小麦梭条斑花叶病毒(WSSMV)提纯、粒子性质和血清学

 采用冰冻患病小麦叶片,经0.5M磷酸钾缓冲液pH7.0抽提,1/4体积四氯化碳澄清,6%聚乙二醇,3% NaCl和1% Triton X-100沉淀,二次差速离心(含20%蔗糖垫,0.3% Triton X-100).一次10-50%蔗糖密度梯度离心,最后经一次超速离心浓缩,成功地获得部分提纯制剂。经电镜观察,可见大量线状病毒粒子,很少有杂质。病毒粒子在中性条件下稳定,在酸性条件下变形。病毒粒子直径13-14nm.长度250-1800nm,其中在300-400nm,550-700nm之间具有二个主要分布峰。经紫外读数,病毒制剂表现为典型的核蛋白吸收特性.其中OD260/OD244为1.19,OD260/OD280为1.29。病毒粒子外壳蛋白有二个组分,分子量分别约为30kd和27kd。
在免疫电镜实验中,小麦梭条斑花叶病毒(WSSMV)和小麦黄花叶病毒(WYMV)抗血清不仅均能显著而有效地捕获汁液中的WSSMV粒子,而且还可在粒子外面包上一层厚厚的抗血清外套。     

我国不同地区小麦梭条斑花叶病毒的比较鉴定

 河南、四川、陕西、江苏、浙江等地发生的小麦土传病毒经SDS-琼脂双散扩及间接ELISA法测定,在血清学上是强相关的,SDS-聚丙烯酰胺凝胶电泳测定其外壳蛋白分子量均为36.6kd.以上比较研究结果,结合已有的研究资料,初步澄清了我国豫、川、陕、江浙等地发生的小麦土传病毒为小麦梭条斑花叶病毒。     

发生在我国的小麦黄花叶病毒病

本文对山东荣成流行的一种小麦病毒病进行了鉴定。提纯的病毒颗粒为长线状,13×100—300nm及13×350—650nm。汁液接种感染小麦,但不感染烟草、苋色藜等植物。病土、病根以及含有禾谷类多粘菌(POlymyxa graminis)游动孢子的浸液可以传毒于小麦、大麦及黑麦。此病毒与大麦黄花叶病毒(BYMV)、小麦梭条花叶病毒(WSSMV)有血清学关系,与小麦土传花叶病毒(WSBMV)无血清学关系。病叶表皮细胞中有无定形内含体。超薄切片可见风轮状内含体。实验结果表明,荣城地区发生的这种小麦病毒病是小麦黄花叶病毒所致。     

用聚乙二醇沉淀法粗提纯植物病毒

南方菜豆花叶病毒(SBMV)经两次PEG沉淀提纯的粗提纯病毒制剂和经密度梯度提纯的精提纯病毒制剂,在电镜下均能观察到大量的球状病毒,且均具有典型的核蛋白吸收图谱。两种方法提纯的病毒制剂免疫家兔,都能制备出效价高达1/1024(免疫双扩散法)的抗血清。此外,烟草花叶病毒(TMV),黄瓜花叶病毒(CMV),马铃薯Y病毒(N株系)(PVY~N),苜蓿花叶病毒(AMV)和烟草坏死病毒(TNV)用PEG沉淀法粗提纯病毒也均获得良好的效果。     

我国草莓斑驳病毒研究鉴定

 作者采集6省13市病样73株。经鉴定带SMoV的67株,占样本的91.78%。采取指示植物小叶嫁接法及蚜虫分离和传播病毒试验,并辅以病毒提纯,人工摩擦接种草本植物,病毒粒子电镜及超薄切片观察等手段加以确证。在辽宁省田间获得一种草莓中瘤钉毛蚜(Chaetosiphon sp.)以及棉蚜和桃蚜均传播本病毒,传播率依次为70%,40%及33%(最高率),系半持久性传播。病毒提纯制荆最大吸收值在260nm处,最小吸收值在240nm处。A260/280=1.21。电镜观测病毒粒子为球形,直径28~30nm,病叶超薄切片观察,在细胞质里有晶状球形颗粒,直径约25nm,排列整齐。以分离物(M-3)提纯制剂为抗原所制备的抗血清与有关病样呈阳性反应。试验证实了国内SMoV的发生和分布。     

江苏小麦条纹病毒病和中国小麦花叶病的初步鉴定

江苏位于长江中下游,地处中国的南北交界处,小麦上病毒病种类较多。先后发现的有小麦丛矮病(病原为北方禾谷花叶病毒,Northern cereal mosaic virus,NCMV)、小麦梭条花叶病(病原为小麦黄花叶病毒,Wheat yekllow mosaic virus, WYMV)、小麦黄矮病(病原为大麦黄矮病毒, Barley yellow dwarf virus,BYDV)、小麦“绿矮”病 (病原为水稻黑条矮缩病毒,Rice black streaked dwarf virus,RBSDV),其中小麦梭条花叶病曾大面积流行。2005年春天以来,江苏小麦上又发生了多种病毒病,除了以上4种,还出现了2种新的     

黄瓜绿斑驳花叶病毒磁免疫层析试纸条的研制

用磁性纳米颗粒标记黄瓜绿斑驳花叶病毒的兔多克隆抗体建立了简便快速的磁免疫层析试纸条检测方法。该方法检测提纯病毒的灵敏度为0.1 μg/mL,检测葫芦病汁液可稀释10 000倍(重量／体积)。试纸条和黄瓜绿斑驳花叶病毒具有特异性反应,和同属的烟草花叶病毒、番茄花叶病毒、辣椒轻斑驳病毒和齿兰环斑病毒,未出现交叉反应。该试纸条可用于田间病害检测和诊断。     

大麦黄花叶病毒(BaYMV)的提纯

 本文提出了一个改进了的大麦黄花叶病毒提纯方法。病大麦叶片在高浓度(0.5M)磷酸钾缓冲液pH7.0(内含0.1% ME,0.01M EDTA)中匀浆,经1/4体积四氯化碳澄清后,病汁液用6% PEG,3% NaCl和1% TritonX-100混合物沉淀,高浓度(0.5M)同种缓冲液(含0.5M尿素,0.1% ME和0.01M EDTA)悬浮,接着进行20%蔗糖垫(含0.3% Triton X-100)超迷离心去除寄主细胞成分,10-40%蔗糖密度梯度离心进一步纯化。所获得的BaYMv提纯制剂A260/A280,A260/A240比值分别为1.20和1.04,纯病毒产量约为5.5-8.0mg/kg病叶。提纯病毒制剂在电镜下看不到有寄主杂质存在。     

湖北省小麦黄花叶病病原的部分序列鉴定

 从湖北省真菌传小麦黄花叶病病毒分离物中抽提病毒总RNA,应用逆转录-聚合酶链式反应(RT-PCR)方法,合成病毒外壳蛋白(CP)基因cDNA。对所获得的cDNA克隆进行序列测定及分析,结果表明,湖北省真菌传小麦黄花叶病病毒分离物与小麦梭条斑花叶病毒(WSSMV)、大麦黄花叶病毒(BaYMV)、大麦和性花叶病毒(BaMMV)等病毒CP基因相应序列的同源性均低于70%,而与报道的小麦黄花叶病毒(WYMV)分离物CP基因核苷酸及编码氨基酸序列同源性均超过97%。表明湖北省真菌传小麦黄花叶病病原应为小麦黄花叶病毒(WYMV)。     

玉米弯孢霉叶斑病菌生物学特性的研究

 利用小麦黄花叶病毒(Wheat yellow mosaic virus,WYMV)和小麦梭条花叶病毒(wheat spindle streak mosaic virus,WSSMV)的特异性序列为引物,逆转录-PCR方法并配合免疫电镜观察,对采自我国四川陕西、河南、安徽、江苏5省9个县、市的27份感病田小麦样品进行检测。除2份样品未检测到病毒外,其余25份样品均为小麦黄花叶病毒所侵染,没有发现小麦梭条花叶病毒,由此表明在中国长江中、下游、淮河及渭河流域严重危害小麦的真菌传线状病毒应主要为小麦黄花叶病毒。     

中国小麦黄花叶病毒(WYMV)分布的RT-PCR鉴定

 镰刀菌产生的单端孢霉烯毒素,包括DON、DAS和T-2毒素,是一类重要的植物病原真菌毒素。这类毒素不仅加强病菌的为害程度,而且人畜食用罹病谷物及其制成品易造成中毒。近年来,单端孢霉烯毒素的生物合成途径、关键酶及其基因克隆,毒素在致病过程中的作用,以及利用毒素作为选择压鉴定小麦抗病性和筛选抗病突变体均取得了明显进展。本文对此作一综述。     

A novel putative member of the family Benyviridae is associated with soilborne wheat mosaic disease in Brazil

Soilborne wheat mosaic disease (SBWMD), originally attributed to infections by Soilborne wheat mosaic virus (SBWMV) and Wheat spindle streak mosaic virus (WSSMV), is one of the most frequent virus diseases and causes economic losses in wheat in southern Brazil. This study aimed to characterize molecularly the viral species associated with wheat plants showing mosaic symptoms in Brazil. Wheat leaves and stems displaying mosaic symptoms were collected from different wheat cultivars in Passo Fundo municipality, Rio Grande do Sul State, southern Brazil. Double-stranded RNA was extracted and submitted to cDNA library synthesis and next-generation sequencing. No sequences of SBWMV and WSSMV were detected but the complete genome sequence of a putative new member of the family Benyviridae was determined, for which the name wheat stripe mosaic virus (WhSMV) is proposed. WhSMV has a bipartite genome with RNA 1 and RNA 2 organization similar to that of viruses belonging to Benyviridae. WhSMV RNA 1 has a single open reading frame (ORF) encoding a polyprotein with putative viral replicase function. WhSMV RNA 2 has six ORFs encoding the coat protein, the major protein (read-through), triple gene block movement proteins (TGB 1, 2 and 3) and ORF 6 (hypothetical protein). In addition to the genomic organization and nucleotide and amino acid sequence identities, phylogenetic analyses also corroborated that WhSMV is a virus species of the Benyviridae. However, isolates of WhSMV formed a clade distinct from members of the genus Benyvirus. It was also demonstrated that the plasmodiophorid Polymyxa graminis is associated with wheat roots showing SBWMD symptoms and infected by WhSMV.     

小麦黄色花叶病毒不同分离物外壳蛋白(CP)基因序列的比较分析

 核苷酸序列分析结果表明,小麦黄色花叶病毒(W YMV)不同分离物的外壳蛋白基因存在一定的差异。邓州分离物CP基因在其31~33nt处均缺失了3个核苷酸,其余分离物与潢川分离物及日本分离物长度一致,均为882nt。不同分离物CP基因核苷酸序列同源性为97.3%~98.9%,由此推导的氨基酸序列同源性为97.6%~99.3%,外壳蛋白N末端的110个氨基酸和C末端的55个氨基酸在各个分离物间是高度保守的。潢川分离物有5个氨基酸与其它5个分离物明显不同。WYMV不同分离物外壳蛋白序列分析结果进一步确认了WYMV与WSSMV为Bymovirus属的2种不同病毒。     

Genome-wide association analysis of resistance to wheat spindle streak mosaic virus in bread wheat

Wheat spindle streak mosaic virus (WSSMV) is a major concern for cereal crops in Europe and North America. A strong increase in the occurrence of WSSMV has been observed in each French region where susceptible cultivars are cultivated. Most European bread wheat cultivars are resistant, but assessing the status of newly registered cultivars or breeding lines regarding WSSMV resistance is of importance. This paper describes a genome-wide association study carried out on a panel of 163 cultivars and tested for their resistance to WSSMV. Two regions on chromosomes 5B and 7D showed minor effects on WSSMV resistance. More importantly, a large genomic region on chromosome 2D explained most of the resistance to WSSMV. More than 99% of the cultivars carrying the AA genotype at the most associated marker (Excalibur_c15426_661) were resistant to WSSMV, while 100% of the cultivars showing the GG genotype were susceptible. This large genomic region of 45.8 Mb was found distal to the centromere and showed very high linkage disequilibrium. It is hypothesized that this region may be an alien introgression originating from a wild related species. This region contains a total of 2605 predicted genes based on the Chinese Spring IWGSC RefSeq v. 1.0 including genes potentially involved in plant disease resistance. A kompetitive allele-specific PCR (KASP) single-nucleotide polymorphism (SNP) marker was designed in order to identify breeding lines or registered cultivars resistant to WSSMV.     

Characterization and partial sequence of a new furovirus of wheat in China

A soil-borne wheat virus causing severe mosaic and stunting symptoms on wheat in China has been characterized. It had been considered to be soil-borne wheat mosaic virus (SBWMV) because of its rod-shaped virions and similarities to epidemiology and host range. In this study, the virions purified from infected wheat tissue were approximately 20 nm in diameter and of two lengths (140–160 nm and 280–300 nm), with a coat protein of 19 kDa and two RNA components of approximately 7 and 3.5 kb. A rabbit antiserum was produced against the virus and a serological relationship to SBWMV from the USA (Oklahoma) was demonstrated. However, the coat protein was not recognized by most monoclonal antibodies against Oklahoma SBWMV in either ELISA, ISEM or Western blot analysis, indicating epitope differences. In RT-PCR experiments the viral nucleotide sequences were significantly different from those of SBWMV, and this was confirmed by partial sequencing of the cloned PCR fragments generated from RNA1 ( c . 1100 nt) and RNA2 ( c . 1400 nt), which showed homologies of about 79 and 63%, respectively, to corresponding regions of SBWMV. Because of these significant differences in serology and nucleotide sequence it is suggested that it is a new furovirus for which the name Chinese wheat mosaic virus (CWMV) is proposed.     

Molecular comparisons amongst wheat bymovirus isolates from Asia, North America and Europe

To study the variation between wheat bymovirus isolates and to resolve uncertainties about the identity of the virus in some countries, leaves of infected plants were obtained from nine sites in China and from one each in Italy, Germany, USA and Canada. The German isolate was obtained from rye and the Canadian isolate was the type strain of wheat spindle streak mosaic virus (WSSMV). In RT-PCR, using primers designed from a partial sequence of a French isolate (tentatively described as WSSMV), genome fragments were obtained from the Italian and the French isolates but not from the Chinese ones. Conversely, products were consistently obtained from the Chinese isolates, but not from the Italian or French ones, when primers were designed from the sequence of a Japanese isolate of wheat yellow mosaic virus (WYMV). Nucleotide sequences were obtained from regions at or near the 3'-terminus of RNA1 of six Chinese isolates and the four from Europe and North America, usually including the coat protein. Nucleotide and amino acid sequence comparisons demonstrated that the European and North American isolates were extremely similar and were therefore WSSMV, while the Chinese isolates were close to the Japanese isolate and were thus WYMV.     

Response of UK winter wheat cultivars to <Emphasis Type="Italic">Soil-borne cereal mosaic</Emphasis> and <Emphasis Type="Italic">Wheat spindle streak mosaic viruses</Emphasis> across Europe.

Twenty-one UK winter wheat cultivars were grown over three seasons at sites with natural inoculum sources of Soil-borne cereal mosaic virus (SBCMV) and Wheat spindle streak mosaic virus (WSSMV) located in France, Italy and the UK. Plants were assessed visually for virus symptoms and leaf extracts were tested for the presence of each virus using enzyme-linked immunosorbent assays (ELISA). Cultivars showing little or no foliar symptoms and low levels of virus in leaf tissue were classified as resistant to each virus. All the trials were taken to harvest and agronomic data collected. At the most heavily infected sites, severe symptoms of SBCMV were observed in all UK cultivars except Aardvark, Charger, Claire, Cockpit, Hereward and Xi 19. The latter cultivars exhibited either light or no symptoms and little or no SBCMV infection in leaves. In fields with WSSMV, the virus failed to develop in Italy, but was detected in the leaves of all the susceptible control cultivars at a site in France. However, no UK cultivar tested positive for WSSMV. Multi-site analysis indicated that the presence of WSSMV did not increase the impact of SBCMV on the height, thousand-grain weight or yield of UK cultivars. The wheat cultivars on test gave a similar response to SBCMV across three European countries. Possible sources of SBCMV resistance are discussed.