Characterization of Sclerotinia and mycoparasite Coniothyrium minitans interaction by microscale co-culture

Aims:  To characterize the interaction of Sclerotinia sclerotiorum and S. minor with strains of the mycoparasite and commercial biocontrol agent Coniothyrium minitans using novel perfusion chamber gasket co-culture.
Methods and Results:  Sclerotinia were cultured in perfusion chamber gaskets and then flooded with Coniothyrium conidia. After germination, Coniothyrium failed to show any form of directed growth, making contact with Sclerotinia hyphae in a random manner. In turn, some Coniothyrium hyphae coiled round Sclerotinia counterparts and although no intracellular growth was observed, Coniothyrium proliferated, while the hyphae of Sclerotinia became vacuolated and lost the cytoplasm. When co-cultures of Sclerotinia with Coniothyrium were flooded with FITC-lectins, small difference in fluorescence between the fungi was found with FITC-Con A suggesting that cell walls of both the species exposed mannose. In contrast, Coniothyrium fluoresced poorly in comparison with Sclerotinia when FITC-wheat germ agglutinin was used, indicating a marked paucity of N -acetylglucosamine exposure by cell walls of Coniothyrium, hence reduced exposure to chitinolytic enzyme action.
Conclusions, Significance and Impact of the Study:  The approach employed supported direct sequential microscopic observation of Coniothyrium and Sclerotinia as well as the utilization of representative fluorescent moieties to characterize relative carbohydrate cell wall exposure.     

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.     

Expression of barley oxalate oxidase confers resistance against Sclerotinia sclerotiorum in transgenic Brassica juncea cv Varuna

Transgenic Research - Sclerotinia Stem Rot (SSR) caused by the oxalic acid (OA)-secreting necrotrophic fungal pathogen Sclerotinia sclerotiorum, causes significant yields losses in the crop...     

植病生防菌盾壳霉的分子生物学研究进展

盾壳霉是一种重要的核盘菌寄生茵.近年来,该菌在分子水平的研究取得了一定的进展.本文概述了盾壳霉在产孢调控、与核盘菌互作、遗传转化以及动态检测和遗传多样性等方面的研究现状,并对研究中存在的问题进行了讨论.希望在此基础上能够促进该茵分子生物学研究的不断深入,更好地开发利用该菌的基因资源.     

核盘菌属一新种——人参核盘菌

本文报道了采自人参(Panax ginseng C. A．Mey (保留名nom. Coiiscry))的核盘菌属一新种——人参核盘菌(Sclerotinla．Ginseng Wang C. R.,C. F. Chen et J. Chen)。该种在形态学以及可溶性蛋白、果胶(甲)酯酶和多聚半乳糖醛酸酶谱带等方面,均不同于已知种核盘菌(S. sclerotiorum (Lib．)de Bary),小核盘菌(S. minor Jagger)车轴草核盘菌(S．Trifoliorum Erikss．)和细辛核盘菌(S．Asari Wu et C．R. Wang)。模式标本(800719)保存于沈阳农业大学植物免疫研究室。     

核盘菌可溶性蛋白质及同工酶电泳研究*

为了研究核盘菌的分类和生理分化问题,对供试的28个核盘菌株进行了可溶性蛋白质,芳香基酯酶和酸性磷酸酯酶同工酶电泳谱带以及紫外光吸收峰图形的试验研究,其结果,可分为五种类型。其中属于核盘菌属有四个种,即核盘菌Sclerotinia sclerotiorum(Lib.)de Bary(=Whetzelinia sclerotiorum(Lib.)Korf & Dumont),三叶草核盘菌S.trifoliorum Erikss.,细辛核盘菌(S.asari Wu et C.R.Wang和人参菌核病菌Sclerotinia sp。人参菌核病的另一分离菌和油菜菌核病的一个分离菌其谱带与灰葡萄孢(Botrytis cinerea)谱带相似,应归属于与葡萄孢属(Botrytis)相应的核盘菌。在核盘菌S.sclerotiorum种群中,南方菌系与北方菌系的可溶性蛋白质稍有差异,芳香基酯酶和酸性磷酸酯酶活性强度也稍有不同,可能存在生理分化现象。     

Biology and mycovirus-assisted biological control of Sclerotinia sclerotiorum infecting vegetable and oilseed crops

Abstract

Plant disease caused by pathogenic fungal infection causes maximum crop damage. Among different fungal diseases, rot caused by Sclerotinia spp.; is a primary concern for vegetables and oilseed industry. Disease management using Chemical fungicides is a potential hazard and leads to the development of many fungicide-resistant strains. Hypovirulence associated mycoviruses is a possible environment-friendly solution, and current studies are aiming to exploit their potential as biocontrol agents. The use of the mycovirus mediated hypovirulent approach has emerged as a new technique to identify successful biocontrol agents. Most mycoviruses are known to have RNA genomes, double-stranded RNA (dsRNA) or single-stranded RNA (ssRNA). A total of six dsRNA mycoviruses and a one ssDNA mycovirus have been reported from Sclerotinia sclerotiorum till date which includes the most recent entry as published by Hamid and his group in 2018. In contrast to dsRNA mycovirus, ssDNA mycovirus reported from Sclerotinia sclerotiorum has significant potential to be used as a biocontrol agent in the fields. Despite several reports on mycoviruses of Sclerotinia, not much could be done to explore its commercial importance. The present review describes the recent developments in the area of mycoviruses of Sclerotinia sclerotiorum and associated biocontrol potential.     

植病生防菌盾壳霉的研究进展

盾壳霉是世界性病原真菌核盘菌的主要拮抗真菌之一。许多研究表明盾壳霉具有控制温室和田间多种作物菌核病的潜力。目前欧洲一些国家已有盾壳霉商品制剂销售。为了加速盾壳霉产业化进程和更好地发挥其控制菌核病的作用 ,以及为了使人们对盾壳霉的研究不断深入 ,综述了盾壳霉生态学特性、遗传改良、对核盘菌的生防机制、在菌核病防治上的应用及基因工程研究等方面的研究进展。     

核盘菌可溶性蛋白质及同工酶电泳研究

为了研究核盘菌的分类和生理分化问题,对供试的28个核盘菌株进行了可溶性蛋白质,芳香基酯酶和酸性磷酸酯酶同工酶电泳谱带以及紫外光吸收峰图形的试验研究,其结果,可分为五种类型。其中属于核盘菌属有四个种,即核盘菌Sclerotinia sclerotiorum(Lib.)de Bary(=Whetzelinia sclerotiorum(Lib.)Korf & Dumont),三叶草核盘菌S.trifoliorum Erikss.,细辛核盘菌(S.asari Wu et C.R.Wang和人参菌核病菌Sclerotinia sp。人参菌核病的另一分离菌和油菜菌核病的一个分离菌其谱带与灰葡萄孢(Botrytis cinerea)谱带相似,应归属于与葡萄孢属(Botrytis)相应的核盘菌。在核盘菌S.sclerotiorum种群中,南方菌系与北方菌系的可溶性蛋白质稍有差异,芳香基酯酶和酸性磷酸酯酶活性强度也稍有不同,可能存在生理分化现象。     

An approximation to the phylogeny of Sclerotinia and related genera

Phylogenies are constructed based on nuclear ribosomal internal transcribed spacer (ITS) DNA sequences from an ingroup consisting of 50 isolates representing 24 species of the discomycete family Sclerotiniaceae and an outgroup consisting of five related taxa of the same family. The ingroup taxa are: three Botrytis spp., two Botryotinia spp., one Ciborinia sp., one Dumontinia sp., one Grovesinia sp., six Myriosclerotinia spp., nine Sclerotinia spp. and one Sclerotium sp. The outgroup taxa are: one Ciboria sp., one Encoelia sp. and three Monilinia spp. The type species is included for all taxa except for Ciborinia and Encoelia. Several of the included taxa are important plant pathogens. The resulting phytogenies are discussed with regard to morphology, life history and taxonomy. A suspected relationship between Sclerotinia borealis and S. tetraspora , and Myriosclerotinia is rejected, while a suspected relationship between Ciborinia ciborium and Myriosclerotinia is strongly supported. Sclerotinia ulmariae , previously synonymized with Dumontinia tuberosa , is reinstated as an independent species of Dumontinia. Two new combinations, Dumontinia ulmariae and Myriosclerotinia ciborium , are proposed. The imperfectly known taxon Sclerotium cepivorum seems most closely related to Dumontinia. We conclude that Dumontinia , and Myriosclerotinia , as currently conceived, are monophyletic, and that Botryotinia along with Botrytis anamorphs probably also constitute a monophyletic lineage. The genus Sclerotinia is probably polyphyletic and characterized by symplesiomorphies rather than synapomorphies. Two putatively new taxa, Sclerotinia sp. 1, and Sclerotinia sp.2 are most closely related to S. minor, S. sclerotiorum and S. trifoliorum , and to S. borealis , respectively.     

Evaluation of polyclonal-antibody-based immunoassays for detection of Sclerotinia sclerotiorum on canola petals, and prediction of stem rot

The potential for using polyclonal-antibody-based immunoassays for detection of Sclerotinia sclerotiorum on canola petals as part of a disease prediction model was investigated. A commercial ELISA kit designed for Sclerotinia homoeocarpa was evaluated for specificity to S. sclerotiorum in comparison to other Sclerotinia spp., and known phyllosphere fungi. This polyclonal-antibody-based kit cross-reacted with antigens from other Sclerotinia spp., and fungi, and absorbance values obtained from S. sclerotiorum-infested canola petals were poorly correlated with percentages of infested petals as determined by plating on semi-selective medium, except when petals were incubated at high humidity for 24 h at 20 degrees C-22 degrees C prior to ELISA evaluation. Additional polyclonal antibodies were prepared from mycelial and semi-purified cell wall antigens, and these antibodies were more specific to S. sclerotiorum than the ELISA kit. However, absorbance values obtained from S. sclerotiorum-infested canola petals were poorly correlated with percentages of infested petals as determined by plating on semi-selective medium. The results are discussed in relation to the use of polyclonal-antibody-based immunoassays for the prediction of epidemics or crop risk from sclerotinia stem rot of canola.     

孜然种子提取物枯茗醛和枯茗酸抑菌作用研究

以番茄早疫病菌、棉花黄萎病菌、小麦纹枯病菌、烟草赤星病菌、小麦全蚀病菌、油菜菌核病菌、辣椒疫霉病菌、水稻纹枯病菌、小麦白粉病菌和番茄灰霉病菌等为供试菌种,采用离体与活体相结合的方法系统地测定了枯茗醛和枯茗酸的抑菌活性。离体抑菌活性测定结果表明,枯茗醛和枯茗酸对多种病原菌具有一定的抑制效果,其中对油菜菌核病菌菌丝生长的抑制效果高于其它供试病原菌,有效中浓(EC50)分别为2.1和7.3 mg/L;枯茗醛和枯茗酸对小麦白粉病的防治实验结果表明,供试浓度为1000 mg/L时,两种药剂的保护防效均高于50%;相同处理浓度下,枯茗酸对油菜菌核病的保护防效与速克灵处理相当,达到57.52%。     

Enhancing resistance to Sclerotinia minor in peanut by expressing a barley oxalate oxidase gene

Sclerotinia minor Jagger is the causal agent of Sclerotinia blight, a highly destructive disease of peanut (Arachis hypogaea). Based on evidence that oxalic acid is involved in the pathogenicity of many Sclerotinia species, our objectives were to recover transgenic peanut plants expressing an oxalic acid-degrading oxalate oxidase and to evaluate them for increased resistance to S. minor. Transformed plants were regenerated from embryogenic cultures of three Virginia peanut cultivars (Wilson, Perry, and NC-7). A colorimetric enzyme assay was used to screen for oxalate oxidase activity in leaf tissue. Candidate plants with a range of expression levels were chosen for further analysis. Integration of the transgene was confirmed by Southern-blot analysis, and gene expression was demonstrated in transformants by northern-blot analysis. A sensitive fluorescent enzyme assay was used to quantify expression levels for comparison to the colorimetric protocol. A detached leaflet assay tested whether transgene expression could limit lesion size resulting from direct application of oxalic acid. Lesion size was significantly reduced in transgenic plants compared to nontransformed controls (65%-89% reduction at high oxalic acid concentrations). A second bioassay examined lesion size after inoculation of leaflets with S. minor mycelia. Lesion size was reduced by 75% to 97% in transformed plants, providing evidence that oxalate oxidase can confer enhanced resistance to Sclerotinia blight in peanut.     

Studies on Biological Activity of Cyclic Imide Compounds

The structure-activity relationships of l-phenylpyrrolidine-2,5-dione derivatives were investigated on Sclerotinia sclerotiorum by the agar dilution method. In addition, several representative compounds were tested for antimicrobial spectra in vitro with 15 pathogenic microbes and for foliage protection activity in green house tests with rice blast, rice brown spot, rice sheath blight and kidney bean stem rot. It was found that 3,5-dihalo-substituents on the benzene moiety are essential to high antifungal activity against Sclerotinia sclerotiorum. Generally, l-(3′,5′-dihalophenyl)pyrrolidine-2,5-diones are active against Corticiaceae, Dematiaceae, Pleosporaceae and Sclerotiniaceae, especially active against Sclerotinia sclerotiorum and Botrytis cinerea (the conidia form of Sclerotinia fuckeliana). N-(3,5-Dichlorophenyl)itaconimide showed a peculiarly broad antimicrobial spectrum. In green house tests, these compounds showed high activity against rice brown spot, rice sheath blight and kidney bean stem rot. Results of green house tests on the above-mentioned diseases correlate fairly well with those of in vitro tests.     

Mycoflora and Potential for Mycotoxin Production of Freshly Harvested Black Bean from the Argentinean Main Production Area

A mycological survey was carried out, for the first time, on black bean samples from the northwestern Argentinean province of Salta in the 1999 harvest season. Ten varieties of black beans were evaluated at three locations. Species of the genus Alternaria were the most prevalent component of the black bean mycoflora. Species of Fusarium, Sclerotinia, Rhizoctonia and Acremonium were also recorded. The predominant species of the genera isolated were Alternaria alternata, Sclerotinia sclerotiorum, Rhizoctonia solani, Fusarium semitectum and Acremonium strictum. An analysis of variance was applied to determine possible differences between black bean varieties. Variety FT88/519 was the most susceptible to Sclerotinia sclerotinia infection, while the variety DOR 604 was the least susceptible. As toxigenic species were recovered, Alternaria toxins, zearalenone and trichothecenes may pose a contamination risk for black bean.     

Candidate gene association mapping of Sclerotinia stalk rot resistance in sunflower (Helianthus annuus L.) uncovers the importance of COI1 homologs

Key message

Functional markers for Sclerotinia basal stalk rot resistance in sunflower were obtained using gene-level information from the model species Arabidopsis thaliana.

Abstract

Sclerotinia stalk rot, caused by Sclerotinia sclerotiorum, is one of the most destructive diseases of sunflower (Helianthus annuus L.) worldwide. Markers for genes controlling resistance to S. sclerotiorum will enable efficient marker-assisted selection (MAS). We sequenced eight candidate genes homologous to Arabidopsis thaliana defense genes known to be associated with Sclerotinia disease resistance in a sunflower association mapping population evaluated for Sclerotinia stalk rot resistance. The total candidate gene sequence regions covered a concatenated length of 3,791 bp per individual. A total of 187 polymorphic sites were detected for all candidate gene sequences, 149 of which were single nucleotide polymorphisms (SNPs) and 38 were insertions/deletions. Eight SNPs in the coding regions led to changes in amino acid codons. Linkage disequilibrium decay throughout the candidate gene regions declined on average to an r ² = 0.2 for genetic intervals of 120 bp, but extended up to 350 bp with r ² = 0.1. A general linear model with modification to account for population structure was found the best fitting model for this population and was used for association mapping. Both HaCOI1-1 and HaCOI1-2 were found to be strongly associated with Sclerotinia stalk rot resistance and explained 7.4 % of phenotypic variation in this population. These SNP markers associated with Sclerotinia stalk rot resistance can potentially be applied to the selection of favorable genotypes, which will significantly improve the efficiency of MAS during the development of stalk rot resistant cultivars.     

A Simple Method for the Mating of Sclerotinia trifoliorum

Rehnstrom, A. L., and Free, S. J. 1993. A simple method for the mating of Sclerotinia trifoliorum. Experimental Mycology 17, 236-239. A simple method which allows for the controlled mating of L and S mating type strains of Sclerotinia trifoliorum is described. Using the method, we have been able to mate L and S strains and have demonstrated the segregation of the genetic markers involved in mycelial incompatibility into the progeny.     

Microsatellite markers for Sclerotinia subarctica nom. prov., a new vegetable pathogen of the High North

Eight polymorphic microsatellite loci were isolated from the ascomycete fungus Sclerotinia subarctica nom. prov. In Alaska, this pathogen causes white mold vegetable diseases sympatrically with the cosmopolitan and closely related Sclerotinia sclerotiorum. Eighteen alleles were observed across the 41 isolates tested and ranged from two to three alleles per locus. Together, the alleles from the eight polymorphic loci yielded only four haploid multilocus genotypes and exhibited significant linkage disequilibrium, reflecting extreme selfing and clonal vegetative reproduction.     

Studies on Biological Activity of Cyclic Imide Compounds

The structure-activity relationships of 3-phenyloxazolidine-2, 4-diones, 3-phenyl-4-imino-oxazolidine-2-ones and n-phenylcarbamates were investigated on Sclerotinia sclerotiorum by the agar medium dilution method. In addition, antimicrobial spectra of several compounds against other 15 pathogenic microbes were investigated by the same method. In each series, 3, 5-dihalo-substituents on benzene ring are essential to high antifungal activity against Sclerotinia sclerotiorum and in the case of n-phenylcarbamates, it is necessary that the α-position of alcohol moiety is substituted by such a group as cyano group, ethoxy-carbonyl group or carbamoyl group. α-Cyanoisopropyl N-(3, 5-dichlorophenyl) carbamate, 3-(3′, 5′-dichlorophenyl)-5, 5-dimethyl-4-iminooxazolidine-2-one and 3-(3′, 5′-dichlorophenyl)-5, 5-dimethyloxazolidine-2,4-dione were the most effective and completely inhibited the mycelial growth of Sclerotinia sclerotiorum at 3.2 γm (about l.0 ppm). In general, 3-(3′, 5′-dichlorophenyl) oxazolidine-2, 4-diones and related compounds are highly active against Sclerotinia sclerotiorum and Botrytis cinerea, and fairly active against Cochliobolus miyabeanus, Pellicularia filamentosa, Pellicularia sasakii and Alternaria kikuchiana.     

酿酒酵母INVSC1对油菜菌核病菌抑制作用的研究

以油菜菌核病菌为供试病原菌,在摇瓶发酵备件下,研究了酿酒酵母菌INVSC1在各种培养条件下对油菜菌核病菌的抑菌效果,确定了酿酒酵母的最佳培养条件：最佳培养温度为28℃,培养时间为20h,接种量为5％。酿酒酵母能抑制油菜菌核病可能与它发酵产乙醇有关。酿酒酵母抑制油菜菌核病的发现使生物防治又多了一类新的可供选择的生防菌。