The contribution of tomato and alternative host plants to tomato leaf curl virus inoculum pressure in different areas of South India

Indian tomato leaf curl virus (ToLCV) (Geminiviridae: Sub-group III) was detected both in field-collected and laboratory-reared B. tabaci using a triple-antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA). ToLCV was detected in six of the 10 group samples of field collected B. tabaci. ToLCV was also identified in 13 weed species commonly found in Karnataka, both by symptom expression and TAS-ELISA. ToLCV from c. 61% of infected plants was transmitted successfully to tomato by B. tabaci. Tomato plots were planted at three locations on the University of Agricultural Sciences Campus, Bangalore. Indian tomato leaf curl virus disease (ToLCVD) incidence increased most rapidly when the tomato plot was situated adjacent to an older ToLCVD-infected tomato field. When the plots were positioned in a dryland or a wetland area, at least 500 m away from any infected tomato fields, the ToLCVD incidence increased less rapidly, although in all sites it was 100% by 11 wk after transplanting. The numbers of B. tabaci caught on yellow traps in all sites increased during weeks 1–3 after transplanting and thereafter remained at between 10–15 adults trap^-1 24 h^_1. Adult numbers recorded on tomato plants by direct counts remained approximately constant at 2–4 adults plant“”¹. Tomato fields were planted in three taluks (administrative areas) of Karnataka, that had different current and previous histories of tomato production. ToLCVD incidence increased most and least rapidly, respectively, in Kolar taluk where tomato is grown continuously and Doddaballapur tuluk where tomato was grown in the area for the first time. In Malur tuluk, where tomato was grown discontinuously (once a year), the incidence of ToLCVD increased at an intermediate rate. Weed host-plant species growing near the experimental sites had averages of between 1.5–10.0 B. tabaci nymphs per plant, whereas the tomato plants had only 0.3 nymphs per plant. The percentage parasitism of B. tabaci nymphs on tomato and weed species, respectively, was 0.7% and 2–6%. Nymphs and pupae were parasitised by an Encarsia sp. and Eretmocerus mundus Mercet. The relevance and implications of these findings for the epidemiology and management of ToLCVD in Karnataka State, South India is discussed.     

Transmission of tomato leaf curl geminiviruses by Bemisia tabaci: effects of virus isolate and vector biotype

Cultures of Bemisia tabaci from Ivory Coast (IC), Pakistan (PK) and USA (US B-type) were compared for the frequency with which they transmitted three tomato geminivirus isolates: Indian tomato leaf curl virus from Bangalore (ITmLCV), and tomato yellow leaf curl viruses from Nigeria (TYLCV-Nig) and Senegal (TYLCV-Sen). Frequency of transmission from tomato to tomato depended both on the whitefly culture and the virus isolate. US B-type and IC whiteflies transmitted TYLCV-Sen more frequently than ITmLCV whereas PK whiteflies transmitted ITmLCV more frequently than TYLCV-Sen. US B-type whiteflies transmitted both viruses four to nine times more frequently than IC whiteflies. TYLCV-Nig was transmitted rarely by US B-type and not at all by IC whiteflies. Previous work indicates that the geminivirus coat protein controls vector transmissibility. The differential adaptation of TYLCV-Sen to transmission by US B-type whiteflies and of ITmLCV to PK whiteflies was associated with a large difference in epitope profile of the coat proteins of the two viruses. Also, the readily transmissible TYLCV-Sen differed appreciably in epitope profile from the poorly transmissible TYLCV-Nig, which reached a consistently greater concentration in source tissues but lacked epitope 18. However, the lack of epitope 18 in ITmLCV did not prevent its transmission by US B-type whiteflies. Differences in frequency and specificity of geminivirus transmission by whitefly cultures from different countries therefore were associated with differences among epitope profiles of the coat proteins of the viruses, but the structural features of the proteins that control transmission remain to be determined.     

Particle purification, properties and epitope variability of Indian tomato leaf curl geminivirus

A whitefly-transmissible stock isolate of Indian tomato leaf curl geminivirus (ITmLCV) was cultured in graft-inoculated tomato plants and its particles purified from chloroform-clarified extracts in citrate buffer by precipitation with 70 g/litre polyethylene glycol, ultracentrifugation and sucrose density gradient centrifugation. Contaminating helical filaments were eliminated by banding in caesium sulphate gradients. ITmLCV particles had the shape typical for geminiviruses, measured c. 30 × 20 nm and contained a single major protein of estimated mol. wt c. 32 000. They reacted in immunosorbent electron microscopy with antisera to four other whitefly-transmitted geminiviruses. ITmLCV reacted with one out of 17 monoclonal antibodies specific for different epitopes in the particle protein of African cassava mosaic geminivirus and five or six out of 10 monoclonal antibodies to the particle protein of Indian cassava mosaic geminivirus. Virus isolates from tomato at nine locations in Karnataka State showed only slight differences in epitope profile, and isolates from four weed species in tomato fields were similar or identical to those from tomato.     

烟粉虱传播双生病毒研究进展

综述了烟粉虱Bemisia tabaci对双生病毒的获取、传播及存留等方面的特性。烟粉虱最短的获毒和接种时间为15～30 min;双生病毒在烟粉虱体内可存留1至数周,有的终身存在。烟粉虱对双生病毒的传毒效率除了随其获毒及传毒时间的延长、传毒烟粉虱个体数量的增加以及病毒体浓度的增加而提高外,还与烟粉虱的龄期及性别有关。双生病毒除了在植物与粉虱之间直接传播外,还可通过烟粉虱交配及经卵携带的途径在烟粉虱个体和代别间进行传播。寄主植物、双生病毒的一些特殊蛋白以及烟粉虱内共生菌产生的GroEL蛋白,都可影响烟粉虱携带的双生病毒种类及传毒的可能性。双生病毒可对烟粉虱的发育、存活和生殖产生不利或有利的影响。雌成虫携带番茄黄化曲叶病毒（tomato yellow leaf curl virus, TYLCV）后,存活力和生殖力均下降; 而携带番茄斑驳病毒（tomato mottle virus, ToMoV）后,生殖力提高。此外,植物感染双生病毒后,其对烟粉虱的适合性可能提高。     

Tomato yellow leaf curl virus infection of tomato does not affect the performance of the Q and ZHJ2 biotypes of the viral vector Bemisia tabaci

Abstract To better understand the etiology of begomovirus epidemics in regions under invasion we need to know how indigenous and invasive whitefly vectors respond to virus infection. We investigated both direct and indirect effects of infection with Tomato yellow leaf curl virus (TYLCV) on the performance of the invasive Q biotype and the indigenous Asian ZHJ2 biotype of whitefly Bemisia tabaci. The Q biotype performed better than the ZHJ2 biotype on either uninfected or virus‐infected tomato plants. However, virus‐infection of host plants did not, or only marginally affected, the performance of either biotype of whiteflies in terms of fecundity, longevity, survival, development and population increase. Likewise, association of the vectors with TYLCV did not affect fecundity and longevity of the Q or ZHJ2 biotypes on cotton, a non‐host of TYLCV. These results indicate that the alien Q biotype whitefly, but not the indigenous ZHJ2 biotype, is likely to become the major vector of TYLCV in the field and facilitate virus epidemics.     

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。     

番茄玉米间套作对烟粉虱的屏障效应及控制番茄黄化曲叶病毒病的效果

为探讨番茄与玉米间套作对烟粉虱的趋避效应及控制番茄黄化曲叶病毒病的效果,设置番茄品种‘金山511'以固定株行距与玉米品种‘先玉335'分别以10、20、30 cm的播种株距进行间作套种,调查3种栽培方式对烟粉虱屏障效应及黄化曲叶病毒病发生的影响。结果表明: 与番茄单作相比,玉米植株建立起的自然屏障使番茄生长环境相对稳定,玉米播种株距为10、20、30 cm时,番茄植株在6:00—20:00的平均温度分别降低3.01、2.26和1.45 ℃,平均相对湿度分别提高13.0%、8.8%、6.0%,平均光照强度分别降低26.1%、20.4%和14.5%;在每日的高温时段可显著降低番茄温度,提高湿度,减少光照强度,有效缓解强光、高温和干旱等有利于病毒病发生的不良环境条件,且在玉米播种距离为10 cm时效果最好。番茄间作玉米对烟粉虱有趋避效应,能抑制番茄黄化曲叶病毒病的发生,玉米株距为10、20、30 cm时,烟粉虱虫口数分别比单作减少88.7%、82.0%、73.9%,对病毒病的抑制作用呈减弱趋势,间作病情指数分别显著降低67.3%、59.4%和44.5%。玉米/番茄间作的种植模式有利于番茄植株生长和坐果,可提升番茄产量,玉米种植密度越高,效果越好,本试验条件下以玉米种植株距10 cm效果最好。     

感染球孢白僵菌的烟粉虱若虫免疫应答转录组分析

【目的】筛选烟粉虱Bemisia tabaci(Gennadius)若虫应对白僵菌Beauveria bassiana侵染的应答基因,以进一步研究烟粉虱免疫反应的分子机制。【方法】采用新一代高通量测序技术对感染和非感染白僵菌的烟粉虱4龄若虫进行了测序分析,并筛选了差异表达基因;利用生物信息学工具对转录组测序得到的基因进行了功能注释、分类以及参与的信号通路展示。【结果】组装得到非冗余Unigene 232 554个,其N50和N90分别为1 153 bp和260 bp,平均长度为67 424 bp。对所有的基因进行差异表达分析发现:以P<0.05为标准筛选得到了1 166个差异表达基因,其中上调表达基因有474个,下调表达基因有692个,其中,与免疫相关的基因有405个;GO富集分析发现:有416个GO term有富集现象,包括156个生物学过程(66 402个Unigenes),89个细胞组分(27 645个Unigenes)和154个分子功能(73 417个Unigenes);KEGG代谢通路分析将烟粉虱转录组1 145个DEG匹配到309个通路上,其中76个通路得到了富集。【结论】对405个可能参与到烟粉虱若虫对白僵菌侵染的免疫识别和防御的基因进行了测序。研究结果为从分子水平上开展应用虫生真菌防治烟粉虱的研究奠定信息学基础。     

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。     

烟粉虱体内木尔坦棉花曲叶病毒的LAMP快速检测技术

木尔坦棉花曲叶病毒(Cotton leaf curl Multan virus,CLCu Mu V)是引起世界范围内棉花曲叶病流行的主要病原之一,目前已入侵我国广东、广西、海南、福建、云南等地区。该病害由烟粉虱传播,随着烟粉虱扩散范围增加、危害不断加重,棉花曲叶病对我国棉花生产的潜在危害也日益增加。加强该病毒传播介体携带病毒的快速、特异性检测技术研发,对该病害的有效检疫与防控具有重要意义。本研究基于木尔坦棉花曲叶病毒(CLCu Mu V)的基因序列设计出LAMP检测4条特异性引物,建立LAMP扩增体系并优化扩增条件,扩增试验证明引物组合的特异性高,LAMP检测所需时间短,仅29 min即可定性检测CLCu Mu V扩增产物;该方法较普通PCR的扩增灵敏度高,可用于检测1头烟粉虱体内是否携带CLCu Mu V,简便易行,只通过颜色变绿或浊度变浑浊即可定性判断。建立的LAMP检测技术可被用于苗木上烟粉虱携带CLCu Mu V的早期检测,为介体昆虫带毒的检疫监测提供一种快速、准确和易操作的新技术。     

Serological studies on the accumulation and localisation of three tomato leaf curl geminiviruses in resistant and susceptible Lycopersicon species and tomato cultivars

Accessions of wild Lycopersicon spp. and selected Fl hybrid tomato cultivars were compared for their resistance to three whitefly-transmissible geminiviruses: Indian tomato leaf curl virus (ITmLCV) and tomato yellow leaf curl viruses from Sardinia (TYLCV-Sar) and Senegal (TYLCV-Sen). The resistance of different plant lines was expressed in different ways but in most instances a given line reacted similarly to graft inoculation with the three viruses. L. pimpinellifolium LA1478 produced as much virus antigen, assessed by triple antibody sandwich-ELISA, as the susceptible cv. Moneymaker but developed only very mild symptoms and is therefore tolerant of infection. In L. hirsutum LA1777 and L. peruvianum CMV-INRA, very mild or no symptoms developed but antigen concentrations were substantially less than in Moneymaker. L. chilense LA1969 remained symptomless and its antigen concentration was < 1% of that in Moneymaker. Symptoms were mild or barely evident in the Fl hybrid cultivars. Cultivars Tyking and Fiona had antigen concentrations about 5–10% of those of Moneymaker, whereas TY20, Top 21 and Tyger had intermediate antigen concentrations. In a few instances, the extent to which virus accumulation was restricted depended on the challenge virus. Accumulation of TYLCV-Sen in TY20, Top 21 and Tyger was less affected than that of the other two viruses, and accumulation of TYLCV-Sar in accessions LA1777 and CMV-INRA was less affected than that of TYLCV-Sen or ITmLCV. Tissue-printing tests showed that ITmLCV and TYLCV-Sen antigens were confined to phloem tissue. In Tyking, the number of virus antigen-containing phloem traces and the antigen content of individual traces were less than in Moneymaker but the partitioning of antigen between internal and external phloem was unaffected.     

观察球孢白僵菌侵染烟粉虱若虫过程的新方法——荧光显微法

【目的】介绍一种观察白僵菌Beauveria bassiana侵染烟粉虱Bemisia tabaci(Gennadius)若虫的荧光显微方法。【方法】将被白僵菌侵染的烟粉虱若虫用荧光素二乙酸酯(FDA)染色,并于波长为450~490 nm的蓝光下显微观察。【结果】在接种白僵菌12 h和24 h的烟粉虱若虫上分别观察到昆虫表皮上分生孢子的萌发和芽管穿透表皮。结合裸眼和荧光显微观察结果证实了被真菌侵染的烟粉虱若虫体色变红和菌体在虫体内增殖是同时发生的。【结论】应用荧光显微方法能观察到白僵菌在烟粉虱若虫体表和体内的侵染。     

Screening additional wild tomatoes for resistance to the whitefly-borne tomato yellow leaf curl virus

Plants of 25 wild Lycopersicon accessions were screened in the greenhouse for resistance to the whitefly-borne tomato yellow leaf curl virus (TYLCV). High levels of resistance were detected in 7 of 9 accessions of L. peruvianum and in all 5 accessions of L. chilense tested. In contrast, plants of 7 accessions of L. hirsutum and 3 of 4 accessions of L. pimpinellifolium were highly susceptible. Plants of accession CIAS 27 (L. pimpinellifolium) showed moderate resistance to TYLCV.     

烟粉虱携带番茄黄化曲叶病毒检测及其传入山东省的考证

【背景】番茄黄化曲叶病毒（TYLCV）是由媒介昆虫烟粉虱传播的一种双生病毒,对蔬菜及烟草等经济作物造成严重危害。前人资料表明,该病毒于2006年传人我国南方地区,2007年传人山东省,2008年后在山东各地逐渐蔓延扩散。【方法】为了考证TYLcV传人山东省的时间,本研究利用mtCOI基因对于2005和2006年7—8月份在山东省不同地区作物上共采集的15份烟粉虱样品进行了生物型鉴定,并进一步检测了烟粉虱携带TYLCV情况,同时对PCR扩增产物进行了测序分析。【结果】2005年的4份样品烟粉虱生物型均为B型,均不携带TYLCV。2006年的11份烟粉虱样品为B型与Q型混合样品,其中,2份烟粉虱样品检测到TYLCV,进一步证实该病毒为TYLCV。【结论与意义】本研究首次证实了TYLCV早在2006年就已经传入山东省。研究结果不仅对于防控该病毒具有重要指导意义,而且对于其入侵生物学研究也具有重要参考价值。     

Detection and relationships of cotton leaf curl virus and allied whitefly-transmitted geminiviruses occurring in Pakistan

A stock culture of cotton leaf curl virus from Pakistan (CLCuV-PK), was transmitted by whiteflies (Bemisia tabaci) to seven plant species, including French bean, okra, tobacco and tomato, and caused vein thickening and leaf curl symptoms. It was readily detected in triple antibody sandwich ELISA (TAS-ELIS A) by 11 out of 31 monoclonal antibodies raised against the particles of three other geminiviruses: African cassava mosaic, Indian cassava mosaic and okra leaf curl viruses. Reaction strength was enhanced when the tissue extraction fluid contained sodium sulphite. Minor variations in epitope profile were found among virus isolates from cotton (Gossypium hirsutum) collected from different districts in Pakistan over a 5-year period. These epitope profiles were distinguishable from that of cotton leaf curl virus from G. barbadense in southern India but indistinguishable from the profiles of viruses causing yellow vein disease of okra in India or Pakistan, or leaf curl of okra {Abelmoschus esculentus), Hibiscus tiliaceus, radish or sunflower in Pakistan, suggesting that these plants are putative natural hosts of CLCuV-PK. The viruses in cotton, and in okra with leaf curl or yellow vein symptoms, were also detected by PCR with three pairs of CLCuV-PK-specific primers. Five additional whitefly-transmitted geminiviruses were found among isolates from 11 other naturally-infected species in Pakistan, and were distinguished by their epitope profiles. These viruses were associated, respectively, with tobacco leaf curl, squash yellow blotch, tomato yellow leaf curl, watermelon leaf crinkle and soybean yellow mosaic diseases. The first four of these viruses were detected readily by PCR with geminivirus general primers but only weakly, if at all, with two pairs of CLCuV-PK-specific primers. Pakistani crops are infected with a range of distinguishable but relatively closely related whitefly-transmitted geminiviruses, some of which resemble those found in India.