新疆地区烟粉虱生物型的区域分布及其携带的番茄黄化曲叶病毒检测

【目的】明确不同烟粉虱Bemisia tabaci (Gennadius)生物型在新疆地区的区域分布及其携带番茄黄化曲叶病毒的情况。【方法】本文利用烟粉虱mtDNA COⅠ基因片段作标记以及番茄黄化曲叶病毒(tomato yellow leaf curl virus, TYLCV)特异性引物, 鉴定新疆不同地区烟粉虱生物型, 并检测其携带TYLCV情况。【结果】石河子市、 博乐市、 岳普湖县、 泽普县、 叶城县、 莎车县、 墨玉县、 和田市与和田县烟粉虱属于B型烟粉虱, 且720 bp COⅠ基因序列一致性为100%; 阿克苏市、 哈密市、 鄯善县、 乌鲁木齐市、 克拉玛依市与伊宁市烟粉虱属于Q型烟粉虱, 720 bp COⅠ基因序列一致性为99.9%~100%; 吐鲁番市与库尔勒市兼有B型烟粉虱和Q型烟粉虱。伊宁市、 乌鲁木齐市、 鄯善县、 哈密市、 阿克苏市、 库尔勒市、 莎车县、 和田县和墨玉县的烟粉虱样本携带TYLCV, 除2011年底已经暴发危害的莎车县外, 其他区域均为番茄黄化曲叶病毒病发生的高风险区域。【结论】本研究为新疆地区烟粉虱及番茄黄化曲叶病毒病的科学防控提供依据。     

辽宁省冬季温室内越冬粉虱伪蛹的种类鉴定及烟粉虱体内番茄黄化曲叶病毒的检测

为了揭示辽宁省冬季温室内越冬粉虱伪蛹的种类及烟粉虱Bemisia tabaci （Gennadius）携带番茄黄化曲叶病毒（tomato yellow leaf curl virus, TYLCV）情况, 于2012年1月份在辽宁省不同县市区的温室作物上采集了17份粉虱伪蛹样品（每样品含30头粉虱伪蛹） , 镜检鉴别粉虱种类并利用mtCOI基因对烟粉虱生物型进行了鉴定; 检测了烟粉虱携带TYLCV情况并对其PCR扩增产物进行了测序分析。结 果表明： 辽宁省冬季温室内存在越冬温室白粉虱Trialeurodes vaporariorum （Westwood）与烟粉虱。17份粉虱样品中, 11份样品为烟 粉虱样品, 6份样品为温室白粉虱和烟粉虱混合样品。混合样品中, 温室白粉虱仅在锦州凌海（LH）样品中占优势。17份烟粉虱样品（包 括混合样品）中, 仅有4份样品为B型与Q型混合样品, 其他13份样品烟粉虱生物型均为Q型。17份烟粉虱样品中有3份Q型烟粉虱样品检测 到TYLCV, 系统树分析进一步证实该病毒是TYLCV。调查结果为辽宁省粉虱与TYLCV的早期测报和防控提供了科学依据。     

烟粉虱传毒相关蛋白基因的克隆及其表达量分析

【目的】烟粉虱Bemisia tabaci(Gennadius)是一种世界性的入侵性害虫,其传播的番茄黄化曲叶病毒(Tomato yellow leaf curl virus,TYLCV)给我国番茄生产造成巨大经济损失。为了阐明烟粉虱传播双生病毒的机理,本文拟研究明确B和Q烟粉虱体内传毒相关蛋白Gro EL基因及其表达量。【方法】采用特异性引物克隆了B和Q烟粉虱内共生菌编码的传毒相关蛋白Gro EL基因,并进行序列分析;并利用荧光定量PCR技术检测两种生物型及其获取双生病毒前后该基因的相对表达量。【结果】烟粉虱内共生菌Hamiltonella编码的Gro EL基因全长为1 668 bp,编码555个氨基酸;B、Q烟粉虱该基因的核苷酸序列相似性为99.94%,氨基酸同源性为99.82%;带毒烟粉虱中Gro EL基因的表达量显著高于未带毒的对应生物型烟粉虱;无论带毒与否,Q烟粉虱该基因的表达量均显著高于B(P<0.05)。【结论】烟粉虱携带TYLCV后可诱导Gro EL的表达量升高,B和Q烟粉虱中Gro EL基因及其表达量均存在差异,这可能是B和Q烟粉虱传毒效率存在显著差异的原因之一。     

泛素-蛋白酶体系统对烟粉虱体内番茄黄曲叶病毒的影响

【目的】泛素化修饰广泛参与细胞周期、信号传导、转录调控、免疫应答等多个方面,是细胞内一种非常重要的蛋白水平可逆修饰。但目前关于泛素-蛋白酶体系统是否参与介体昆虫传播病毒还鲜有报道。本研究旨在探索泛素-蛋白酶体系统在烟粉虱Bemisia tabaci传播番茄黄曲叶病毒(Tomato yellow leaf curl virus,TYLCV)过程中所起的作用。【方法】通过RT-qPCR分析取食感染TYLCV和不感染TYLCV番茄植物的烟粉虱成虫体内泛素和蛋白酶体亚基基因的表达,采用Western blot检测TYLCV侵染后烟粉虱成虫体内泛素蛋白含量的变化;饲喂抑制剂和注射双链RNA的方法抑制烟粉虱成虫体内蛋白酶体活性后,通过RT-qPCR测定其体内TYLCV含量的变化。【结果】烟粉虱成虫携带TYLCV后,泛素以及26S蛋白酶体亚基4、6B和β基因的表达量没有发生显著变化,体内游离泛素和缀合泛素的含量以及两者的比例也都没有显著改变,表明TYLCV侵染不会影响烟粉虱体内泛素-蛋白酶体的活性。但饲喂抑制剂(Bortezomib或MG132)或沉默Rpn11抑制蛋白酶体活性后,烟粉虱成虫体内的TYLCV含量显著升高。【结论】泛素-蛋白酶体系统对烟粉虱体内的TYLCV起负调控作用,该系统可能通过直接降解病毒或激活免疫反应等方式抑制病毒含量,进而帮助烟粉虱应对TYLCV带来的不利影响。     

基于16S rDNA基因的中国部分地区非B型烟粉虱系统发育关系分析

【目的】烟粉虱Bemisia tabaci是一个快速进化的复合种。由于近年来烟粉虱危害不断增加, 其生物型问题也越来越受到关注。在我国, 不仅存在危害严重的B型烟粉虱, 同时也已发现多种非B型烟粉虱。通过鉴定我国部分地区目前发生的烟粉虱生物型种类, 分析它们与世界各地不同生物型之间的关系, 可为我国烟粉虱的生物型检测和综合防治提供依据。【方法】利用16S rDNA基因作为分子标记, 鉴定了2005年与2006年在我国6个省份（自治区）采集的22个非B型烟粉虱样本的生物型, 并探讨了各生物型之间的系统进化关系。【结果】所研究的22个非B型烟粉虱归属于Q型、Nauru型和An型, 3种生物型之间的遗传距离在10%以上, 但是Q生物型与B生物型之间的亲缘关系最近, 遗传距离在2.8%~4.0%范围内; 进化分歧数据还表明, 不同生物型之间的遗传距离明显大于同一生物型内遗传距离, 其中Q型内部差异最小, 在0.9%以内; 同时结果表明目前在我国多个生物型共同存在是一个普遍的现象, 其中Nauru型的分布较广泛; 在云南地区检测到的非B型烟粉虱生物型类型最多。【结论】烟粉虱生物型遗传分化复杂, 利用16S rDNA基因能有效鉴定烟粉虱的生物型。     

田间系统调查表明山东省农区烟粉虱优势种为Q隐种

【目的】为了揭示山东省农区烟粉虱 Bemisia tabaci 隐种的分布情况,对2013年7-8月份期间在该省不同农区采集的烟粉虱隐种组成进行了系统调查研究。【方法】在山东省15市22个地点作物上采集了69份烟粉虱样品,利用mtCOI基因PCR-RFLP方法进行了分子鉴定,并分析了烟粉虱Q隐种在不同寄主作物及地理区域内的分布状况。【结果】22个采集地点中,17个地点的采集样品全部为烟粉虱Q隐种;其他5个地点（潍坊、菏泽、泰安、淄博和临沂）的烟粉虱Q隐种所占比例均大于90%,B隐种比例较低（<10%）。烟粉虱Q隐种比例在5种寄主（茄子、辣椒、番茄、黄瓜和棉花）种群间以及在山东省东部丘陵、中部山地和西部平原地区间均没有显著差异。【结论】2013年田间调查发现烟粉虱Q隐种在山东各地已广泛取代B隐种成为该地区优势种群。     

下调烟粉虱MED隐种BtabCSP6表达对番茄黄化曲叶病毒传播的影响

【目的】番茄黄化曲叶病毒(tomato yellow leaf curl virus, TYLCV)是对农业生产造成威胁的主要病毒之一,自然条件下通过媒介昆虫烟粉虱Bemisia tabaci传播。已有研究表明烟粉虱雌成虫比雄成虫具有更强的获毒与传毒能力。本研究旨在探明烟粉虱化学感受蛋白(chemosensory protein, CSP)基因BtabCSP6表达对病毒传播的影响,为控制病毒发生寻找新途径。【方法】使用TYLCV侵染性克隆方法获得带毒番茄植株,微虫笼收集不带毒烟粉虱MED隐种成虫固定在感染TYLCV的番茄植株叶片获毒48 h;利用RT-qPCR技术测定分别取食感染和未感染TYLCV番茄植株的烟粉虱MED隐种雌雄成虫体内BtabCSP1-8基因表达量变化;通过饲喂法利用RNAi对烟粉虱MED隐种雌成虫BtabCSP6基因进行干扰48 h后饲喂TYLCV感染的番茄植株,测定烟粉虱MED隐种雌成虫的获毒率和传毒率。【结果】RT-qPCR检测结果表明,与未侵染的烟粉虱MED隐种雌成虫相比,侵染TYLCV的雌成虫体内BtabCSP3和BtabCSP6基因的表达量变化最为显著。同样地,在侵染TYLCV的雄成虫体内,BtabCSP4和BtabCSP6表达量的变化最为明显。饲喂dsBtabCSP6 48 h后,烟粉虱MED隐种雌成虫体内BtabCSP6表达量降低;取食感染TYLCV番茄植株不同时间烟粉虱MED隐种雌成虫的获毒率和不同数量雌成虫对未感染TYLCV的番茄植株的传毒率与对照相比均显著降低。【结论】下调烟粉虱MED隐种雌成虫体内BtabCSP6基因的表达,可显著降低烟粉虱MED隐种雌成虫的获毒率和传毒率,说明BtabCSP6可能影响TYLCV传播。     

番茄褪绿病毒对MED烟粉虱成虫在不同寄主植物上的寄主选择性和取食行为的影响

【目的】由粉虱传播的番茄褪绿病毒(tomato chlorosis virus, ToCV)具有较强的暴发性和流行性,目前该病毒现已蔓延至世界各地,可为害多种作物,对农林经济生产造成严重危害。病毒侵染会影响介体昆虫的寄主选择性和取食行为,从而影响病毒传播。本研究旨在明确ToCV对MED烟粉虱Bemisiatabaci在不同寄主上的寄主选择性及取食行为的影响。【方法】利用Y型嗅觉仪测定携带和未携带ToCV的MED烟粉虱雌成虫的寄主选择性,利用刺吸电位(electrical penetration graph, EPG)技术比较携带和未携带ToCV的烟粉虱雌成虫在健康的番茄、辣椒、棉花和豇豆4种寄主植株上的取食行为差异。【结果】未携带ToCV的MED型烟粉虱雌成虫对于4种寄主植物的选择偏好性排序为番茄、辣椒＞棉花＞豇豆;携带ToCV烟粉虱雌成虫对4种寄主植物的选择性偏好性降低,排序为番茄、辣椒、棉花＞豇豆。与未携带ToCV烟粉虱雌成虫相比,携带ToCV烟粉虱雌成虫取食4种植物所产生的刺探次数均显著增加,第1次到达韧皮部的时间有明显延后,取食总时间与韧皮部的取食时间明显减少。【结论】ToCV使烟粉虱对寄主的选择偏好性和取食行为发生显著改变,从而增加了病毒在寄主植物间传播的可能。     

江苏地区烟粉虱发生危害及其生物型监测

【目的】随着Q型和B型烟粉虱Bemisia tabaci(Gennadius)在全世界范围内的传播和入侵,两种入侵烟粉虱的发生危害和种群扩张越来越受到人们的关注。本文对2010—2013年江苏省内烟粉虱的发生危害程度及其生物型的分布进行了全面的调查研究。【方法】应用室内镜检鉴定烟粉虱的发生危害程度,同时结合mt COⅠ分子标记快速检测法鉴定烟粉虱的生物型。【结果】从时间来看,烟粉虱的发生危害程度在2011年最严重,而在2013年最低,呈"先上升后下降"的趋势。从不同地区来看,苏南和苏北地区呈现出"先上升后下降"的趋势,而苏中地区与之相反。从寄主来看,烟粉虱在茄子,黄瓜和南瓜上的发生危害程度最严重。【结论】烟粉虱在江苏省内的发生危害程度因时间,地区和寄主的不同而差异显著,这种差异与烟粉虱不同生物型之间的竞争取代没有直接联系。虽然Q型烟粉虱在江苏省内占据了优势地位,但是B型烟粉虱在苏南和苏北地区仍然占据了一定的比例,并且有逐步上升的趋势。     

两个入侵烟粉虱隐种MEAM1与MED对番茄黄曲叶病毒水平传播能力的比较

【目的】烟粉虱 Bemisia tabaci 是番茄黄曲叶病毒（Tomato yellow leaf curl virus, TYLCV）在自然界的唯一传播媒介,除了可以直接取食获取TYLCV,烟粉虱还可以通过交配获取此植物病毒。虽然前人研究证明了烟粉虱Middle East-Asia Minor 1 （MEAM1）和Mediterranean （MED）隐种都可以通过交配在两性个体之间水平传播TYLCV,但有关MEAM1与MED隐种水平传播TYLCV的能力是否存在差异,不同研究的结果却并不一致。另外,目前尚无关于烟粉虱的水平传播行为是否有助于TYLCV在田间扩散的相关研究。【方法】从浙江、广东、云南和河南4个省份的田间采集MEAM1和MED隐种烟粉虱种群,在室内应用分子标记对各种群所属隐种鉴定后分别建立7个供试种群,然后观察每个种群内带毒成虫与不带毒成虫通过交配在不同性别个体之间水平传播TYLCV的能力,并选用采自广东的MEAM1隐种烟粉虱模拟群体交配实验探究通过交配获毒的烟粉虱个体是否具有传播TYLCV致健康番茄植株发病的能力。【结果】4个省内的MEAM1和MED隐种烟粉虱都可通过交配对TYLCV进行水平传播,但传播频率一般在10%以下。不同省份的MEAM1隐种种群之间在水平传播TYLCV的能力上无显著差异,不同省份的MED隐种种群之间也不存在显著差异。另外,同一省份的MEAM1隐种与MED隐种之间在水平传播TYLCV的能力上也不存在显著差异,而且在两个隐种中,带毒雄虫将病毒水平传播给不带毒雌虫与带毒雌虫将病毒水平传播给不带毒雄虫的频率没有显著差异。研究还表明,采自广东的MEAM1隐种烟粉虱个体通过交配水平获毒后不能致健康番茄植株发病。【结论】综合以上结果,我们推测TYLCV在中国境内两个入侵烟粉虱隐种MEAM1和MED各自种群内个体之间的水平传播概率较低,对该病毒在田间的扩散可能没有作用或作用不大。     

Efficiency of insect exclusion screens for preventing whitefly transmission of tomato yellow leaf curl virus of tomatoes in Israel

Tomato yellow leaf curl virus (TYLCV) is the most frequently occurring virus in tomatoes in the Middle East, and the most harmful one. It is transmitted solely by the whitefly Bemisia tabaci (Gennadius). Within 4-6h of inoculative feeding, a whitefly can transmit TYLCV to a healthy plant with 80% probability. The symptoms are apparent after two to three weeks whereupon fruit-set is effectively terminated. The only means of controlling TYLCV is by controlling the whitefly. Until 1990 this was exclusively by insecticides. Starting in 1990, growers of greenhouse tomatoes in Israel began adopting insect exclusion screens to prevent inoculation of TYLCV. This article reports on the methods used in the search for efficient screening materials and presents data on their relative efficiencies in excluding B. tabaci and several other greenhouse pests. Ten materials were tested, of which five were found to be effective in excluding B. tabaci under laboratory conditions. This number was reduced to three following field trials and trials in commercial tomato greenhouses. These materials are now in widespread use in Israel: by 2000 practically all table tomatoes in Israel were grown under exclusion screens. The use of exclusion screens has been shown to be an economically viable pest management method.     

亚致死剂量溴氰虫酰胺对MED烟粉虱取食行为及传播TYLCV的影响

取食是烟粉虱Bemisia tabaci(Gennadius)传播植物病毒最重要的途径之一.本研究利用昆虫刺探电位(EPG)技术研究了杀虫剂溴氰虫酰胺亚致死浓度(LC15)对携带番茄黄化曲叶病毒(TYLCV)MED烟粉虱取食行为和传毒效率的影响.结果表明,溴氰虫酰胺对未携毒和携毒MED烟粉虱的亚致死浓度(LC15)分别为1.52 mg/kg和1.75 mg/kg,差异不显著.携毒烟粉虱在溴氰虫酰胺亚致死浓度处理的番茄苗上取食,第一次刺探起始时间显著推迟,是对照番茄苗上的3.7倍.在韧皮部取食阶段,处理组烟粉虱在韧皮部分泌唾液平均时间、吸食汁液总时间和平均时间均显著缩短,分别是对照组的0.10倍、0.14倍和0.10倍,韧皮部取食能力显著降低.qPCR试验研究表明,溴氰虫酰胺亚致死浓度处理极显著的降低了烟粉虱的传毒效率,携毒烟粉虱取食48 h的番茄苗培养10 d后,TYLCV相对含量仍接近于0,表明溴氰虫酰胺对烟粉虱传播TYLCV具有良好的控制潜力.研究结果为科学合理的使用化学农药防治烟粉虱及其传播的双生病毒病提供理论依据.     

Phylogenetic analysis and inflow route of <Emphasis Type="Italic">Tomato yellow leaf curl virus</Emphasis> (TYLCV) and <Emphasis Type="Italic">Bemisia tabaci</Emphasis> in Korea

Tomato yellow leaf curl virus (TYLCV) is a member of the genus Begomovirus of the family Geminiviridae, members of which are characterized by closed circular single-stranded DNA genomes of 2.7-2.8 kb in length, and include viruses transmitted by the Bemisia tabaci whitefly. No reports of TYLCV in Korea are available prior to 2008, after which TYLCV spread rapidly to most regions of the southern Korean peninsula (Gyeongsang-Do, Jeolla-Do and Jeju-Do). Fifty full sequences of TYLCV were analyzed in this study, and the AC1, AV1, IR, and full sequences were analyzed via the muscle program and bayesian analysis. Phylogenetic analysis demonstrated that the Korea TYLCVs were divided into two subgroups. The TYLCV Korea 1 group (Masan) originated from TYLCV Japan (Miyazaki) and the TYLCV Korea 2 group (Jeju/Jeonju) from TYLCV Japan (Tosa/Haruno). A B. tabaci phylogenetic tree was constructed with 16S rRNA and mitochondria cytochrome oxidase I (MtCOI) sequences using the muscle program and MEGA 4.0 in the neighbor-joining algorithm. The sequence data of 16S rRNA revealed that Korea B. tabaci was closely aligned to B. tabaci isolated in Iran and Nigeria. The Q type of B. tabaci, which was originally identified as a viruliferous insect in 2008, was initially isolated in Korea as a non-viruliferous insect in 2005. Therefore, we suggest that two TYLCV Japan isolates were introduced to Korea via different routes, and then transmitted by native B. tabaci.     

Differential indirect effects of two plant viruses on an invasive and an indigenous whitefly vector: implications for competitive displacement

The role of vector–begomovirus–plant interactions in the widespread invasion by some members of the whitefly species complex Bemisia tabaci is poorly understood. The invasive B biotype of B. tabaci entered China in the late 1990s and had become the predominant or only biotype of the whitefly in many regions of the country by 2005–2006. Meanwhile epidemics of begomoviruses have been observed in many crops including tomato for which Tomato yellow leaf curl China virus (TYLCCNV) and Tomato yellow leaf curl virus (TYLCV) have been identified as two major disease-causing agents. Here, we conducted laboratory experiments to compare the performance of the invasive B and indigenous ZHJ1 whitefly biotypes on uninfected, TYLCCNV-infected and TYLCV-infected plants of tomato cv. Hezuo903, a cultivar that has been widely cultivated in many regions of China. The infection of tomato plants by either of the viruses had no or only marginal effects on the development, survival and fecundity of the B biotype. In contrast, survival and fecundity of the ZHJ1 biotype were significantly reduced on virus-infected plants compared to those on uninfected plants. Populations of the B biotype on uninfected and TYLCCNV-infected plants increased at similar rates, whereas population increase of the ZHJ1 biotype on TYLCCNV-infected plants was affected adversely. These asymmetric responses to virus infection of tomato plants between the B and ZHJ1 biotypes are likely to offer advantages to the B biotype in its invasion and displacement of the indigenous biotype.     

番茄褪绿病毒TaqMan荧光定量PCR快速检测方法的建立与应用

【目的】本研究旨在建立TaqMan实时荧光定量PCR(TaqMan RT-qPCR)技术,快速检测单头烟粉虱Bemisia tabaci体内的番茄褪绿病毒(tomato chlorosis virus,ToCV)。【方法】根据ToCV外壳蛋白保守序列设计了1对特异性引物和1条TaqMan探针,建立了TaqMan RT-qPCR方法;与常规PCR检测进行比较,检测该方法的灵敏度与特异性;并应用该方法对单头烟粉虱成虫体内ToCV进行了快速检测。【结果】本研究构建的TaqMan RT-qPCR检测ToCV的标准曲线,其循环阈值(Ct值)与模板浓度具有良好的线性关系,扩增效率为98%。该方法对ToCV的最低检测浓度为8.3×10 copies/μL,灵敏度是常规RT-PCR的1000倍。该方法与田间番茄两种重要病毒番茄黄化曲叶病毒(tomato yellow leaf curl virus,TYLCV)和番茄斑萎病毒(tomato spotted wilt virus,TSWV)检测无交叉反应。单头烟粉虱成虫ToCV检测结果表明,温室内ToCV侵染植株上烟粉虱携毒率为100%,田间烟粉虱的携毒率为30%。【结论】本研究建立的TaqMan RT-qPCR检测方法,可快速有效检测单头烟粉虱体内ToCV携毒情况,为该病毒病的防控提供了技术支撑。     

Upregulation of temperature susceptibility in Bemisia tabaci upon acquisition of Tomato yellow leaf curl virus (TYLCV)

Acquisition of plant viruses has various effects on physiological mechanisms in vector insects. Bemisia tabaci is the only known vector of Tomato yellow leaf curl virus (TYLCV), which is a serious virus affecting tomato cultivars. In this study, the lifespan of Q1 biotype was compared between non-viruliferous (NV) and TYLCV-viruliferous (V) whiteflies. Total lifespan from egg to adult death of NV whiteflies was 62.54days but 10.64days shorter in V whiteflies. We investigated the temperature susceptibility of B. tabaci by comparing mortalities as well as heat shock protein (hsp) mRNA levels between NV and V whiteflies. For this, NV and V whiteflies were exposed for either 1 or 3h at 4, 25, and 35°C. The mortality of V whiteflies was higher than NV ones following exposure at either 4 or 35°C, but there was no significant difference at 25°C. Analysis of the expression level of heat shock protein (hsp) genes using quantitative real-time PCR showed that both cold and heat shock treatments stimulated higher expression of hsps (hsp40, hsp70, and hsp90) at various rates in V whiteflies than NV ones, but there was no difference at 25°C. All together, our results show that TYLCV acquisition accelerated the developmental rate and increased susceptibility to thermal stress in B. tabaci. Therefore, this modification may result in reduced vector longevity due to increased metabolic energy utilization. Our results provide insights into the complex interaction between vector fitness and thermal stress in relation to the acquisition and transmission of plant viruses.