甘薯病毒病的研究Ⅰ甘薯羽状斑驳病毒的分离、鉴定

本文从寄主范旧,蚜传特性、病毒颗粒形态、免疫电镜等方面对嫁接获得的甘薯羽状斑驳病毒廊坊分离物(SPFMV—LF)进行了鉴定,并和SPFMV—RC(美国株)进行了实验比较。SPFMV—LF易汁液摩擦传播和嫁接传播,并以蚜虫非持久性传播,SPFMV—LF系统侵染巴西牵牛(Ipomoea setosa)、牵牛(I.nil);不侵染Chenopodium quinoa、C.ama-ranticolor、Nicotiana benthamiana及其它烟草。尚未发现其局部斑寄主。SPFMV—LF及SPFMV—RC感染I.setosa后引起相似的症状,但感染I.nil后症状差别较大。病毒颗粒长860—830nm,经两次蔗糖垫超速离心从SPFMV—LF感染的I.setosa叶片提取病毒并在BALB/C小鼠中制备抗SPFMV—LF的腹水多克隆抗体,免疫电镜表明,SPFMV—LF的抗体和SPFMV—RC株系有反应。上述实验结果表明SPFMV—LE是SPFMV的一个株系,但不同于SPFMV—RC。     

甘薯病毒病的研究Ⅰ甘薯羽状斑驳病毒的分离、鉴定

 本文从寄主范旧,蚜传特性、病毒颗粒形态、免疫电镜等方面对嫁接获得的甘薯羽状斑驳病毒廊坊分离物(SPFMV-LF)进行了鉴定,并和SPFMV-RC(美国株)进行了实验比较。SPFMV-LF易汁液摩擦传播和嫁接传播,并以蚜虫非持久性传播,SPFMV-LF系统侵染巴西牵牛(Ipomoea setosa)、牵牛(I.nil);不侵染Chenopodium quinoa、C.ama-ranticolor、Nicotiana benthamiana及其它烟草。尚未发现其局部斑寄主。SPFMV-LF及SPFMV-RC感染I.setosa后引起相似的症状,但感染I.nil后症状差别较大。病毒颗粒长860-830nm,经两次蔗糖垫超速离心从SPFMV-LF感染的I.setosa叶片提取病毒并在BALB/C小鼠中制备抗SPFMV-LF的腹水多克隆抗体,免疫电镜表明,SPFMV-LF的抗体和SPFMV-RC株系有反应。上述实验结果表明SPFMV-LE是SPFMV的一个株系,但不同于SPFMV-RC。     

制备免疫吸附电镜检测甘薯羽状斑驳病毒样品

 甘薯羽状斑驳病毒(Sweet potato feathery mottle virus,SPFMV)可经蚜虫、摩擦、嫁接方式传播,是Y病毒组的一个成员。受此病毒感染的甘薯叶片上形成羽状褪绿斑,脉间褪绿斑点以及紫色环斑,有的品种出现紫色条纹。在甘薯块根上,有的呈严重纵向褐色龟裂,有的呈横向螺纹状木质化,有的块根内部形成木栓化,是危害甘薯最严重的病毒病害,可使甘薯严重退化及减产。无论是甘薯的抗病毒育种、病毒病害的防治,还是脱毒、无病毒甘薯种薯生产都离不开病毒的检测。灵敏的免疫吸附电镜(ISEM)检测方法被应用于各种病毒的检测中。     

甘薯羽状斑驳病毒外壳蛋白基因的分子变异

应用单链构象多态性(single-strand conformation polymorphism,SSCP)技术结合核苷酸序列测定的方法,对我国甘薯主产区11个省份的甘薯羽状斑驳病毒(Sweet potato feathery mottle virus,SPFMV)外壳蛋白(CP)基因的分子变异情况进行了研究.结果表明,SPFMV CP基因的RT-PCR产物表现了较丰富的图谱类型,50个分离物共产生9种主要的SSCP带型;对显示不同带型的20个样品的CP基因进行了序列测定和进化树分析,CP基因核苷酸序列一致性为77.2％～99.9％.说明这些样品的SPFMV的CP基因存在较大的分子变异,可划分为EA、RC、O和C4个株系.     

中国甘薯病毒的血清学检测

 作者用4种甘薯病毒抗体(IgG),3种血清学方法(DAS-ELISA、Dot-blot-ELISA和ISEM)对北京,江苏、四川、山东四省(市)的253份甘薯病毒病样品进行了检测。结果表明:上述地区甘薯中普遍存在甘薯羽状斑驳病毒(SPFMV)和甘薯潜隐病毒(SPLV),尚难确定是否存在甘薯轻斑驳病毒(SPMMV)和甘薯花叶菜花叶状病毒(Sweet Potato Caulimo-like Virus,SPCLV)。21%的显症样品同上述4种病毒的抗血清不产生反应,显示我国甘薯上尚存在其它病毒。用Dot blot-ELISA和ISEM检测甘薯病毒比用DAS-ELISA灵敏准确。     

甘薯病毒病害(SPVD)的多重RT-PCR检测方法及其应用

根据甘薯褪绿矮化病毒(Sweet potato chlorotic stunt virus,SPCSV)热激蛋白(Hsp70)基因和甘薯羽状斑驳病毒(Sweet potato feathery mottle virus,SPFMV)外壳蛋白(CP)基因核苷酸序列的保守区域设计了4对引物,以单-RT-PCR反应体系为基础,分别对影响多重RT-PCR扩增的退火温度、Taq DNA聚合酶浓度、dNTPs浓度、Mg2+浓度、引物浓度等条件进行了优化,建立了能同时检测SPVD两种病原的多重RT-PCR方法.该方法能有效区分SPFMV的主要株系类型.灵敏性试验表明,建立的多重RT-PCR方法对SPFMV-CH2、SPFMV-CH和SPCSV的最低检测浓度分别为1.42×104、1.32×104拷贝/μL和2.47×104拷贝/μL.该方法可用于甘薯叶片和薯块中病毒的检测,为SPVD的监测预警提供了一个有用的工具.     

山东甘薯主要病毒的鉴定及多样性分析

为明确山东省甘薯病毒病发生现状,在重病区调查采样,通过鉴别寄主、电镜和分子检测技术明确主要病毒种类;并克隆病毒外壳蛋白基因序列,利用Mega 5.0构建系统进化树进行遗传分析。结果显示,巴西牵牛嫁接甘薯染病枝条后叶片黄化、褪绿及皱缩;病样组织中存在大量600~900 nm的线状病毒粒子和柱状内含体。24份病样中检测到甘薯羽状斑驳病毒、甘薯潜隐病毒、甘薯G病毒、甘薯曲叶病毒和甘薯褪绿矮化病毒5种病毒,其中23份为复合侵染,存在11种侵染类型。遗传分析显示山东省甘薯羽状花叶病毒主要为EA、O和C株系,甘薯潜隐病毒与周边省份分离物相近,甘薯G病毒与中国海南和美国分离物相近,甘薯曲叶病毒分属3个株系。表明山东地区甘薯病毒种类繁多,侵染模式复杂,病毒遗传结构具有多样性。     

中国甘薯病毒种类的血清学和分子检测

 2009～2010年,从我国18个省（市）采集了176份表现病毒病症状的甘薯样品。利用血清学、PCR和核苷酸序列测定的方法,对上述样品中的病毒种类进行了鉴定。血清学检测结果表明,供试样品中甘薯羽状斑驳病毒（SPFMV）的阳性率最高,达56.3%,其次为甘薯G病毒（SPVG）和甘薯类花椰菜花叶病毒(SPCaLV),阳性率分别为34.1%和33.5%。PCR和核苷酸序列测定结果表明,我国甘薯上至少存在SPFMV、SPVG、甘薯潜隐病毒（SPLV）、甘薯褪绿斑病毒（SPCFV）、甘薯褪绿矮化病毒（SPCSV）、黄瓜花叶病毒（CMV）、甘薯脉花叶病毒（SPVMV）和甘薯卷叶病毒（SPLCV）8种病毒。此外,供试样品中没有检测出甘薯轻斑驳病毒（SPMMV）,是否存在甘薯轻斑点病毒(SPMSV)、SPCaLV和C 6病毒尚不能确定。     

湖北甘薯病毒病的检测与鉴定

2013—2015年采集了湖北黄冈、鄂州、武汉、荆州以及宜昌等5个地区的甘薯病毒病样品,通过双生病毒通用引物PCR扩增、ds RNA技术和序列分析等方法,鉴定了这5个地区甘薯病毒病的病原。结果显示,甘薯羽状斑驳病毒(Sweet potato feathery mottle virus,SPFMV)、甘薯褪绿矮化病毒(Sweet potato chlorotic stunt virus,SPCSV)、黄瓜花叶病毒(Cucumber mosaic virus,CMV)和甘薯卷叶病毒(Sweet potato leaf curl Georgia virus,SPLCGV)等4种病毒被检出。其中,SPFMV SPLCGV这两种病毒在湖北皆为首次报道。     

三种甘薯病毒多重RT-PCR检测技术的建立

本文根据GenBank中甘薯G病毒(SPVG)、甘薯卷叶病毒(SPLCV)和甘薯羽状斑驳病毒(SPFMV)外壳蛋白(CP)基因序列设计特异引物,对多重RT-PCR退火温度、延伸温度、模板浓度、引物浓度进行改良优化,建立能同时检测3种甘薯病毒的多重RT-PCR方法。该方法能同时扩增出SPVG、SPLCV和SPFMV特异片段,其大小分别是800、276和570bp。测序结果表明,扩增出的3种病毒序列与相应参考序列相似性达到98%以上。灵敏度分析结果表明,多重RT-PCR方法能够检测cDNA的量为0.1ng。应用建立的多重RT-PCR检测方法对田间样品进行检测,结果显示该方法可以特异、快速、灵敏地同时检测3种甘薯病毒。这些研究结果可为甘薯病毒检测提供参考。     

The Helper Component-Proteinase of Sweet potato feathery mottle virus Facilitates Systemic Spread of Potato virus X in Ipomoea nil

ABSTRACT When Ipomoea nil was coinfected with Sweet potato feathery mottle virus (SPFMV), a member of the genus Potyvirus, and Potato virus X (PVX) typical symptoms caused by PVX were observed on those by SPFMV on the first upper true leaves at 14 days postinoculation (dpi). On the other hand, no PVX-induced symptoms were observed on the first upper true leaves at 14 dpi when plants were infected with PVX alone. In the case of coinfection with PVX and SPFMV, PVX RNA was detected not only in the inoculated cotyledonary leaves but also in the first upper true leaves at 14 dpi. In the case of single infection with PVX, PVX RNA was detected in the inoculated cotyledonary leaves but not in the first upper true leaves at 14 dpi. The accumulation of SPFMV remained unchanged, regardless of whether the inoculum consisted of SPFMV alone or a mixture of SPFMV and PVX. Although recombinant PVX engineered to express the helper component-proteinase (HC-Pro) of SPFMV (PVX.HC) enhanced symptoms severity in Nicotiana benthamiana, PVX.HC induced the synergism characterized by an enhanced viral movement in Ipomoea nil. Immunofluorescence microscopic examination revealed that the HC-Pro was present in phloem of SPFMV-infected I. nil. These results suggest that SPFMV HC-Pro acts as an enhancer of long distance movement for PVX in I. nil.     

甘薯无病毒苗培育技术研究

１９９１￣１９９６年,甘薯茎尖离体培养采用ＭＳ为基本培养基,添加６－苄基氨基嘌呤（６－ＢＡ）０．５ｍｇ／Ｌ＋萘乙酸（ＮＡＡ）０．２ｍｇ／Ｌ＋腺素（Ａｄ）５ｍｇ／Ｌ,培育出１２个甘薯品种的脱毒苗,茎尖培养成苗率５０．０％￣９５．６％,成苗期４３￣６０ｄ。切取的茎尖大小与脱毒效果呈显著的负相关（ｒ＝－０．９８７５）,茎尖带１个叶原基时,ＳＰＦＭＶ脱毒率为３８．１％,培养基中添加适量的植物病毒钝化剂可使     

Characterization of an isolate of beet mosaic virus from South Kazakhstan

A filamentous virus isolated from a sugar-beet plant showing systemic mosaic collected in South Kazakhstan was identified as an isolate of beet mosaic virus (BMV-K). BMV-K was transmitted by the green peach aphid Myzus persicae in a non-persistent manner, and by sap inoculation to 11 out of 19 species from seven families tested. The virus could not be transmitted to Nicotiana tabacum, N. debneyi, N. glutinosa and N. clevelandii, cither mechanically or with M. persicae. The thermal inactivation point of BMV-K in sugar-beet sap was 55-60 C, dilution end point 1:1000 and longevity in vitro 2 days at 20 C. A purification procedure produced 1-5-3 mg of purified virus from 100 g of infected Stellaria media plants. Purified virus contained a single protein species of molecular weight 34 700 Da. In ELISA tests, BMV-K reacted positively with BMV-specifc antisera obtained from Japan. Germany and Portugal. By competitive DAS- ELISA, the virus isolate was shown to be closely serologically related to all the three isolates of BMV, and very distantly related to bean yellow mosaic and soy bean mosaic viruses.     

草莓伪轻型黄边病毒的鉴定

 从6省13市约18品种草莓样本、分离纯化并研究鉴定,初步证实辽宁、哈尔滨及南京一些栽培田,有草莓伪轻型黄边病毒(SPMYEV)的发生分布。在春香、宝交早生、丹东鸡心,诺宾卡及80-3.1等品种上发病。病样先以小叶嫁接技术,接于草莓UC-4、EMC及"Alpine",约3~5周显症、下部叶片呈现黄色斑块,接UC-5、EMB不太敏感仅于老叶上现轻斑驳或褪绿斑,一种草莓中瘤钉毛蚜(Chaetosiphon sp.)及棉蚜(Aphis gossypii)作半持久性传播本病毒,桃蚜(Myzus persicae)不传播。UC-4对本病毒的繁殖、保存和嫁接鉴别最为适合,提纯病毒电镜观察其粒子线状,大小约630~680×12~13nm,县典型核蛋白吸收光谱,其最大吸收值位于2.63nm处;最小吸收值位于243nm处。A260/280=1.24。经SDS-聚丙烯酰胺凝胶电泳,测其外壳蛋白分子量约为37,000d。病叶制做超薄切片也观察到线形晶状病毒粒子,分散或聚集于寄主的薄壁组织细胞质中。本病毒系国内首报。     

北京地区一种侵染菊花的线条病毒

 从北京地区菊花病毒病株上分离到一种线条状病毒CA分离物。经寄主范围,传播方式、汁液体外抗性,外壳旧白分子量、粒体大小和在细胞中产生的内含体研究结果分析,此病毒为Potyvirus成员,沉淀反应,免疫双扩散反应和免疫电镜技术检测证明CA分离物与PVY在清学相关性。CA分离物已制备抗血清和提纯IgG,并应用于品种菊组培苗脱毒检测工作。     

Parietaria mottle virus, a possible new ilarvirus from Parietaria officinalis (Urticaceae)

A previously undescribed virus, probably a new member of the ilarvirus group, was isolated from Parietaria officinalis showing symptoms of yellow mosaic or mottling. This virus, for which the name parietaria mottle virus (ParMV) is proposed, differs in host range from other ilarviruses. ParMV was purified from Chenopodium quinoa by sap clarification with chloroform, and differential and sucrose density gradient centrifugation.
Purified particles were quasi-isometric to ovoid with diameters of about 24, 29 and 36 nm; no bacilliform particles were detected. Buoyant density in caesium chloride, determined on glutaraldehyde-fixed virus, was 1.35 g/cm³.
An antiserum to ParMV was obtained with a titre of 1:32 in agar gel double-diffusion tests. ParMV did not react with eight other viruses or virus strains belonging to the ilarvirus group or to pelargonium zonate spot virus.
Polyacrylamide gel electrophoresis indicated that ParMV contains a single major protein species of estimated molecular weight 24300 Da and four RNA species of estimated molecular weight 1 37, 1.19, 0.86 and 0.36 × 10⁶ Da.     

Natural wild hosts of sweet potato feathery mottle virus show spatial differences in virus incidence and virus-like diseases in Uganda

Sweet potato feathery mottle virus (SPFMV, genus Potyvirus) is globally the most common pathogen of sweetpotato. An East African strain of SPFMV incites the severe 'sweetpotato virus disease' in plants co-infected with Sweet potato chlorotic stunt virus and threatens subsistence sweetpotato production in East Africa; however, little is known about its natural hosts and ecology. In all, 2,864 wild plants growing in sweetpotato fields or in their close proximity in Uganda were observed for virus-like symptoms and tested for SPFMV in two surveys (2004 and 2007). SPFMV was detected at different incidence in 22 Ipomoea spp., Hewittia sublobata, and Lepistemon owariensis, of which 19 species are new hosts for SPFMV. Among the SPFMV-positive plants, approximately 60% displayed virus-like symptoms. Although SPFMV incidence was similar in annual and perennial species, virus-like diseases were more common in annuals than perennials. Virus-like diseases and SPFMV were more common in the eastern agroecological zone than the western, central, and northern zones, which contrasted with known incidence of SPFMV in sweetpotato crops. The data on a large number of new natural hosts of SPFMV detected in this study provide novel insights into the ecology of SPFMV in East Africa.     

Tulip veinal streak,a disorder probably caused by tobacco ringspot virus

A newly recognized disease of tulip called veinal streak is described. The disorder occurs more often in tulips grown under glass than in the field, but totally diseased stocks are found under both conditions. When sap from tulip leaves with veinal streak was manually inoculated to test species, no virus isolate was obtained whereas the sap clarified with a mixture of n-butanol and chloroform and concentrated by centrifugation consistently gave isolates of tobacco ringspot virus (TRSV). However, TRSV was also obtained from symptomless and diseased tulips infected with other viruses. Isolates were propagated inNicotiana tabacum White Burley and sap from systemically infected leaves was used for serological identification in microprecipitin tests. It is postulated that TRSV is the cause of tulip veinal streak. Generally the virus is latent in tulips. The development of symptoms is associated with particular environmental growing conditions (Asjes and Muller, 1972).Samenvatting Een nieuw herkende ziekte in tulpen, genoemd de nerven- of strepenziekte, wordt beschreven. De ziekte treedt meer op in tulpen in de kas dan te velde, doch totaal zieke partijen worden onder beide omstandigheden gevonden. Wanneer sap van tulpebladeren met de nervenziekte mechanisch geïnoculeerd werd op toetsplanten, kon geen virus-isolatie worden verkregen. Het sap gedeeltelijk gezuiverd met een mengsel van n-butanol en chloroform en door centrifugering geconcentreerd gaf steeds weer isolaties van het tabakskringvlekkenvirus (TRSV). Het virus werd echter ook verkregen van symptoomloze planten en zieke tulpen die geïnfecteerd waren met andere virussen. Isolaties werden vermeerderd inNicotiana tabacum White Burley en sap van systemisch geïnfecteerde bladeren werd gebruikt voor serologische identificatie in microprecipitatietoetsen. Aangenomen wordt dat TRSV de oorzaak is van de nerven- of strepenziekte van tulpen. Gewoonlijk is het virus latent in tulpen aanwezig. De ontwikkeling van de symptomen gaat samen met bijzondere groeiomstandigheden (Asjes and Muller, 1972).     

侵染番红花的芜菁花叶病毒

 从表现花叶症状的德国进口番红花上获得一线状病毒分离物SA,线状病毒粒子长度为700~800nm。其在人工接种的11科39种植物中能侵染6科14种植物,在克里芙兰烟上产生系统坏死,在小白菜、花椰菜等十字花科植物上产生系统花叶或为隐症,在昆诺藜等藜科植物上为局部侵染。在番红花病叶、球茎及克里芙兰烟病叶组织中观察到卷轴状和片层状聚集的风轮状内含体。免疫吸附和免疫修饰电镜观察结果显示,SA与芜菁花叶病毒((TuMV)具有紧密的血清学关系。根据这些特征将SA鉴定为TuMV的一个分离物。这是TuMV侵染番红花的首次报道。