum sanictum L., Azadirachta indica Juss., Zingiber officinale Roscoe. And Curcuma longa L.) substrata on the germination of ascospores io Sclerotinia sclerotiorum (Lib.) de Bary has been observed. Maximum germination was noticed on flower petals of gram (C.arietinum) with minimum time (2.5 h) for germ tube initiation. Among the non-host substrate germination was completely inhibited on ginger rhizome peeling whereas delayed germination (after 12h) and lowest germination percentage (48%) as compared with other non-hosts, were observed on turmeric rhizome peeling. It is suggested that ginger extract may be effective in controlling stem rot and wilt of gram incited by S. sclerotiorum in the field.     

Agrobacterium-mediated transformation of Sclerotinia sclerotiorum

Ascospores from the phytopathogenic fungus Sclerotinia sclerotiorum were transformed to hygromycin B resistance by co-cultivation with Agrobacterium tumefaciens. Transformed spores germinated and grew on PDA supplemented with 100 ug/ml hygromycin B. The presence of mitotically stable hph gene integration at random sites in the genome was confirmed by PCR and Southern blot analysis. A transformation frequency of 8 x 10(-5) was achieved in five separate experiments. This study is the first report of success co-cultivating A. tumefaciens with S. sclerotiorum. This report of a reproducible Agrobacterium-mediated transformation method should allow the development of T-DNA tagging as a system for insertional mutagenesis in S. sclerotiorum and provide a simple and reliable method for genetic manipulation.     

Mycoparasitism by Coniothyrium minitans on Sclerotinia sclerotiorum and its Effect on Sclerotial Germination

Scanning electron microscopy showed that hyphae of Coniothyrium minitans produced appressorium-like swellings when they came in contact with Sclerotinia sclerotiorum in dual culture on PDA. The parasitized hyphae gradually skrank and collapsed, and hyphae of the mycoparasite were found inside the host hyphae. The mycoparasite hyphae grew inter- and intracellularly within the sclerotia of S. sclerotiorum. In the later stages of parasitism, hyphae of the mycoparasite proliferated extensively within the sclerotia and formed pycnidia near the sclerotial surface. At this stage, the sclerotia became flattened, soft and disintegrated. Sclerotia parasitized by C. minitans failed to germinate either myceliogenically or carpogenically.     

Organic acid metabolism in Sclerotinia sclerotiorum

Summary Citrate synthase (EC 4.1.3.7), aconitate hydratase (EC 4.2.1.3), NADP specific isocitrate dehydrogenase (EC 1.1.1.42), fumarate hydratase (EC 4.2.1.2) and malate dehydrogenase (EC 1.1.1.37) were detected in cell-free preparations of Sclerotinia sclerotiorum (Lib.) D By. grown on liquid glucose-salts medium in stationary culture. Isocitrate lyase (EC 4.1.3.1) was present when the fungus grew on a carbohydrate-free medium but was not detected when the cultures grew on the glucose-salts medium. The amount of oxalate in the culture filtrate declined as the specific activity of citrate synthase and malate dehydrogenase in the mycelium declined. Increasing the initial pH of the medium resulted in an increase of the dicarboxylic acids in the culture filtrate and the specific activity of malate dehydrogenase in the mycelium. The specific reaction(s) leading to oxalic acid formation were not identified.     

Factors Affecting the Parasitic Activity of Gliocladium virens on Sclerotia of Sclerotinia sclerotiorum and a Note on its Host Range

Aspects of the biology of Gliocladium virens and parasitism of sclerotia of Sclerotinia sclerotiorum in soil were studied. G. virens parasitized and decayed sclerotia of S. sclerotiorum, S. minor, Botrytis cinerea, Sclerotium, rolfsii and Macrophomina phaseolina on laboratory media and caused a reduction in survival of sclerotia of S. sclerotiorum in soil. It was active over a broad range of soil moisture levels and over the entire agricultural soil pH range. The main factor limiting its use as a biological control agent was its temperature requirements.     

Structural Aspects of the Parasitism of Sclerotia of Sclerotinia sclerotiorum (Lib.) de Bary by Coniothyrium minitans Campb.

Sclerotia of Sclerotinia sclerotiorum inoculated with pycnidiospores of Coniothyrium minitans were studied by means of light microscopy and transmission and scanning electron microscopy. The hyperparasite penetrated the walls of the rind cells by means of physical pressure and destroyed the cell contents. Penetration of medullary hyphae was by enzymic lysis and physical pressure; there was evidence to suggest that the hyperparasite may coil around the host cells before inserting infection hyphae.     

粉红黏帚霉67-1菌株寄生核盘菌菌核的酶学动态研究

对粉红黏帚霉67-1菌株侵染核盘菌菌核过程的多种细胞壁降解酶活性进行了连续测定,以研究几丁质酶等在这一寄生互作体系中的可能作用。结果表明:葡聚糖酶活性变化表现活跃,且随寄生过程呈增加趋势,配对法T检验结果表明,第10d的处理与对照酶活性差异达到最大;几丁质酶、蛋白酶活性变化表现较低,而纤维素酶未检测得到。酶学动态变化与之前石蜡切片显微观察的结果在时间上表现一致;认为葡聚糖酶可能是粉红黏帚霉67-1菌株寄生核盘菌菌核的关键酶。     

Purification of the beta-glucosidase from Sclerotinia sclerotiorum

A beta-glucosidase (EC 3.2.1.21) has been isolated from culture filtrates of the fungus Sclerotinia sclerotiorum. The protein was purified by gel filtration on a column of Bio-Gel P-300 and by ion exchange chromatography on DEAE-Bio-Gel A. The molecular weight, determined by gel filtration, was 240,000. Km values for the enzyme towards p-nitrophenyl-beta-D-glucoside and cellobiose were respectively 0.10 mM and 1.23 mM. The beta-glucosidase activity was found to be strongly associated with a beta-xylosidase (EC 3.2.1.37) activity, suggesting that both activities could be represented in a single protein complex.     

Enzymatic oxalate decarboxylation in isolates of Sclerotinia sclerotiorum

Abstract Sclerotinia sclerotiorum isolates B24 (virulent) and SS41 (hypovirulent) possess oxalate decarboxylase. Production was regulated by composition and pH of culture medium and required the presence of oxalate or its precursor, succinic acid, as inducers. Mycelia of both isolates contain equivalent amounts of enzyme.     

Plant Growth Promoting Metabolites of Sclerotinia sclerotiorum

In the research on the plant growth regulators produced by phytopathogenic fungi, two active metabolites, sclerotinin A and B, in addition to sclerin have been isolated from the culture filtrate of S. sclerotiorum. Sclerotinin A and B have been shown to be 3,6,8-tri-hydroxy-3,4,5,7-tetramethyl-3,4-dihydroisocoumarin and 3,6,8-trihydroxy-3,5,7-trimethyl-3,4- dihydroisocoumarin, respectively.     

菌核病防治研究进展

菌核病是一种寄主种类广泛的重大植物病害,可侵染450多种重要作物和草类,在我国每年给油菜、大豆以及多种蔬菜带来10～30亿元的损失.介绍了菌核病的症状、危害以及致病机理等,概述了主要的防治措施,并报道了国内外在关于菌核病生物防治、转基因育种、分子机理等方面的研究进展.     

Secretion of pectic isoenzymes by Sclerotinia sclerotiorum

Abstract Cultures of Sclerotinia sclerotiorum grown on different pectin-related polysaccharides (citrus pectin, apple pectin, sodium polygalacturonate), carboxymethylcellulose (CMC) or glucose as the only carbon source were examined daily for polygalacturonase and pectinase activities. Electrophoretic forms of polygalacturonase and pectin methylesterase activities were revealed using analytical IEF and sodium polygalacturonate and citrus pectin as substrates in overlay gels. A sequence in the production of pectic enzymes and isoenzyme synthesis was found in pectic-polymer cultures corresponding to the induction of several isoenzymes. Enzyme activities in glucose media were associated with three polygalacturonase and two pectinmethylesterase isoforms which were produced constitutively. Sodium-dodecyl-sulphate polyacrylamide-gel electrophoresis followed by immunoblotting with polyclonal antibodies against an exo-polymethylgalacturonase and an exo-polygalacturonase revealed that these exo-enzymes were secreted from the beginning of cultivation in the different culture media showing characteristics of constitutive enzymes.     

Soil temperatures and inoculation techniques affect emergence and reisolation of Sclerotinia sclerotiorum from soybean

Emergence of Amsoy soybean (Glycine max) seed inoculated withSclerotinia sclerotiorum was significantly reduced below noninoculated seed at soil temperatures of 25°, 30°, and 35 °C, but not at 20 °C.S. sclerotiorum was readily reisolated from wound-inoculated stems of seedlings and nearly mature plants above the point of inoculation and below to the crown area, but not from roots. The fungus was recovered from stems but not roots of 15-day seedlings grown in sterile soil before infestation of the soil surface with a suspension of mycelium and sclerotia and assayed at 15 days after soil infestation. When compared to healthy, seeds, infected seeds withS. sclerotiorum were characterized by appearing flattened.Supported in part by the Illinois Agricultural Experiment Station; Regional Project S-72; and U.S. Agency for International Development, grant csd-1922.     

Characterization of the Sclerotinia sclerotiorum cell wall proteome

We used a proteomic analysis to identify cell wall proteins released from Sclerotinia sclerotiorum hyphal and sclerotial cell walls via a trifluoromethanesulfonic acid (TFMS) digestion. Cell walls from hyphae grown in Vogel's glucose medium (a synthetic medium lacking plant materials), from hyphae grown in potato dextrose broth and from sclerotia produced on potato dextrose agar were used in the analysis. Under the conditions used, TFMS digests the glycosidic linkages in the cell walls to release intact cell wall proteins. The analysis identified 24 glycosylphosphatidylinositol (GPI)‐anchored cell wall proteins and 30 non‐GPI‐anchored cell wall proteins. We found that the cell walls contained an array of cell wall biosynthetic enzymes similar to those found in the cell walls of other fungi. When comparing the proteins in hyphal cell walls grown in potato dextrose broth with those in hyphal cell walls grown in the absence of plant material, it was found that a core group of cell wall biosynthetic proteins and some proteins associated with pathogenicity (secreted cellulases, pectin lyases, glucosidases and proteases) were expressed in both types of hyphae. The hyphae grown in potato dextrose broth contained a number of additional proteins (laccases, oxalate decarboxylase, peroxidase, polysaccharide deacetylase and several proteins unique to Sclerotinia and Botrytis) that might facilitate growth on a plant host. A comparison of the proteins in the sclerotial cell wall with the proteins in the hyphal cell wall demonstrated that sclerotia formation is not marked by a major shift in the composition of cell wall protein. We found that the S. sclerotiorum cell walls contained 11 cell wall proteins that were encoded only in Sclerotinia and Botrytis genomes.     

黑龙江省大豆菌核病菌核盘菌遗传多样性分析

了解黑龙江省不同地区侵染大豆核盘菌菌株分离物间的主要特性差异,利用PDA培养基对核盘菌进行分离和纯化,同时利用RAPD和rDNA-ITS标记方法对核盘菌进行遗传多样性分析,获得了50株纯化的核盘菌,用RAPD标记确定的遗传相似系数范围为0.54-0.98,平均相似系数为0.76,说明供试的核盘菌菌株的基因型具有一定的差异。对50个测定序列有差异的32个核盘菌ITS和5.8S rDNA片段的多序列对位分析,在ITS1区域的1-40bp种间变化较大,主要以碱基颠换和转换为进化形式。ITS2区域非常保守没有变异位点。黑龙江省核盘菌菌株在DNA水平上和ITS间隔区上具有较显著的遗传变异,显示出丰富的遗传多样性。