核盘菌致病机理研究进展

菌核病(Sclerotinia stem rot,SSR)是我国油料作物生产中主要病害之一,严重制约长江中下游地区油菜主产区的产业发展。菌核病的病原是子囊菌门的核盘菌,是一种世界性分布的重要植物病原真菌。其寄主范围广泛,引起的菌核病对多种作物的产量和品质造成重要影响。核盘菌作为典型的死体营养型病原真菌,侵染寄主植物时通过直接杀死细胞和破坏组织攫取生长所需的营养物质。核盘菌的致病机理相对复杂,前期研究主要集中在其分泌的水解酶类(角质酶、细胞壁降解酶和蛋白酶等)和草酸在核盘菌侵染寄主植物中的作用。近些年来,越来越多的研究证实了分泌蛋白在核盘菌的致病过程中同样发挥着重要作用。分泌蛋白(效应蛋白)主要通过诱导植物细胞死亡或抑制寄主细胞的免疫反应,促进核盘菌的侵染和定殖。综述了水解酶类、草酸和分泌蛋白等在核盘菌致病机制中的作用,并对核盘菌致病机理研究进行了展望,以期为菌核病的安全防控提供理论参考。     

茶活性成分对几种植物致死型病原真菌的抑制效果分析

已有研究表明茶叶对细菌和有些真菌有显著抑制作用。为探讨茶的活性物质对几种植物致死型病原真菌的抑制效果,我们购买了两种重要的茶活性物质茶皂素和绿茶提取物茶多酚,测试它们对核盘菌、镰刀菌和灰霉菌的抑菌效果。结果表明,茶皂素对3种植物致死型病原真菌的抑制效果与浓度成正比关系。绿茶提取物茶多酚对灰霉菌有明显抑制效果,但对核盘菌1980和镰刀菌PH-1仅在浓度为100 mg/m L,液体体积为100μL时有抑菌效果。同时还发现不同品种的茶叶乙醇浸提时间不同,对几种病原真菌的抑制效果不同。红茶、绿茶和乌龙茶浸提4 h、8 h、16 h的乙醇提取液对核盘菌1980都有抑制效果;8 h红茶、绿茶浸提液和16 h红茶浸提液对镰刀菌PH-1有明显抑菌效果。该研究结果为进一步在控制植物致死型病原真菌病害方面开发利用茶活性物质提供了理论依据。     

核盘菌和灰葡萄孢基因组中的简单重复序列分析

利用公共的真菌基因组数据库资源, 对核盘菌（Sclerotinia sclerotiorum）和灰葡萄孢（Botrytis cinerea）基因组中SSRs的结构类型、分布、丰度及最长序列等进行了系统分析, 并与已经研究过的禾谷镰孢菌（Fusarium graminearum）, 稻瘟病菌（Magnaporthe grisea）和黑粉菌（Ustilago maydis）等几种植物病原真菌基因组中的SSRs进行了比较。结果表明: 核盘菌和灰葡萄孢基因组中的SSRs非常丰富, 分别为6 539和8 627个, 并且在结构类型和分布规律上具有一定的相似性; 与其他几种病原真菌相比, 核盘菌和灰葡萄孢基因组中长重复的四、五、六核苷酸基序更为丰富, 从而使得这两种真菌具有更高的变异性。同时, 我们发现真菌基因组中SSRs的丰度与基因组的大小及GC含量没有必然的关系。文章对核盘菌和灰葡萄孢基因组中SSRs的丰度、出现频率及最长基序的分析为快速、便捷地设计多态性丰富的SSRs引物提供了有益的信息。     

单克隆抗体在植物病理学中的应用及其研究进展

植物病理学家的特殊任务包括植物病害的诊断、病原物的鉴定和检测、病原物的特性研究以及寄主-病原物相互关系的研究。植物病理学家的根本目的在于保持植物健康,也就是防治病害。病害的准确诊断无疑是防治的前提条件。病原物被鉴定得越快速、越准确,就越能快速地实施正确的防治。病害诊断和病原检测的常规方法主要包括病害的症状学、显微镜技术、微生物学技术、生物鉴定技术和血清学技术。免疫化学技术对病害的快速准确的常规诊断极其有用,对植物病原的鉴定和定量分     

利用SRAP分析核盘菌遗传多样性

核盘菌(Sclerotinia sclerotiorum)是危害油菜等多种经济作物的重要病原真菌.研究不同地区、相同或不同寄主核盘菌的遗传多样性对了解核盘菌的遗传演化过程和指导病害防控具有重要意义.我们采用序列相关扩增多态性(sequence-related amplified polymorphism,SRAP)标记对不同地理来源、不同寄主来源的76个核盘菌菌株的遗传多样性进行了分析.7对SRAP引物共获得260个位点,其中114个为多态位点,占43.85%.UPGMA聚类结果显示,在相似性系数为0.64时,76个核盘菌菌株分为4个组,每组包含的菌株数分别为54、18、2和2.聚类及主成分分析结果显示,来源于春油菜生态区和冬油菜生态区油菜上的核盘菌菌株可以明显分为两簇,而油菜、大豆、莴苣等不同寄主植物上的核盘菌菌株没有明显的遗传分化.分子变异分析(AMOVA)结果显示.不同地理来源、不同油菜生态区和不同寄主来源的核盘菌群体内的变异率分别为75.2%、81.2%和97.6%,均达到极显著水平(P<0.001);不同地理来源和不同油菜生态区的核盘菌群体间的变异率分别为24.8%和18.8%,也达到极显著水平(P<0.001);不同寄主来源的核盘菌群体间的变异率仅为2.4%,变异不显著(P=0.8673).研究结果表明,来源于春油菜生态区的核盘菌的遗传多样性高于冬油菜生态区.     

PCR技术在植物病原细菌研究中的应用*

利用rep-PCR、AFLP和RAPD等多种基因组指纹分析方法,指纹图谱结合计算机辅助分析,用于研究植物病原细菌群体遗传多样性;遗传多样性图谱有助于理解病原细菌的分类、群体结构,还有利于设计特异、灵敏、快速检测策略用于植物病原检测和病害诊断。已有许多以PCR为基础的检测技术如rDNA-PCR、ITS-PCR、ARDRA等,有很多特异性引物及检测程序,已在植物病原检测和病害诊断中广泛应用。PCR技术的应用和计算机的辅助分析,为植物病原细菌群体动态和生态学知识提供了基本框架,使植物病理学进入一个新时代。     

PCR技术在植物病原细菌研究中的应用

利用rep-PCR,AFLP和RAPD等多种基因组指纹分析方法,指纹图谱结合计算机辅助分析,用于研究植物病原细菌群体遗传多样性;遗传多样性图谱有助于理解病原细菌的分类,群体结构。还有利于设计特异,灵敏,快速检测策略用于植物病原检测和病害诊断。已有许多以PCR为基础的检测技术如rDNA-PCR,ITS-PCR,ARDRA等,有很多特异性引物及检测程序,已在植物病原检测和病害诊断中广泛应用,PCR技术的应用和计算机的辅助分析。为植物病原细菌群体动态和生态学知识提供了基本框架,使植物病理学进入一个新时代。     

捕食线虫丝孢菌的菌寄生性测定

研究了节丛孢Arthrobotrys、单顶孢Monacrosporium和隔指孢Dactylella三个捕食线虫丝孢菌属16个菌株,对水稻立枯丝核菌RhizoctoniasolaniAG1、大豆核盘菌Sclerotiniasclerotiorum、茄科镰刀菌Fusariumsolani和恶疫霉Phytophthoracactorum四种常见土壤植物病原真菌的菌寄生性。结果表明供试菌可以通过弹簧式菌丝圈缠绕、类附着胞结构吸附、简单的菌丝缠绕或者贴附寄主菌丝生长四种方式寄生病原菌。其中,绝大多数菌株对立枯丝核病菌有寄生作用,一些供试真菌对其它三种病原真菌有寄生现象。利用孢子液浸泡法测定了其中5种捕食线虫真菌对核盘菌菌核的寄生能力,显示有较高寄生率。     

植物病原真菌的DNA转化

一、绪论 所有植物都易受真菌侵害。全世界由植物病原真菌引起的植物病害对栽培植物和森林树木造成了严重的损害。已知的植物病原真菌大约8000种,分属于真菌所有的主要分类类群。它们的生理特性及生活方式各不相同,包括从营专性寄生到兼性寄生等类型。 过去,由于成功地培育和采用了抗病性增强的植物品种,加之运用多种化学防治方法,真菌病害得到了控制,但未能被消除。最近分子遗传学作为一种强有力的手段已被用于研究真菌寄生物与其寄主相互作     

内生真菌感染对宿主羽茅及邻生植物抗病性的影响

Epichloë内生真菌能够影响宿主植物的抗病性, 但目前关于内生真菌感染对宿主邻生植物抗病性影响的研究很少。该研究首先调查了内蒙古呼伦贝尔天然草地中植物病害发生情况, 发现在常见牧草中禾本科植物病害最为严重, 其中感染内生真菌的羽茅(Achnatherum sibiricum)的总病情指数最低。进一步比较了与羽茅邻生及非邻生植物的病害差异, 结果显示羽茅显著降低了邻生羊草(Leymus chinensis)、狼针草(Stipa baicalensis)和冰草(Agropyron cristatum)的褐斑病病情指数。为探究羽茅对邻生植物病害的影响是否与其内生真菌有关, 分别从内生真菌发酵液、离体叶片和植株层面进行了室内验证试验。结果表明: 羽茅的2种内生真菌Epichloë gansuensis和Epichloë sibiricum发酵液显著抑制了新月弯孢(Curvularia lunata)、根腐离蠕孢(Bipolaris sorokiniana)、核盘菌(Sclerotinia sclerotioru)和三叶草核盘菌(Sclerotinia trifoliorum)的生长; 对于离体叶片, E. gansuensis和E. sibiricum显著减小了宿主叶片受4种病原真菌侵染后的病斑长度。在新月弯孢和根腐离蠕孢的植株接种条件下, 内生真菌显著降低了宿主羽茅及邻生植物羊草的病害程度。该研究首次发现并验证了羽茅内生真菌对邻生禾草抵御褐斑病有一定的增益作用。     

Limitations of In Vitro Strain Screening Methods for the Selection of Sclerotinia spp. as Potential Mycoherbicides against the Perennial Weed Ranunculus acris

Nineteen isolates of Sclerotinia sclerotiorum and three isolates of S. minor were inoculated on to excised tissues and intact plants of Ranunculus acris (giant buttercup), to evaluate their pathogenicity. All isolates proved pathogenic, with S. sclerotiorum being more pathogenic than S. minor on both excised tissues and intact plants. Seven of the S. sclerotiorum isolates were more pathogenic than the others on excised tissues, but no significant differences in pathogenicity were found between any of the isolates when they were inoculated on to intact plants. The results of this study indicate that the excised tissue method cannot be used to predict whole plant mortality, nor, therefore, the mycoherbicide potential of strains of S. sclerotiorum for this perennial weed.     

Physiological and Molecular Features of the Pathosystem Arabidopsis thaliana L.-Sclerotinia sclerotiorum Libert

The fungal pathogen Sclerotinia sclerotiorum Libert causes rot diseases on many crops worldwide and large economic losses occur frequently because of a lack of resistant varieties. The pathogenesis of S. sclerotiorum and the molecular basis of plant responses to the pathogen are poorly understood. In the present investigation, the process of S. sclerotiorum infection in Arabidopsis thaliana L., a plant that is highly susceptible to this fungus, was analysed. In addition, the defense activation in the host was investigated. A convenient inoculation method using millet grain was developed for S. sclerotiorum in Arabidopsis. The fungus rapidly infected the plants, probably through ball- or cushion-like infection structures. Visible symptoms developed within 24 h and plants were killed 72 h after inoculation. Cellulase, the main enzyme that caused host tissues to rot, was secreted by S. sclerotiorum in a pH-dependent manner. Oxalic acid, another pathogenic factor secreted by the fungus, induced necrotic lesions on the leaves, infection with S. sclerotiorum strongly induced the production of the pathogenesis-related （PR） proteins β-1,3-glucanase and chitinase in Arabidopsis. Furthermore, the PR gene PDF. 1 was induced, but not PR1, indicating that the pathogen activated basal defense of jasmonic acid/ethylene dependence, which is consistent with its necrotrophic characteristics. This pathosystem for Arabidopsis-S. sclerotiorum could provide an approach for the analysis of the interactions between S. sclerotiorum and other crops, thereby facilitating genetic manipulation techniques for controlling this pathogen.     

Genomic analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea

Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38-39 Mb genomes include 11,860-14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared to <1% of B. cinerea. The arsenal of genes associated with necrotrophic processes is similar between the species, including genes involved in plant cell wall degradation and oxalic acid production. Analysis of secondary metabolism gene clusters revealed an expansion in number and diversity of B. cinerea-specific secondary metabolites relative to S. sclerotiorum. The potential diversity in secondary metabolism might be involved in adaptation to specific ecological niches. Comparative genome analysis revealed the basis of differing sexual mating compatibility systems between S. sclerotiorum and B. cinerea. The organization of the mating-type loci differs, and their structures provide evidence for the evolution of heterothallism from homothallism. These data shed light on the evolutionary and mechanistic bases of the genetically complex traits of necrotrophic pathogenicity and sexual mating. This resource should facilitate the functional studies designed to better understand what makes these fungi such successful and persistent pathogens of agronomic crops.     

Defense against Sclerotinia sclerotiorum in Arabidopsis is dependent on jasmonic acid, salicylic acid, and ethylene signaling

Genotypic differences in susceptibility of Arabidopsis thaliana to Sclerotinia sclerotiorum have not been reported due to the extreme susceptibility of this cruciferous plant. To overcome this limitation, we have established inoculation conditions that enable evaluation of differences in susceptibility to S. sclerotiorum among Arabidopsis mutants and ecotypes. Two coil mutant alleles conferred hypersusceptibility to S. sclerotiorum. The plant defensin gene PDF1.2 was no longer induced after challenging the coi1-2 mutant with S. sclerotiorum. Hypersusceptibility of the coi1-2 mutant to S. sclerotiorum was not correlated with oxalate sensitivity. The mutants npr1 and ein2 were also hypersusceptible to S. sclerotiorum. Induction of PDF1.2 and the pathogenesis-related gene PR1 was reduced in ein2 and npr1 mutants, respectively. Actigard, a commercial formulation of the systemic acquired resistance inducer benzothiadiazole, reduced susceptibility to S. sclerotiorum. Based on histochemical analysis of oxalate-deficient and wild-type strains of S. sclerotiorum, oxalate caused a decrease in hydrogen peroxide production but no detectable changes in plant superoxide production or gene expression.     

Identification and characterization of Sclerotinia sclerotiorum NADPH oxidases

Numerous studies have shown both the detrimental and beneficial effects of reactive oxygen species (ROS) in animals, plants, and fungi. These organisms utilize controlled generation of ROS for signaling, pathogenicity, and development. Here, we show that ROS are essential for the pathogenic development of Sclerotinia sclerotiorum, an economically important fungal pathogen with a broad host range. Based on the organism's completed genome sequence, we identified two S. sclerotiorum NADPH oxidases (SsNox1 and SsNox2), which presumably are involved in ROS generation. RNA interference (RNAi) was used to examine the function of SsNox1 and SsNox2. Silencing of SsNox1 expression indicated a central role for this enzyme in both virulence and pathogenic (sclerotial) development, while inactivation of the SsNox2 gene resulted in limited sclerotial development, but the organism remained fully pathogenic. ΔSsnox1 strains had reduced ROS levels, were unable to develop sclerotia, and unexpectedly correlated with significantly reduced oxalate production. These results are in accordance with previous observations indicating that fungal NADPH oxidases are required for pathogenic development and are consistent with the importance of ROS regulation in the successful pathogenesis of S. sclerotiorum.     

Type 2A phosphoprotein phosphatase is required for asexual development and pathogenesis of Sclerotinia sclerotiorum

Sclerotinia sclerotiorum is a necrotrophic, omnivorous plant pathogen with worldwide distribution. Sclerotia of S. sclerotiorum are pigmented, multihyphal structures that play a central role in the life and infection cycles of this pathogen. Plant infection depends on the formation of melanin-rich infection cushions, and secretion of hydrolytic enzymes and oxalic acid. Type 2A Ser/Thr phosphatases (PP2As) are involved in the regulation of a variety of cellular process. In the presence of cantharidin, a PP2A-specific inhibitor, hyphal elongation and sclerotia numbers were impaired whereas sclerotial size increased. We partially inactivated PP2A by antisense expression of the gene (pph1) encoding the PP2A catalytic subunit. When antisense expression was induced, almost complete cessation of fungal growth was observed, indicative of a crucial role for PP2A in fungal growth. RNAi-based gene silencing was employed to alter the expression of the 55-kDa R2 (B regulatory subunit). Isolates in which rgb1 RNA levels were decreased were slow growing, but viable. Melanin biosynthesis, infection-cushion production, and pathogenesis were significantly impaired in the rgb1 mutants, yet theses mutants were pathogenic on wounded leaves. Reduced ERK (extracellular signal-regulated kinases)-like mitogen-activated protein kinase (MAPK) function conferred a reduction in NADPH oxidase and PP2A activity levels, suggesting a functional link between MAPK, reactive oxygen species, and PP2A activity in S. sclerotiorum.     

Pathogenicity of Sclerotinia sclerotiorum on Ranunculus acris in Dairy Pasture

Fifty-four isolates of Sclerotinia sclerotiorum from Ranunculus acris and other natural hosts were applied as mycelial infested kibbled wheat onto 6 month-old R. acris plants in two glasshouse screening experiments. Most isolates (90%) did not differ in their pathogenicity towards R. acris. One isolate, S. sclerotiorum G45, was selected based on its ability to cause severe disease and suppress regeneration of R. acris. A field experiment was conducted to determine the efficacy of S. sclerotiorum (G45) against R. acris in infested dairy pastures in the Takaka Valley, Golden Bay, New Zealand. Isolate G45 was formulated as a wettable powder and was applied as a slurry at 20 and 40 ml/plant in December 1995. After 10 weeks, regeneration from the crown of treated plants was apparent and a second application of S. sclerotiorum was made in February 1996. Best control of R. acris was obtained when the plants were inoculated in full flower in December. At the first time of treatment, the 40 ml application of S. sclerotiorum slurry reduced the total dry weight of R. acris by an average of 57%. The second application had no effect on total dry weight, possibly because moisture levels were not sufficient for S. sclerotiorum infection. This study confirmed S. sclerotiorum to be an aggressive pathogen of R. acris under both glasshouse and field conditions. As a result, this pathogen has potential as a mycoherbicide for R. acris. Further experiments are required to explore ways of enhancing the efficacy of S. sclerotiorum against R. acris by manipulation of the host, pathogen and environment.     

Calcineurin is required for sclerotial development and pathogenicity of Sclerotinia sclerotiorum in an oxalic acid-independent manner

Sclerotinia sclerotiorum is a necrotrophic, omnivorous plant pathogen with worldwide distribution. Sclerotia of S. sclerotiorum are pigmented, multihyphal structures that play a central role in the life and infection cycles of this pathogen. Calcineurin, a Ser/Thr phosphatase linked to several signal-transduction pathways, plays a key role in the regulation of cation homeostasis, morphogenesis, cell-wall integrity, and pathogenesis in fungi. We demonstrate that calcineurin expression in S. sclerotiorum is altered in a phase-specific manner during sclerotial development. Inhibition of calcineurin by FK506, cysclosporin A, or inducible antisense calcineurin expression impaired sclerotial development at the prematuration phase and increased germination of preformed sclerotia. Induction of antisense calcineurin expression in S. sclerotiorum resulted in reduced pathogenesis on tomato and Arabidopsis. However, secretion of oxalic acid, a key virulence factor of S. sclerotiorum, was not altered. Inhibition of calcineurin conferred a reduction in cell wall beta-1,3-glucan content and increased sensitivity to cell-wall-degrading enzymes and to the glucan synthase inhibitor caspofungin. Thus, calcineurin plays a major role in both sclerotial development and pathogenesis of S. sclerotiorum and, most likely, other phytopathogens.