【作者】 于晓庆；

【导师】 李向东；

【作者基本信息】 山东农业大学 ， 植物病理学， 2008， 博士

【摘要】 马铃薯X病毒(Potato virus X,PVX)是危害烟草的一种常见病毒,单独侵染造成的危害较轻,而当与马铃薯Y病毒属(Potyvirus)病毒复合侵染时则使症状加重并造成严重损失。烟草脉带花叶病毒(Tobacco vein banding mosaic virus,TVBMV)属于Potyvirus,近几年在我国烟草上的发生有上升和蔓延的趋势。本研究分别测定了PVX和TVBMV的全基因组序列,分析了二者复合侵染对烟草植株生长和光合作用的影响以及辅助成分-蛋白酶(helper component-proteinase,HC-Pro)定点突变对PVX/HC-Pro协生作用的影响。最后,对病毒病害的生物防治进行了初步研究。根据GenBank已发表的PVX全基因组序列,设计简并和特异引物,克隆并测定了我国PVX-FX21和PVX-1985两个分离物的全基因组序列,这是我国大陆第一次扩增得到PVX分离物全基因组序列。两个分离物的基因组全长均为6435 bp ,3′-末端具poly(A),包含5个开放阅读框(open reading frames,ORFs)。基因组全序列分析表明,PVX-FX21和PVX-1985均与欧亚组9个分离物一致率较高,而与美洲组4个分离物一致率较低。系统进化分析与一致率分析的结果一致,说明PVX的分布表现一定的地域性。基于衣壳蛋白(CP)序列的系统进化分析表明,38个PVX分离物分为两个大组:组I和组II,PVX-FX21和PVX-1985位于组I。PVX-FX21与荷兰X3分离物(D00344)的一致率最高(核苷酸一致率为96.9 %,氨基酸一致率为100 %),而PVX-1985与英国的2个分离物Roth1、XS聚为一簇(核苷酸一致率分别为97.6%、97.3%,氨基酸一致率均为97.5%)。遗传多样性分析表明,不同地理来源的PVX种群所受的选择压力没有显著差别;全基因组5个开放阅读框均处于负向或纯化选择,其中RdRp所受的选择压力最大;进一步分析显示RdRp中间编码区在进化过程中处于正向或多样化选择。利用针对马铃薯Y病毒属病毒的简并或特异引物,通过RT-PCR的方法扩增得到TVBMV云南分离物YND的全基因组序列。除了3′-端poly(A)外,YND基因组全序列包括9570个核苷酸,含有一个大的开放阅读框,编码一个由3079个氨基酸组成的多聚蛋白,分子量约为348.6 kDa。全基因组序列系统进化分析表明,TVBMV是Potyvirus中一个独立的种。TVBMV-YND分离物基因组不同部分与Potyvirus其它病毒相应部分有不同的一致率水平,然而,多数部分与南美红辣椒脉斑驳病毒(Chilli veinal mottle virus,ChiVMV)一致率最高。比较特别的是,该分离物的NIb/CP切割位点为Q/N,这在马铃薯Y病毒属病毒中非常少见。另外,HC-Pro中含有比较罕见的参与蚜传的RITC基序。本研究通过接种试验证明TVBMV与PVX复合侵染烟草表现出协生作用。在供试的三生烟和普通烟上,二者复合侵染不但使病害症状加重,而且能显著抑制烟草植株的生长,并使光合作用下降。在本氏烟上,二者复合侵染甚至导致植株的死亡。但与以往研究不同的是,两种病毒在烟草植株体内的浓度都没有显著增加。利用基因定点突变技术获得了PVX-(HC-Pro)的12个突变体,应用反向遗传学技术证明HC-Pro的锌指结构、糖基化位点等多个保守基序中单个氨基酸的缺失或替换能影响其协生作用。而参与蚜传的KITC基序中K突变成A以后,并不影响其协生功能。本研究在大田作物和蔬菜根际共分离得到295个根际细菌菌株,通过对峙培养初筛、温室盆栽复筛得到一株对多种植物病原菌有较好拮抗活性的PGPR菌株Lyc2。温室盆栽试验表明,Lyc2对棉花立枯病的防治效果达到48.8%;水培棉花苗试验表明,Lyc2能显著增加棉苗的鲜重和干重,但对株高的影响不显著。TMV枯斑寄主接种试验证明,Lyc2菌悬液处理的三生烟不但枯斑数量明显减少而且枯斑的直径明显比对照小。大田试验结果表明,Lyc2处理对株高的促进作用较明显,但对茎粗及叶面积几乎没有什么影响,对病毒病的防效为33.7%。该菌经形态、生理生化试验测定及16S rDNA和ITS序列分析,初步确定为洋葱伯克霍尔德氏菌(Burkholderia cepacia);进一步通过种特异的recA基因序列分析,证明Lyc2菌株属于B. cepacia复合物中基因型IX,B. pyrrocinia。更多还原

【Abstract】 Potato virus X (PVX) is one of the most common plant viruses infecting solanaceous plants, including potato, tobacco, eggplant and tomato. PVX alone causes mild symptoms in solanaceous plants. However, it causes much more severe symptoms when it mixedly infects with potyviruses. Tobacco vein banding mosaic virus (TVBMV) is a species of the genus Potyvirus and has been detected frequently in tobacco plants in Shandong, Henan, Anhui and Yunnan provinces. The losses caused by TVBMV had been increasing in the past few years. In this paper, we reported the complete genomic sequence of two Chinese PVX isolates and the first complete genomic sequence of TVBMV in the world, confirmed synergism between PVX and TVBMV. And analyzed the effects of site-directed mutagenesis in HC-Pro protein on PVX/HC-Pro synergism. Finally, we investigated the use of plant growth-promoting rhizobacteria (PGPR) to induce resistance to infection by plant viruses.The complete genomic sequences of two Chinese PVX isolates (PVX-FX21 and PVX-1985) were determined from four overlapping cDNA clones. The genome of both isolates are 6435 nucleotides (nt) in length excluding the poly(A) tail and contains five open reading frames (ORFs). Both the entire sequences shared higher identities with nine European isolates (group I), and lower identities with four South American isolates (group II). Phylogenetic analysis of complete genomic sequence of 15 isolates and the coat protein genes of 38 isolates revealed two major groups. All PVX isolates from Asia are clustered to group I, while isolates from Europe and America are clustered to both groups. PVX-FX21 had the highest identities of 96.9% and 100.0 % with PVX-X3 (group I) at nt and amino acid (aa) levels, respectively. While PVX-1985 was clustered with two isolates (Roth1 and XS ) from United Kingdom, sharing identities of 97.6% and 97.3% at nt level, respectively, and identity of 97.5% at aa level. Nucleotide sequence diversity analyses showed that constraint on the ORF encoding RdRp is much higher than those on the other four ORFs. The central coding region of RdRp was under positive seletion, although the encoding RdRp was under negative selection.The complete genomic sequence of one Tobacco vein banding mosaic virus isolate from Yunnan China, TVBMV-YND, was determined by sequencing overlapping cDNA fragments obtained by RT-PCR with degenerate and/or specific primers. The genome is composed of 9,570 nt excluding the 3′-terminal poly(A) tail and contains one single open reading frame of 9,240 nt encoding a large polyprotein of 3,079 amino acids with predicted Mr of 348.6 kDa. Phylogenetic analysis of complete genomic sequences confirmed that TVBMV is a distinct species of the genus Potyvirus. Different parts of TVBMV-YND genome shared different levels of identity with other potyviruses, while most parts showed greatest identity with Chilli veinal mottle virus among the potyviruses with available complete genomic sequences. TVBMV-YND had a rare Q/N cleavage site for NIb/CP and uncommon RITC motif in HC-Pro that is crucial for aphid transmission of potyviruses.Mixed infections of Nicotiana tabacum cvs NC89 and Samsun plants by PVX and TVBMV resulted in synergistic interaction. NC89 and Samsun plants coinfected by both viruses displayed remarkably inhibited plants growth and significantly decreased photosynthesis. N. benthamiana plants coinfected by both viruses also displayed stronger synergistic interaction, progressing from severe mosaic and eventually to plant death. Differenting from previous study, the concentration of both viruses did not increase significantly in any of the infected tobacco plants. When the HC-Pro of potato virus A (PVA) was transiently expressed in N. benthamiana via potato virus X (PVX) vector, a highly synergistic response of leaf necrosis and plant death was observed in PVX-HC infected plants. Twelve sites in the HC-Pro of PVX-HC were mutated successfully through site-directed mutagenesis. Eleven of these mutants loss the synergism function. However, mutation lysine (K) to alanine acid (A) in the“KITC”motif, which is involved in aphid transmission of potyviruses, had no obvious influence on synergism.Two hundred and ninety-five bacterial isolates were obtained from rhizosphere of plants.Among which five potential biocontrol isolates showed broad and strong antagonistic effects on plant pathogens. One bacterial strain, Lyc2, was selected for further study. It showed remarkable inhibition to the mycelial growth of multiple pathogenic fungi in dual culture. Lyc2 showed 48.8% efficiency to control cotton seedling damping-off in greenhouse, and significantly increased the fresh weight and dry weight of cotton seedlings in solution culture experiment. It could also induce resistance to TMV in Samsun plants. In field, Lyc2 could promote the growth of tobacco plants, and showed 33.7% efficiency to control plant viral disease. The morphological, physio-chemical characteristics, homology of 16S rDNA and ITS sequences showed that Lyc2 was closely related to Burkholderia cepacia. Further comparison of recA gene sequence showed that Lyc2 belonged to B. pyrrocinia, genomovar IX of B. cepacia complex.更多还原