【作者】 张蕾；

【导师】 李国庆；

【作者基本信息】 华中农业大学 ， 植物病理学， 2010， 博士

【摘要】 本文研究了灰葡萄孢菌弱毒菌株CanBc-1的生物学特性、致病力衰退相关RNA病毒(Botrytis cinerea debilitation related RNA virus,简称BcDRV)的基因组结构及其分类属性、致病力衰退的分子机理。最后还以弱毒将菌株CanBc-1及其恢复致病力的单分生孢子后代CanBc-1c-66为材料,初步分析了菌株CanBc-1中差异表达的基因。取得了如下研究结果：首先,对源于6个地区和14种寄主植物的23个灰葡萄孢菌菌株进行了生物学特性分析。鉴定出一个异常菌株CanBc-1,其生长缓慢、菌丝异常分枝、仅产生少量分生孢子和菌核、完全丧失了致病能力。在菌株CanBc-1菌丝中检测到一种3.0 kb大小的dsRNA分子。通过对菌株CanBc-1单分生孢子后代的致病力,发现该菌株单分生孢子后代的致病力发生分化,有强毒后代出现。通过对不同类型杀菌剂的抗性测定,发现弱毒菌株CanBc-1对多菌灵、菌核净、农利灵等具有抗性。其次,对灰葡萄孢菌弱毒菌株CanBc-1中的致病力衰退相关RNA病毒(Botrytis cinerea-debilitation related RNA virus,缩写为BcDRV)基因组的分子生物学特性进行了研究。通过cDNA克隆和RT-PCR技术获得了BcDRV基因组的全长序列,共2806 bp,GC含量为33.3%。通过与网上已知序列的分析比对,确定CanBc-1中的dsRNA分子是一个线粒体病毒,属于裸露病毒科(Narnaviridae)、线粒体病毒属(Mitovirus)。此外,还对灰葡萄孢菌菌丝的超微结构进行了透射电镜观察,发现弱毒菌株CanBc-1菌丝线粒体出现异常,包括皱缩、溃解、内部没有瘠等特征。可见,BcDRV可能以灰葡萄孢菌的线粒体作为攻击目标。再次,对灰葡萄孢菌弱毒菌株CanBc-1的致病力衰退机制进行了研究。通过对不同灰葡萄孢菌菌株的侵染结构、果胶酶、植物毒素次生代谢产物、酸类物质和漆酶等致病相关因子的研究,发现弱毒菌株CanBc-1能够正常的分泌果胶酶、漆酶和植物性毒素物质,RT-PCR结果也证实弱毒菌株CanBc-1的果胶酶基因(Bcpg1-6)、漆酶基因(Bclcc1,Bclcc2)和毒素合成相关基因(Bcbot1)均能正常表达；但是通过对侵染结构的观察,发现弱毒菌株CanBc-1的菌丝顶端不能正常的形成侵染结构。因此,导致弱毒菌株CanBc-1致病力衰退机理包括：菌丝线粒体发生畸变,菌丝顶端不能正常产生侵染垫结构。最后,对弱毒菌株CanBc-1和强毒菌株CanBc-1c-66(可视为菌株CanBc-1的近等基因系)在转录水平上的差异表达基因进行了分析。采用基因表达谱分析的方法,共得到了2288个差异表达的基因。在弱毒菌株CanBc-1中,1392个基因上调表达,896个基因下调表达。根据KEGG基因功能分类,表明差异表达的基因的功能涉及到糖类代谢(8%)、脂质代谢(5%)、核苷代谢(2%)、氨基酸代谢(4%)、多糖的生物合成与代谢(1%)、辅助因子和维生素代谢(2%)、能量代谢(3%)、聚酮化合物和萜类化合物的生物合成(1%)、外源物质的生物降解(1%)、酶类家族(10%)、转录(2%)、翻译(2%)、聚集和降解(2%)、复制和修复(2%)、膜运输(5%)、信号转导(1%)、配体受体的相互作用(3%)、细胞内加工处理(3%)和未分类蛋白(43%)等19类。其中,可能与灰葡萄孢菌和其它病原菌致病过程相关的差异表达基因有12个。上述结果为进一步挖掘灰葡萄孢菌致病相关基因奠定了基础。更多还原

【Abstract】 In this study, we investigated some biological characteristics, the genome structure and identity of Botrytis cinerea-debilitation related RNA virus (BcDRV) and the mechanisms responsible for pathogenicity debilitation of strain CanBc-1 of Botrytis cinerea, the causal agent of plant grey mould disease. Meanwhile, we performed the expression profiling analysis for the hypovirulent strain CanBc-1 and the virulent strain CanBc-1c-66 (a single-conidium isolate of strain CanBc-1) of B. cinerea. Main results are summarized below.Firstly, twenty-three strains of B. cinerea were isolated from 14 species of plants grown from different places, and were compared for cultural characteristics and pathogenicity on oilseed rape (Brassica napus). Results showed that strains CanBc-1 and CanBc-3 of B. cinerea was severely debilitated in pathogenicity and in mycelial growth, compared with the other virulent strains of B. cinerea. A species of double-stranded (ds) RNA of approximately 3.0 kb in length was detected in strain CanBc-1, but was not detected in strain CanBc-3 and the other 21 strains of B. cinerea. Single-conidium (SC) isolates of strain CanBc-1 differed greatly in pathogenicity. Some SC isolates of strain CanBc-1 (such as CanBc-1c-66) became virulent on leaves of oilseed rape and the 3.0-kb dsRNA was eliminated from these SC isolates. Strains CanBc-1 and CanBc-1c-66 were found to be resistant to fungicides Carbendazim and dimmethachlon.Secondly, the full-length cDNA sequence of Botrytis cinerea debilitation-related virus (BcDRV,3.0-kb dsRNA) was obtanined. It is 2806 bp in length and AU-rich (66.7%). Based on sequence analysis of BcDRV, we found the BcDRV belongs to genus Mitovirus (Narnaviridae). TEM observation showed that strain CanBc-1 shows degenerative symptoms in hyphal cytoplasma and contained numerous malformed mitochondria without formation of cristae inside. It appears that mitochondria of Botrytis cinerea may be the targets of BcDRV.Thirdly, we also analyszed the mechanisms responsible for debilitation of hypovirulent strain CanBc-1 of B. cinerea. Strain CanBc-1 was compared with the virulent strains CanBc-1c-66 and CanBc-2 of B. cinerea for formation of infection cushions on onion scales and leaves of oilseed rape and tomato as well as production of pectinases, toxic metabolites, oxalic acid and laccase in different growth media. Results showed that the three investigated strains of B. cinerea could produce polygalacturonases, toxic metabolites, oxalic acid and laccase in cultures, and the genes coding for endopolygalacturonases (Bcpg1-6), laccases (Bclcc1 and Bclcc2) and botrydial were detected to express normally. Formation of infection cushions was common on epidermis of onion scales and oilseed-rape leaves which inoculated with strains CanBc-1c-66 and CanBc-2, but was rare on the plant tissues inoculated with strain CanBc-1. It is indicated that rare formation of infection cushions is the main cause for the failure of infection of onion and oilseed-rape by the hypovirulent strain CanBc-1 of B. cinerea.Finally, expression profile analysis was performed on hypovirulent for strains CanBc-1 and CanBc-1c-66 of B. cinerea. A total of 2288 differentially expressed genes (DEGs) were identified. Among these genes,1392 were up-regulated and 896 were down-regulated in strain CanBc-1. The 2288 DEGs were then grouped into 19 KEGG functional categories. They include carbohydrate metabolism (8%), lipid metabolism (5%), nucleotide metabolism (2%), amino acid metabolism (4%), glycan biosynthesis and metabolism (1%), metabolism of Cofactors and vitamins (2%), biosynthesis of polyketides and terpenoids (1%), energy metabolism (3%), xenobiotics biodegradation and metabolism (1%), enzyme family (10%), transcription (2%), translation (2%), folding (2%), sorting and degradation (2%), replication and repair (2%), membrane transport (5%), Signal Transduction, Signaling Moleculars and Interaction, Cellular Process and unclassified function (43%). We found 12 DEGs which had been confirmed as the pathogenicity-related factors in B. cinerea or in other plant pathogens. These genes may be involved in the pathogenesis process of B. cinerea.更多还原