The glucose repressor CRE1 from Sclerotinia sclerotiorum is functionally related to CREA from Aspergillus nidulans but not to the Mig proteins from Saccharomyces cerevisiae.

We isolated the putative glucose repressor gene cre1 from the phytopathogenic fungus Sclerotinia sclerotiorum. cre1 encodes a 429 amino acid protein 59% similar to the carbon catabolite repressor CREA from Aspergillus nidulans. In addition to the overall amino acid sequence relatedness between CRE1 and CREA proteins, cre1 can functionally complement the A. nidulans creAd30 mutation as assessed by repression of the alcohol dehydrogenase I gene expression. The CREI region carrying the two zinc fingers is also very similar to the DNA binding domains of the Saccharomyces cerevisiae glucose repressors Mig1p and Mig2p. Despite the presence in the CRE1 protein of several motifs involved in the regulation of Miglp activity, cre1 cannot complement mig deficiencies in S. cerevisiae. These data suggest that glucose repression pathways may have evolved differently in yeasts and filamentous fungi.     

Laccase is upregulated via stress pathways in the phytopathogenic fungus Sclerotinia sclerotiorum

We report on the factors affecting the production of the newly characterized laccase from the phytopathogenic fungus Sclerotinia sclerotiorum (Lib.) de Bary. The carbon/nitrogen ratio appears to be of great importance. Rather than a simple nutrient-rich nitrogen source, yeast extract (YE) behaves as a true laccase upregulator, apparently acting via a stress pathway. Chelidonium majus extract, a known antifungal agent, acts in a similar manner. The compound(s) in the YE responsible for enhancing laccase synthesis are suggested to be hydrolysable choline derivatives. Both extracts reduce biomass and sclerotia development and enhance laccase production, leading to an increase in laccase activity by one order of magnitude compared to controls. The pH of the medium, a well-known virulence regulator for this fungus, also acts as a true laccase regulator, though via a different mechanism. The effect of pH appeared to be linked to the acidification kinetics of the extracellular medium during fungal development. A number of other known laccase inducers were found to enhance laccase production at most twofold.     

Indolyl-3-acetaldoxime dehydratase from the phytopathogenic fungus Sclerotinia sclerotiorum: purification, characterization, and substrate specificity

The purification and characterization of indolyl-3-acetaldoxime dehydratase produced by the plant fungal pathogen Sclerotinia sclerotiorum is described. The substrate specificity indicates that it is an indolyl-3-acetaldoxime dehydratase (IAD, EC 4.99.1.6), which catalyzes transformation of indolyl-3-acetaldoxime to indolyl-3-acetonitrile. The enzyme showed Michaelis-Menten kinetics and had an apparent molecular mass of 44 kDa. The amino acid sequence of IAD, determined using LC-ESI-MS/MS, identified it as the protein SS1G_01653 from S. sclerotiorum. IADSs was highly homologous (84% amino acid identity) to the hypothetical protein BC1G_14775 from Botryotinia fuckeliana B05.10. In addition, similarity to the phenylacetaldoxime dehydratases from Gibberella zeae (33% amino acid identity) and Bacillus sp. (20% amino acid identity) was noted. The specific activity of IADSs increased about 17-fold upon addition of Na(2)S(2)O(4) under anaerobic conditions, but in the absence of Na(2)S(2)O(4) no significant change was observed, whether aerobic or anaerobic conditions were used. As with other aldoxime dehydratases isolated from microbes, the role of IADSs in fungal plant pathogens is not clear, but given its substrate specificity, it appears unlikely that IADSs is a general xenobiotic detoxifying enzyme.     

The effect of polyamine biosynthesis inhibition on growth and differentiation of the phytopathogenic fungus Sclerotinia sclerotiorum

We studied the effects of several polyamine biosynthesis inhibitors on growth, differentiation, free polyamine levels and in vivo and in vitro activity of polyamine biosynthesis enzymes in Sclerotinia sclerotiorum. -Difluoromethylornithine (DFMO) and -difluoromethylarginine (DFMA) were potent inhibitors of mycelial growth. The effect of DFMO was due to inhibition of ornithine decarboxylase (ODC). No evidence for the existence of an arginine decarboxylase (ADC) pathway was found. The effect of DFMA was partly due to inhibition of ODC, presumably after its conversion into DFMO by mycelial arginase, as suggested by the high activity of this enzyme detected both in intact mycelium and mycelial extracts. In addition, toxic effects of DFMA on cellular processes other than polyamine metabolism might have occurred. Cyclohexylamine (CHA) slightly inhibited mycelial growth and caused an important decrease of free spermidine associated with a drastic increase of free putrescine concentration. Methylglyoxal bis-[guanyl hydrazone] (MGBG) had no effect on mycelial growth. Excepting MGBG, all the inhibitors strongly decreased sclerotial formation. Results demonstrate that sclerotial development is much more sensitive to polyamine biosynthesis inhibition than mycelial growth. Our results suggest that mycelial growth can be supported either by spermidine or putrescine, while spermidine (or the putrescine/spermidine ratio) is important for sclerotial formation to occur. Ascospore germination was completely insensitive to the inhibitors.     

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.     

Genetic diversity and mycelial compatibility groups of the plant-pathogenic fungus Sclerotinia sclerotiorum in Brazil

The genetic variability of 40 Sclerotinia sclerotiorum isolates from various fields widely distributed throughout Brazil and different host crops was analyzed using RAPD markers and mycelial compatibility groupings (MCGs). The isolates were characterized using 16 random primers of the OPERON series, which produced 121 DNA fragments. UPGMA cluster analysis using Jaccard's genetic distance and MCGs allowed separation of the isolates into three clusters, with similarity indices of 68.2, 61.8, and 61.8%, and five MCGs. The haplotypes obtained with RAPD markers provided very characteristic groupings of S. sclerotiorum isolates according to MCG, but did not show any relationship with geographic origin or host type. Furthermore, analysis of molecular variance demonstrated that 99.1% of the observed variation was a result of genetic differences between individuals; the host culture did not have a significant effect. This is the first report of high level variability of S. sclerotiorum in Brazil based on the study of isolates of wide geographical origin, supported by RAPD markers and MCGs. These results endorse the prevalence of sexual reproduction in tropical and subtropical regions in contrast to clonal reproduction in temperate regions.     

Structural and functional characterization of the GalNAc/Gal-specific lectin from the phytopathogenic ascomycete Sclerotinia sclerotiorum (Lib.) de Bary

The lectin found in mycelium and sclerotes of the phytopathogenic fungus Sclerotinia sclerotiorum is a homodimer consisting of two identical non-covalently bound subunits of 16,000 Da. CD spectra analysis revealed that the S. sclerotiorum agglutinin (SSA) contains predominantly beta-sheet structures. SSA exhibits specificity towards GalNAc whereby the hydroxyls at positions 4 and 6 of the pyranose ring play a key role in the interaction with simple sugars. The carbohydrate-binding site of SSA can also accommodate disaccharides. The N-terminal sequence of SSA shares no significant similarity with any other protein except a lectin from the Sclerotiniaceae species Ciborinia camelliae. A comparison of SSA and the lectins from C. camelliae and some previously characterized lectins indicates that the Sclerotiniaceae lectins form a homogeneous family of fungal lectins. This newly identified lectin family, which is structurally unrelated to any other family of fungal lectins, is most probably confined to the Ascomycota.     

Clonal dispersal and spatial mixing in populations of the plant pathogenic fungus, Sclerotinia sclerotiorum

Two thousand seven hundred and forty-seven isolates of Sclerotinia sclerotiorum were sampled from four field populations of canola in western Canada. Each field was sampled in a grid of 128 50-m 50-m quadrats plus four intensive quadrats each sampled in a diagonal transect. Sampling was done at two phases of the disease cycle: (1) from ascospore inoculum on petals and (2) from disease lesions in stems. A total of 594 unique genotypes was identified by DNA fingerprinting. In each field, a small group of clones represented the majority of the sample, with a large group of clones or genotypes sampled once or twice. Clone frequencies were compared by χ² tests. The difference in profiles of clone frequencies for the two fields sampled in 1991 was not significant, but in 1992 the difference in profiles was marginally significant, indicating some local population substructure. The difference in profiles of clone frequencies for petals and lesions was not significant in each of the two fields sampled in 1991. In each of the two fields sampled in 1992, however, the difference was highly significant, consistent either with selection for some clones or with waves of immigration during the disease cycle. Nine of the 30 most frequently sampled clones from this study were previously recovered in a macrogeographical sample from western Canada in 1990. For spatial analyses, randomization tests indicated no significant spatial aggregation of either clones on petals or clones from lesions. Also, isolates of a clone on petals were not closer to isolates of the same clone from lesions than could be predicted by chance. Both observations suggest spatial mixing of ascospore inoculum from resident or immigrant sources.     

Thiol redox state and oxidative stress affect sclerotial differentiation of the phytopathogenic fungi Sclerotium rolfsii and Sclerotinia sclerotiorum

Aims:  To investigate the involvement of oxidative stress and thiol redox state (TRS) in sclerotial differentiation of Sclerotium rolfsii and Sclerotinia sclerotiorum.
Methods and results:  Oxidative stress in these fungi was assessed by lipid peroxidation, which was higher in comparison with their nonsclerotiogenic counterpart strains. TRS [measured as glutathione (GSH) and cysteine] was associated with oxidative stress and differentiation using the TRS modulator and antioxidant Ν -acetylcysteine (AcCSH) and the GSH biosynthesis inducer and inhibitor l -2-oxo-thiazolidine-4-carboxylate and l -buthionine- S , R -sulphoximine (BSO) respectively. Differentiation and oxidative stress was decreased by AcCSH in both fungi. The decrease of differentiation by BSO was not associated with oxidative stress in these fungi.
Conclusions:  Differentiation and oxidative stress in both fungi depends on the availability of antioxidant noncytotoxic –SH groups and is not depended on any direct antioxidant role of GSH and its precursor cysteine.
Significance and Impact of the Study:  This study helps to understand the mechanism(s) of sclerotial differentiation in these agriculturally important phytopathogenic fungi and proposes that AcCSH can be used as potent fungicide by (i) acting as growth inhibiting cytotoxic oxidant and (ii) sustaining these fungi in their undifferentiated hyphal stage where they are vulnerable to degradation by soil micro-organisms.     

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。     

Content of acid soluble nucleotides in the microscopic fungus Sclerotinia sclerotiorum at different growth stages

Variations in the content of acid soluble nucleotides at different growth stages of the producer of pectolytic enzymes-the fungus Sclerotinia sclerotiorum were measured. The initial growth stage was characterized by an increased content of adenyl nucleotides whose amount decreased by the 48th hour. In the 48-hour mycelium guanyl nucleoside mono- and diphosphates were the major components of the nucleotide pool. Throughout the entire cultivation cytidyl derivatives occurred in trace quantities.     

Small RNAs from the plant pathogenic fungus Sclerotinia sclerotiorum highlight host candidate genes associated with quantitative disease resistance

Fungal plant pathogens secrete effector proteins and metabolites to cause disease. Additionally, some species transfer small RNAs (sRNAs) into plant cells to silence host mRNAs through complementary base pairing and suppress plant immunity. The fungus Sclerotinia sclerotiorum infects over 600 plant species, but little is known about the molecular processes that govern interactions with its many hosts. In particular, evidence for the production of sRNAs by S. sclerotiorum during infection is lacking. We sequenced sRNAs produced by S. sclerotiorum in vitro and during infection of two host species, Arabidopsis thaliana and Phaseolus vulgaris. We found that S. sclerotiorum produces at least 374 distinct highly abundant sRNAs during infection, mostly originating from repeat-rich plastic genomic regions. We predicted the targets of these sRNAs in A. thaliana and found that these genes were significantly more down-regulated during infection than the rest of the genome. Predicted targets of S. sclerotiorum sRNAs in A. thaliana were enriched for functional domains associated with plant immunity and were more strongly associated with quantitative disease resistance in a genome-wide association study (GWAS) than the rest of the genome. Mutants in A. thaliana predicted sRNA target genes SERK2 and SNAK2 were more susceptible to S. sclerotiorum than wild-type, suggesting that S. sclerotiorum sRNAs may contribute to the silencing of immune components in plants. The prediction of fungal sRNA targets in plant genomes can be combined with other global approaches, such as GWAS, to assist in the identification of plant genes involved in quantitative disease resistance.     

Extracellular proteinases from the phytopathogenic fungus Fusarium culmorum

The growth of Fusarium culmorum fungus on a medium containing thermostable proteins from potato tubers was accompanied by the production of proteinases, exhibiting activity over a broad pH range (from 6.0–10.0). When studied by SDS-PAGE in the presence of β-mercaptoethanol, extracellular proteinases were represented by at least five species with a molecular weight of 30–60 kDa. Inhibitor analysis and studies of enzyme activities with synthetic substrates demonstrated that the culture liquid of Fusarium culmorum contained serine proteinases of various classes. The amount of subtilisin-like proteinases was the highest. A near-complete inhibition of the enzymes was caused by proteinaceous proteinase inhibitors from potato tubers. These data suggest that proteinases of the phytopathogen Fusarium culmorum serve as a metabolic target for natural inhibitors of potato proteinases.     

Proteinases and exopeptidases from the phytopathogenic fungus Ustilago maydis

The proteolytic system of the phytopathogenic and dimorphic fungus Ustilago maydis is not known. In this work, we report the presence of at least four proteases from two haploid strains of U. maydis. Activities of two proteinases pumA and pumB, aminopeptidase pumAPE, and dipeptidylaminopeptidase pumDAP were measured under several nutritional and morphological conditions, including the yeast-mycelium transition. The activity of pumA was found in the intracellular and extracellular fractions, pumAi and pumAe, respectively. The latter activity was detected only during the yeast-mycelium dimorphic transition induced by growth at acid pH in a medium containing ammonium as the sole nitrogen source. Activity of pumAe was partially inhibited by Pepstatin A, which also inhibited mycelium formation. Activity of pumAi was inhibited by this specific inhibitor of aspartyl-proteases. Activity of pumB was detected in intracellular and extracellular fractions, mostly bound to an endogenous inhibitor, which was removed by treatment at acid pH. This fungus contains at least two soluble pumAPE, which might be metallo-proteases, because they were inhibited by EDTA and 1-10, phenanthroline. When the fungus was grown in media containing proline or corn infusion as the nitrogen source, an intracellular pumDAP activity was detected. No carboxypeptidase activity was found with N-benzoyl-l-tyrosine-4-nitroanilide as substrate in any of the conditions tested in any of the U. maydis strains analyzed.     

Expression and regulation of Sclerotinia sclerotiorum necrosis and ethylene-inducing peptides (NEPs)

Successful host colonization by necrotrophic plant pathogens requires the induction of plant cell death to provide the nutrients needed for infection establishment and progression. We have cloned two genes encoding necrosis and ethylene-inducing peptides from Sclerotinia sclerotiorum , which we named SsNep1 and SsNep2 . The peptides encoded by these genes induce necrosis when expressed transiently in tobacco leaves. SsNep1 is expressed at a very low level relative to SsNep2 during infection. The expression of SsNep2 was induced by contact with solid surfaces and occurred in both the necrotic zone and at the leading margin of the infection. SsNep2 expression was dependent on calcium and cyclic adenosine monophosphate signalling, as compounds affecting these pathways reduced or abolished SsNep2 expression coincident with a partial or total loss of virulence.     

Adenylate cyclase from the phytopathogenic fungus Alternaria solani

Abstract Adenylate cyclase activity of Alternaria solani bound to the particulate cell fraction was solubilized by 2.5% Ficoll. The apparent K _m of the solubilized enzyme for adenosine 5'-triphosphate was 2.5 mM, and it required Mn²⁺ for maximum activity. M _r as determined by gel filtration was approximately 500. Fluoride ions at millimolar concentrations, GTP, adenosine, and N⁶-phenylisopropyl adenosine at micromolar concentrations did not stimulate adenylate cyclase activity. The enzyme was inhibited 25–55% by millimolar concentrations of 2'-deoxyadenosine, 2'-0 methyl adenosine, 9-β- d -arabinofuranosyl adenosine and 2'-3'-isopropylidene adenosine. Partially purified enzyme obtained after DEAE-BioGel chromatography was very unstable. The amount of extractable enzyme activity varied during the fungal growth cycle.     

Extracellular proteinases from the phytopathogenic fungus Fusarium culmorum

The growth of Fusarium culmorum fungus on a medium containing thermostable proteins from potato tubers was accompanied by the production of proteinases, exhibiting activity over a broad pH range (from 6.0-10.0). When studied by SDS-PAGE in the presence of beta-mercaptoethanol, extracellular proteinases were represented by at least five species with a molecular weight of 30-60 kDa. Inhibitor analysis and studies of enzyme activities with synthetic substrates demonstrated that the culture liquid of Fusarium culmorum contained serine proteinases of various classes. The amount of subtilisin-like proteinases was the highest. A near-complete inhibition of the enzymes was caused by proteinaceous proteinase inhibitors from potato tubers. These data suggest that proteinases of the phytopathogen Fusarium culmorum serve as a metabolic target for natural inhibitors of potato proteinases.     

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.