【作者】 李波；

【导师】 姜道宏；

【作者基本信息】 华中农业大学 ， 植物病理学， 2016， 硕士

【摘要】 核盘菌是一种寄主范围十分广泛、危害特别严重的植物病原真菌,全世界每年因其引致的菌核病而造成的经济损失十分惨重。真菌病毒是一类寄生真菌的病毒,其中一些真菌病毒能够引起真菌寄主致病力衰退,具有很好的生物防治潜力而备受关注。因此,在核盘菌中筛选和挖掘弱毒资源,不仅可以为核盘菌菌核病的防治提供新思路,同时也有利于探究真菌病毒的分类与进化、真菌病毒与寄主之间的互作机制。分离采集自陕西省的1063个核盘菌菌核进行纯培养,从中筛选出100株菌落形态异常的菌株进行宏转录组测序,结果检测到49种不同病毒的信息,其中病毒核盘菌弹状病毒1(Sclerotinia sclerotiorum Rhabdovirus 1,SsRhV1)存在于菌株SX276中。相对于菌株Ep-1PNA367,菌株SX276菌落形态异常、生长速度慢、致病力完全丧失。进一步对菌株SX276中是否含有其它类型病毒进行了分析,证实菌株SX276携带有4种不同类型的病毒,分别是病毒SsRhV1、内源病毒SsEV2(Sclerotinia sclerotiorum endornavirus 2)、弱毒病毒SsHV1-like(Sclerotinia sclerotiorum hypovirus1-like)及双节段病毒SsBRV2(Sclerotinia sclerotiorum botytivirus 2)。在真菌中首次发现隶属于弹状病毒科的负义链RNA病毒。病毒SsRhV1,基因组大小为11356nt,基因组5,末端没有帽子结构,3,末端也没有poly A尾巴,且5,末端序列和3,末端序列反向互补。病毒SsRhV1基因组推定出5个线性排布的非重叠开放阅读框(open reading frame,ORF),其中ORF1/4/5推定编码的蛋白序列与弹状病毒科病毒的N/G/L蛋白有同源性,但是ORF2/3编码蛋白未在病毒数据库中发现同源序列。病毒SsRhV1的基因间隔区序列是一串富含A/U且相对保守的序列,分析发现病毒的终止密码子为UAA,终止序列为AUAUAA,起始密码子为AUG,起始序列为UCA(U/C/A)(U/C/A)AUG,基因的终止信号和起始信号相互交汇,重合的序列正是富含A/U的保守序列,这段保守序列中包含病毒SsRhV1的TTP保守结构域序列AAA(A/G)A以及TI保守结构域序列AACAGA。系统发育分析表明病毒SsRhV1与动物弹状病毒亲缘关系较近,但独立聚类一个遗传分支,建议以该病毒为模式种建立一个新的病毒属。病毒SsRhV1的病毒粒子可能呈线状,直径在30 nm左右,长度在500 nm-1200 nm不等,病毒粒子表面光滑没有看见明显的突起结构。病毒SsRhV1的传毒效率极低,没有获取传毒成功的转化子。通过脱毒的方法,推测病毒SsRhV1/SX276可能对寄主核盘菌的生物学特性产生一定的影响。病毒SsEV2基因组具有其独特性,且该病毒与寄主弱毒特性相关。根据获得的SsEV2部分cDNA序列(11586 nt),证实该病毒基因组含有2个推定的完整非重叠ORF,其中ORF1编码蛋白含有3486个氨基酸,ORF2编码蛋白含有290个氨基酸,ORF1和ORF2之间的间隔序列长度为206 bp。在病毒SsEV2的ORF1编码蛋白上预测出5个保守的基序(motif),依次为甲基转移酶(MTR)、富含半胱氨酸区域(CRR)、两个解旋酶(DExH和HEL)和S7,而ORF2编码含有8个保守氨基酸位点或结构域的依赖于RNA的RNA聚合酶(RdRp)。系统发育分析表明病毒SsEV2与真菌内源病毒有同源性,但结合其基因组独特性(含有两个ORF),推定是一个新的病毒种。病毒水平传播病试验结果表明SsEV2可以传给强毒力菌株Ep-1PNA367GFP。菌株A367GFP获得病毒SsEV2后,菌落形态发生了显著的变化,生长速度缓慢,致病力也完全丧失。因此,初步推测病毒SsEV2与寄主核盘菌的弱毒特性有相关性。更多还原

【Abstract】 Sclerotinia sclerotiorum is a notorious plant fungal pathogen with a broad host range.Agricultural production suffers great losses because of sclerotiorum disease every year in the world. Hypovirulence-associated mycoviruses have attracted much attention for their potential as biological control agents to combate plant fungal diseases. Therefore, it is imperative to screen hypovirulence-associated mycoviruses in S. sclerotiorum. Moreover,those hypovirulence-associated mycoviruses will not only supply some potential biological resources to control Sclerotinia disease, but also facilitate to research on viral diversity, evolution and interaction between mycovirus and its hosts.1603 sclerotium of S. sclerotiorum were collected from Shanxi Province and 100 strains with abnormal colony morphology were screened for deep sequencing. In total, 49 viruses with different features were obtained, and a novel virus of 49 viruses, named as Sclerotinia sclerotiorum Rhabdovirus 1(SsRhV1), was further reasearched.RT-PCRconfirmed that strain SX276 was infected by SsRhV1. Strain SX276 showed abnormal colony morphology with lower growth rate and no pathogenicity on rapeseed plant. Thus, strain SX276 is a typical hypovirulent strain. DsRNA extraction and sequence clone further confirmed that strain SX276 is co-infected by other three mycovirus of Sclerotinia sclerotiorum endornavirus 2(SsEV2), Sclerotinia sclerotiorum hypovirus 1-like(SsHV1-like) and Sclerotinia sclerotiorum botytivirus 2(SsBRV2) with exception of SsRhV1.SsRhV1 as a member of Rhabdoviridae is firstly characterizated in fungi. The genome of SsRhV1 is consists of 11356 nucleotides(nts) in length with reverse complement of the 5,and 3,terminus sequence, but structures of cap and poly A is lack.SsRhV1 contains five nonoverlapping and linearly arranged ORFs. ORF1/4/5 encodes N/G/L proteins which show conserved sequence with members of Rhabdoviridae, but ORF2/3-encoded proteins have no sequence similarity to any known proteins in the protein database. Termination codon UAA, termination sequence AUAUAA, termination signal sequence, initiation codon AUG, initiation sequence UCA(U/C/A)(U/C/A) AUG,and initiation signal sequence are found among the conserved gene-junction sequence with A/U rich. The conserved overlap sequence between termination signal sequence and initiation signal sequence contains SsRhV1 TTP sequence AAA(A/G) A and TI sequence AACAGA. The phylogenetic analysis indicated that SsRhV1 has a closed relationshipwith rhabdoviruses that infect animals, but formed an independent phylogenetical branch.Thus, we proposed to be created a new genus to accommodate SsRhV1. The virions of SsRhV1 are filamentous with 30 nm in diameter and 500-1200 nm in length. Experiment result prove that SsRhV1 has some effect on biological properties of host S. sclerotiorum.The partial cDNA sequence of SsEV2 was obtained with 11586 nts and two complete ORFs were predicted from the obtained sequence of SsEV2. ORF1-encoded protein contains 5 conserved domains of MTR, CRR, DExH, HEL, and S7, whereas ORF2-encoded protein contains a conserved RdRp domain. This similar phenomenon has not been previously reported in all known endornaviruses. Multiply alignment and phylogenetic analysis indicated that SsEV2 contains 8 conserved amino acid sites or motifs of RdRp, and SsRhV1 may belong to a new endornavirus species, which has a closed relationship with previously reported endornaviruses. Using mixed culture methods, the virulent strain Ep-1PNA367 GFP was successfully obtained SsEV2 from strain SX276 via hyhal fusion. The newly SsEV2-infected isolates displayed debilitation symptoms that were similar to that of strain SX276. Therefore, SsEV2 has great influence on biological properties of host S. Sclerotiorum, which is singnificant different with all previously reported fungal endornavirus.更多还原