【作者】 孙炳剑；

【导师】 谢联辉； 陈剑平；

【作者基本信息】 福建农林大学 ， 植物病理学， 2011， 博士

【摘要】 禾谷多黏菌（Polymyxa graminis L.）传播的小麦病毒病是一类世界性病害,广泛分布于欧洲、北美洲和东亚地区,引起严重的产量损失。在中国禾谷多黏菌传播的小麦病毒有两种,分别是小麦黄花叶病毒（Wheat yellow mosaic virus,WYMV）和中国小麦花叶病毒（Chinese wheat mosaic virus,CWMV）,在我国大部分的冬麦区发生危害。目前生产上唯一有效的防治措施是种植抗病品种。为了指导品种合理布局,有效控制WYMV和CWMV,本研究对WYMV和CWMV的分布变化,WYMV的分子变异,小麦品种抗性评价,病毒病对推广品种产量的影响,抗WYMV机制分析和WYMV-CP功能等方面进行了研究。为了进一步明确目前这两种病毒在我国的分布,特别是重病区、复合侵染区的分布及变化,2008-2010年,采用田间病症观察结合免疫学检测、电镜观察等方法,对WYMV和CWMV的分布进行了研究。结果表明,WYMV重病区呈现向西、向北扩展的趋势,发现山东泰安,河南商丘,湖北丹江口、襄樊、随州等WYMV新病区,江苏省两种病毒复合侵染区也在扩大,四川省仅在内江市零星发生,浙江小麦种植区未检测到病毒。为了研究WYMV种群结构、分子变异和遗传进化特点,我们对不同病区WYMV分离物CP序列和VPg序列进行了分析,结果发现不同分离物之间CP变异比VPg变异更显著。在WYMV-CP氨基酸序列上存在稳定的共变异现象,即Ile¹¹²→Val¹¹²和Asn¹¹⁵→Asp¹¹⁵,按照这种共变异可以将病毒分为三组:A（IEHN）、B（VEHD）和C（IEHD）,病毒类型与分布有一定相关性。在江苏省及周边地区WYMV类型主要是A,在冬小麦种植区北部和西部WYMV类型主要是B,在中国冬小麦区的边沿地带荣成、雅安、杭州和日本一致,类型为C。分子变异是病毒突破小麦抗性的遗传基础,不同地区品种抗性反应可能存在差异。为评价小麦品种在不同病区的抗性差异,筛选抗病小麦品种和种质资源,验证病毒共变异与致病性的关系。在2008-2011年,选择河南西平、江苏扬州、山东烟台和荣成4个有代表性的病区,进行小麦品种田间抗性鉴定,筛选出一批符合不同地区需要的抗病推广品种,适合烟台地区的抗病品种有淮麦20、郑麦366、丰抗1号、0448、小偃5号、陕麦150、西农2208、衡观111、兰天23号和中麦9号等10个。适合荣城地区种植的抗病品种只有烟农23;适合江苏扬州种植的抗病品种有:科优1号、高优503、高优505、花培2号等共24个。适合河南西平种植的抗病品种:阜麦936、高优503、旱抗4118、新麦208、豫麦70、豫麦70-36、郑麦366、郑麦9023等共65个。同时鉴定出大量感病品种,应该避免在病区种植。本研究还筛选出若干适合不同地区,可以用于抗病育种的小麦种质。另外,根据小麦品种抗病反应差异,推测烟台、扬州和西平为不同的致病株系,烟台分离物致病能力更强。为了评价推广品种对WYMV的抗性,于2006—2010年在河南省西平县进行了田间抗性鉴定试验和室内间接ELISA检测,并分析了病害严重程度对产量的影响。结果表明,在供试的62个品种中,仅有新麦208表现为免疫;豫麦70-36、泛麦5号、阜麦936、山东95519、豫麦70、高优503、豫麦9676、郑麦366和陕麦229等9个品种表现为抗病,占供试品种的14.5%;濮优938、兰考矮早8、新原958、花培2号、温优1号、豫麦18、郑麦9023、豫麦47、豫农201、偃展4110、豫麦36、百农878和豫麦49-198等13个品种表现为中抗,占供试品种的21.0%;另外39个品种表现为感病,占供试品种的62.9%。对其中48个品种进行了产量与病害严重度分析,发现随着病害的严重度增加,小麦的穗数、千粒重以及产量都有明显下降,严重度为1级时,平均减产9.6%;严重度达到2和3级时,平均减产分别为30.3%和33.5%。为了研究小麦品种可能的抗WYMV机制,利用Western-blotting及组织免疫印迹技术分析了病毒在抗、感病品种不同组织内分布,结果发现小麦品种的抗病机制存在多样性,有的品种如宁麦资32和临麦7号,对病毒表现高抗或免疫;品种如XK0106-108D6、豫麦70-36和阜麦936,表现为影响病毒的复制及在根部的扩散;品种如陕8242-1、西农889、中麦12号,表现为阻止病毒由根向茎上部的运输;品种如有芒白15、襄麦81、咸农151,表现为降低了病毒在叶部的积累。感病小麦因品种不同,植株内病毒含量也存在明显差异,观0014、扬06-（NX）-71、扬06-129和扬麦11等品种全株病毒含量都很高;陕159、豫农202、绵麦185和烟农22等品种,根和心叶病毒含量高,但是茎含量低;0448和望麦15等品种,根和茎病毒含量低,但叶片含量高。为了研究WYMV编码蛋白与CP的互作关系和CP在植物细胞中的定位,利用酵母双杂交系统（Yeast two-hybrid system）和双分子荧光互补分析（Bimolecular fluorescence complementation,BiFC）技术,结果表明CP-CP,CP-P1存在互作,酵母双杂交的研究结果表明CP缺失C端94个氨基酸后,自身互作增强。同时利用农杆菌瞬时表达体系结合荧光显微技术分析CP蛋白在离体情况下植物细胞内定位特点,发现CP在烟草表皮细胞内的移动与微丝密切相关,与内质网及其形成的囊泡以及微管无关,CP定位在微丝上。更多还原

【Abstract】 Wheat yellow mosaic disease is widespread in winter wheat in China, covering the area of almost 50% of winter wheat planting regions along Huanghuai, and Yangzi River basin as well as Eastern Shandong and Sichuan basin. The causal agents are Wheat yellow mosaic virus (WYMV)and Chinese wheat mosaic virus (CWMV), which are transmitted by plasmodiophorids vector, Polymaxa graminis, a worldwidely spread biotrophic parasite of many plants. Both WYMV and CWMV cause yellow mosaic symptom on wheat, leading to serious yield loss under condition favorable for the disease development. This plasmodiophorids vector is an obligate soil-borne parasite which can survive in soil as resting spores for many years in the absence of the host. Concerning the economic and ecological reasons, currently there are no effective or acceptable chemical for control of virus vector. Therefore, wheat yellow mosaic disease is very difficult to control. Up to now, the only an available way to control this disease is by growing resistant wheat varieties in the field infested with viruliferous vector.In this study, to gain the more detail information on the epidemiology of wheat yellow mosaic disease in winter wheat in China, we examined the prevalence and molecular diversity of viruses in winter wheat growing area. The structure of WYMV population and responses of wheat cultivars to different virus isolates were also analyzed. First, to examine the prevalence of the viruses in winter wheat growing area, we prepared WYMV or CWMV specific antiserum for immunoassay of field samples. From 2007 to 2010, we carried out field survey for wheat yellow mosaic disease in winter wheat growing regions. The results show that the disease had expanded to wider wheat planting area in China during the last 10 years. In several places such as Taian (Shandong province), Shangqiu (Henan province), Danjiangkou, Xiangfan and Suizhou (Hubei province) WYMV occurrence was newly found. Co-infection of WYMV and CWMV had become more serious in Jiangsu province. Neijiang was the only area with disease in Sichuan province whereas wheat yellow mosaic disease was not detected in Zhejiang province.Analysis of genetic variability of viruses can provide important information that leads to understanding of viral pathogenicity and evolutionary process. In this study, the sequences of the CP gene from about 30 independent clones of WYMV from each of a total of 11 different locations in 5 provinces in China were determined. Sequences of the VPg gene from three of these locations were also determined. The CP was generally more variable than the VPg. At some sites, populations appeared relatively uniform, but there was evidence of two or three major subpopulations at three amino acid sites in their CP sequences. Most differences in the VPg were silent but in the CP striking co-variation at amino acid positions 112 and 115 enabled the division of all known WYMV sequences into three types. Based on the consensus sequence of CP gene, WYMV isolates were divided into 3 subgroups. The distribution of these groups is probably correlated with their geographical locations. Almost isolates collected from Jiangsu province belong to A subgroup, whereas isolates from northern and eastern of winter wheat growing area belong to B subgroup.Plant virus commonly breaks host resistances by genetic variation or mutation resulting in big epidemic in the field. To evaluate the resistance of wheat cultivars to WYMV, about 600 wheat cultivars were planted in a infested field at Xiping(Henan province), Yangzhou(Jiangsu province), Yantai and Rongcheng (Shandong province)during 2008 to 2011. The result shows that Huaimai 20,Zhengmai 366,Fengkang 1,0488,Xiaoyan 5,Shaanmai 150,Xinong 2208,Hengguan 111,Lantian 23 and Zhongmai 9 are resistant to Yantai WYMV isolates;Yannong 23 for Rongcheng; Keyou 1,Gaoyou 503,Gaoyou 505, Huapei 2 ect. are resistant to Yangzhou isolates; Fumai 936, Gaoyou 503, Hankang 4118,Xinmai 208, Yumai 70, Yumai 70-36, Zhengmai 366, Zhengmai 902 ect. are resistant to Xiping isolates. This result suggests that those resistant cultivars are potential as germplasmic resources for the breeding of resistant wheat cultivar against WYMV infection.To evaluate resistance of winter wheat cultivars currently grown in Henan Province, China to WYMV, the resistance of 62 commercial wheat cultivars was tested in a infested nursery at Xiping, Henan Province in the years 2006 to 2010. Of the 62 cultivars, only Xinmai 208 was immune to WYMV and nine cultivars (14%) were resistant: Yumai 70-36, Fanmai 5, Fumai 936, Shandong 95519, Yumai 70, Gaoyou 503, Yumai 9676, Zhengmai 366 and Shaanmai 229. Thirteen cultivars (21%) were moderately resistant (Puyou 938, Lankaoaizao 8, Xinyuan 958, Huapei 2, Wenyou 1, Yumai 18, Zhengmai 9023, Yumai 47, Yunong 201, Yanzhan 4110, Yumai 36, Bainong 878 and Yumai 49-198) and the remaining 39 cultivars (63%) were susceptible. Moreover, the effects of disease severity on wheat yield and grain quality were also investigated. The results showed that the average of grain yields, spike numbers and 1000-grain weights are decreased if disease severity was increased. 9.6% of yield was lost when disease severity was grade 1, and 30.3% and 33.5% yield was lost when the disease severity was grade 2 and 3, respectively.Some wheat cultivars were randomly sampled for further analysis of virus distribution in the plants by Western- and tissue blotting. The results showed that WYMV accumulation and movement was inhibited in resistant cultivars differently. In some cultivars, such as Ningzimai 32 and Linmai 7, virus accumulation was not detected in whole plant. In other cultivars such as XK0106-108D6、Yumai 70-36 and Fumai 936, viral replication and spread in the roots were inhibited. Transportation of virus from the root to stem was restricted, in cultivar Shaan 8242-1、Xinong 889、Zhongmai 12, while virus accumulation in leaves was suppressed in cultivar Youmaibai 15、Xiangmai 81、Xiannong 151. The virus content in susceptible wheat cultivar was also different. Some cultivars contained large amount of virus in all tissue, such as Guan 0014、Yang 06-(NX)-71,Yang 06-129 and Yangmai 11. Other cultivars contained more virus in roots and leaves than in stems, such as Shaan 159, Yunong 202, Mianmai 185 and Yannong 22. Some cultivars contained more virus in roots and stems than in leaves, such as 0448 and Wangmai 15.To investigate the intra-viral protein interaction among WYMV-encoded proteins, Bimolecular fluorescence complementation (BiFC) and Yeast two-hybrid system(YTH) assays were carried out to examine the interaction of WYMV CP with other viral-encoded proteins. The results showed that in addition to its self interaction, CP interacted with P1. Analysis of sub-cellular localization indicated that CP was capable to form vesicles which move along microfilament-like structure and endoplasmic reticulum in Nicotiana benthamiana epidermal cell.更多还原