烟粉虱中甘薯褪绿矮化病毒（SPCSV）的快速检测技术

建立了利用NCM-ELISA、RT-PCR和半巢式RT-PCR检测烟粉虱中SPCSV的方法,比较了3种检测方法的灵敏性。结果表明,NCM-ELISA最低能从16头带毒烟粉虱中检测出SPCSV;RT-PCR能稳定地从3头以上带毒烟粉虱中检测出SPCSV;半巢式RT-PCR能稳定地从单头烟粉虱中检测出SPCSV。检测灵敏性研究表明,用体外转录的RNA作为核酸模板,RT-PCR可检测的最低浓度为5.69×104拷贝/μL,半巢式RT-PCR为5.69×101拷贝/μL,说明半巢式RT-PCR检测方法的灵敏性比RT-PCR高1 000倍。     

几种新型杀虫剂防治B型烟粉虱的药效试验

烟粉虱Bemisia tabaci(Gennadius),又称甘薯粉虱、棉粉虱,是热带和亚热带地区的重要害虫之一。该虫可危害棉花、蔬菜和园林花卉等植物,并且能传播双生病毒,平均每年在     

B型烟粉虱携带传播烟草曲茎病毒的能力

B型烟粉虱近20年来入侵许多国家和地区,已成为重要的世界性作物害虫,其主要危害方式之一是传播双生病毒,造成作物病毒病大发生.应用PCR技术,研究了入侵我国的B型烟粉虱个体获取、存留及传播烟草曲茎病毒（Tobacco curly shoot virus,TbCSV）的能力.B型烟粉虱在感染TbCSV的普通烟毒株上饲毒30 min时,就可在40%的个体内检测到TbCSV DNA,个体带毒率随饲毒时间延长而增加,饲毒12 h后,带毒率达100%;TbCSV DNA在B型烟粉虱体内可存留10天左右,但不能终身存留;传毒处理植株的发病症状及PCR检测结果表明,B型烟粉虱是TbCSV的传播媒介.     

福州市发生由木尔坦棉花曲叶病毒引起的朱槿曲叶病

棉花曲叶病是世界棉花生产上最具毁灭性的病毒病害,其主要病原木尔坦棉花曲叶病毒(Cotton leaf curlMultan virus,CLCuMV)已经入侵我国广东、广西、海南等省多个地理区域,扩散危害范围日益加大.2011-2012年调查发现福建省厦门、福州和宁德等多个城市绿化植物朱槿(Hibiscus rosa-sinensis)发生朱槿曲叶病的流行.应用双生病毒通用引物以及烟粉虱生物型鉴定的通用引物,通过PCR扩增、序列分析等方法,分别检测或鉴定了福州市朱槿曲叶病的病原、介体烟粉虱的生物型以及朱槿病株上烟粉虱的带毒率.结果显示:福州市朱槿曲叶病的病原是木尔坦棉花曲叶病毒,与CLCuMV广东分离物和广西分离物的相似性高达99.6％以上;介体烟粉虱生物型为B型,病株上烟粉虱的带毒率为100％.试验结果说明CLCuMV已经扩散至福建省,应警惕和控制木尔坦棉花曲叶病毒的进一步扩散和危害.     

北京地区番茄黄化曲叶病毒病的鉴定及防治对策

番茄黄化曲叶病毒病是一种由烟粉虱传播的病毒病,给番茄生产造成严重威胁。2009年在北京郊区调查时发现部分保护地种植的番茄植株表现典型黄化曲叶症状。通过提取典型症状样品总DNA利用粉虱传双生病毒检测简并引物PA/PB,进行PCR扩增到541bp的特异条带。通过测序和核苷酸序列比对表明该序列与番茄黄化曲叶病毒序列相似性最高为99%。分子检测结果表明北京郊区部分保护地种植的番茄已被烟粉虱传播的番茄黄化曲叶病毒侵染危害。     

番茄黄化曲叶病毒病暴发原因分析及防控对策

<正>番茄黄化曲叶病毒(Tomato yellow leaf curl virus,TYLCV)为双生病毒科(Geminniviridae)菜豆金色花叶病毒属(Begomovirus)成员[1]。该类病毒通过B型烟粉虱和Q型烟粉虱进行传播,同时可经嫁     

SCAR分子标记鉴别烟粉虱和温室白粉虱

在简单重复序列PCR的基础上建立了烟粉虱和白粉虱两个物种DNA特异性片段的SCAR标记。验证表明,新建立的烟粉虱SCAR标记能特异性扩增不同地理种群的烟粉虱而不能扩增温室白粉虱;相反新建立的温室白粉虱SCAR标记则能特异性地扩增不同地理种群的温室白粉虱而不能扩增烟粉虱种群。两种SCAR标记能够快速、准确地鉴定烟粉虱和温室白粉虱。     

广东黄秋葵黄脉曲叶病样中检测到烟粉虱传双生病毒

黄秋葵是近几年来从日本和我国台湾引进的一种蔬菜作物。近期,广东的黄秋葵上发生了黄脉曲叶病。病株的典型症状表现为叶脉黄化,在叶片正面形成网络状,在叶背面叶脉肿大突起明显,病株幼叶小且向下卷曲,甚至整片幼叶黄化。植株早期被感染表现矮化。在发生黄脉曲叶病的黄秋葵田间,其病株率高达60%以上。用烟粉虱传双生病毒简并引物对随机采集的病样进行PCR检测,从这些病样中均能扩增出1条预期大小为570 bp的特异片段;基因克隆及测序分析结果表明,与该特异片段同源的均属双生病毒科菜豆金色花叶病毒属病毒DNA,其中与木尔坦棉花曲叶病毒（Cotton leaf curl Multan virus, CLCuMV）分离物G6相似性最高,为99%。这些研究结果表明,广东黄秋葵黄脉曲叶病中存在烟粉虱传双生病毒,该病害可能也是由CLCuMV侵染引起的。     

近10年我国烟粉虱发生为害及防治研究进展

烟粉虱是我国重要农业害虫,近10年来我国烟粉虱发生为害呈现以下特点:烟粉虱的优势生物型/隐种由B型更替为Q型;传播植物病毒(如黄化曲叶病毒、番茄褪绿病毒)成为了烟粉虱重要的为害方式;烟粉虱抗药性问题逐渐突出;化学农药尤其新烟碱类杀虫剂的广泛使用是Q型烟粉虱取代B型烟粉虱的关键因素。烟粉虱的综合防控措施日益完善,抗药性治理与非化学防控措施受到重视,但是依靠农药的现状并未完全改变。本文综述了近10年来我国烟粉虱的发生为害及防治方面的概况,探讨了今后烟粉虱防控方面的研究内容。     

粉虱传双生病毒的TAS-ELISA及PCR快速检测

 利用粉虱传双生病毒(WTGs)多克隆抗体及单克隆抗体,建立了三抗体夹心ELISA (TAS-ELISA)检测WTGs的方法,并发现了单克隆抗体SCR18可广泛用于我国WTGs的检测。利用根据WTGs基因组上基因间隔区及外壳蛋白基因保守序列设计的引物,建立了PCR特异检测WTGs的方法。对田间病样的TAS-ELISA和PCR检测表明,粉虱传双生病毒在云南省烟草、番茄和南瓜上均存在,2种方法的测定结果是一致的。     

国家种质广州甘薯圃病毒种类鉴定及分析

为明确引起国家种质广州甘薯资源圃中病毒病的病毒种类及优势种,为甘薯种质安全保存提供支持,2017年从甘薯资源圃中未脱毒更新的盆栽苗和大田苗中采集155份具有不同病毒病症状的甘薯资源样品,利用PCR和RT-PCR检测技术对这些样品进行了17种病毒的分子检测.155份样品均有病毒检出,包括甘薯羽状斑驳病毒Sweet pot...     

稻曲病菌薄壁分生孢子制备的影响因素分析

本文研究了培养基、温度、振荡速度等因素对稻曲病菌Ustilaginoidea virens薄壁分生孢子产孢量的影响。结果表明,稻曲病菌薄壁分生孢子在培养第7天基本达到最大孢子量;该菌最适宜产孢的培养基为马铃薯煮汁,在煮汁中添加蔗糖可大幅提高产孢量;适宜的产孢温度为26~28℃;静止培养不利于产孢,振荡培养有利于产孢,并表现为转速越高产孢量越多;光照条件对产孢量没有影响。     

First identification of a sweet potato begomovirus (sweepovirus) in Uganda: characterization,detection and distribution

Sweet potato begomoviruses diverge basally from all other begomoviruses and have been named sweepoviruses. In 2009, a sweepovirus was detected for the first time in sweet potato crops in Uganda by using the indicator plant Ipomoea setosa and generic primers in a polymerase chain reaction (PCR). An isolate was cloned and sequenced, the first fully sequenced genome of a sweepovirus from mainland Africa. At the nucleotide level, this isolate differed from other sweepoviruses by at least 13%, discriminating the Ugandan isolate as a new species which has been tentatively named Sweet potato leaf curl Uganda virus (SPLCUV). In infected sweet potato plants, SPLCUV showed an uneven distribution; it was detected more often in samples from the midrib and lamina of middle and lower leaves, and reversion to healthy tissue occurred, especially in shoots of cv. New Kawogo. This appears to be the first report of resistance to a sweepovirus in sweet potato. While it was only detected at relatively low efficiency by PCR, use of I. setosa plants as an indicator of sweepovirus infection in sweet potato plants was as efficient as using real‐time quantitative PCR (qPCR). Storage of dry leaves for 84 days and dried DNA extracts for 21 days did not affect the ability of PCR and qPCR to detect it. Sweepovirus(es) was detected frequently using generic primers in cultivars Ejumula, New Kawogo and 318L in eastern and central Uganda.     

转Bt基因棉和常规棉对烟粉虱生长发育和繁殖的影响

在实验室恒温和大田自然条件下，通过对转Bt基因棉国抗22和常规棉亲本泗棉3号的对比试验研究，探讨两种棉花对烟粉虱生长发育和繁殖的影响。结果表明：28℃恒温条件下，在花铃期棉花上，国抗22上的B型烟粉虱发育历期（从卵到成虫羽化）比常规棉亲本泗棉3号短17．79％、存活率高4．5％、产卵量高39．62％、雌虫寿命长12．14％、内禀增长率rm大20．18％；在苗期棉花上，国抗22上的B型烟粉虱发育历期比泗棉3号短14．14％、雌虫寿命长17．46％、rm大1．47％，存活率和产卵量差异不显著。在大田自然变温条件下，国抗22上烟粉虱发育历期比泗棉3号短13．6％。在同一品种棉花上，饲养在苗期棉花上烟粉虱的发育历期较花铃期棉花长。结果显示，花铃期棉花比苗期棉花更有利于烟粉虱的生长发育和繁殖；与常规棉亲本相比，转Bt基因棉花上烟粉虱的种群扩增速率更快。     

Optimized loop-mediated isothermal amplification assay for <Emphasis Type="Italic">Tomato leaf curl New Delhi virus-[potato]</Emphasis> detection in potato leaves and tubers

Apical leaf curl disease of potato is caused by a whitefly transmitted begomovirus, Tomato leaf curl New Delhi virus-[potato] (ToLCNDV-[potato]) in India. Detection of this virus is essential to manage the disease, particularly in healthy potato seed production systems. Large scale testing of micro-plants demands a simple, rapid and sensitive assay. Hence, loop-mediated isothermal amplification (LAMP) method was developed for specific detection of ToLCNDV-[potato]. Six primers that recognize the coat protein gene sequence of ToLCNDV-[potato] were designed and LAMP assay was optimized using different concentrations of magnesium sulphate, betaine, dNTPs, Bst DNA polymerase and temperature. The results were assessed by visual observation of turbidity, colour change using SYBR green dye and also by gel electrophoresis. The assay successfully detected the virus in infected plants collected from potato fields whereas no cross-reactions were observed with healthy plants and other potato viruses. The optimized assay was as sensitive as PCR assay and could detect up to 0.002 pg of total DNA. The assay could detect the virus in infected potato tubers and also in asymptomatic plants. Print-capture LAMP assay was developed and its application could reduce the cost and time of the assay in large scale testing under seed production.     

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。     

A more sensitive and rapid multiplex RT-PCR assay combining with magnetic nanobeads for simultaneous detection of viruses in sweet potato

An improved multiplex RT-PCR assay combined with magnetic nanobeads (MNB-RT-PCR) was developed for simultaneous detection of four sweet potato viruses, Sweet potato virus G (SPVG), Sweet potato feathery mottle virus (SPFMV), Sweet potato virus C (SPVC) and Sweet potato chlorotic fleck virus (SPCFV). Four primer pairs specific for each virus were designed and the corresponding PCR products were 169, 357, 516 and 900 bp in length for SPVG, SPFMV, SPVC and SPCFV, respectively. The specificity of the method was tested using different combinations of virus templates, and the identities of the amplification products were confirmed by sequencing. The limits of detection for all four viruses by single and multiplex MNB-RT-PCR assays were comparable. The assay was further evaluated using laboratory and field samples compared with a conventional CTAB-RT-PCR assay, and the comparative results showed that the MNB-RT-PCR assay was more rapid and sensitive. These results suggest that the multiplex MNB-RT-PCR assay is an effective and preferable method for virus detection in sweet potato.     

Detection and molecular characterization of viruses associated with tomato yellow leaf curl disease in cucurbit crops in Jordan

In this study, Tomato yellow leaf curl Sardinia virus (TYLCSV) and the strains Israel and Mild of Tomato yellow leaf curl virus (TYLCV-IL, TYLCV-Mld) were detected for the first time in four cucurbit crops in Jordan by nested polymerase chain reaction (nPCR). These viruses cause the tomato yellow leaf curl disease (TYLCD) in tomato. Cucumber, squash, melon and watermelon plants inoculated with TYLCV-IL[JO:Cuc], TYLCV-Mld, TYLCSV-IT[IT:Sar:88] and the Jordanian isolate of TYLCV (TYLCV-JV) did not show disease symptoms. However, virus-specific fragments were detected in uppermost leaves of symptomless plants by nPCR. A whitefly transmission test showed that Bemisia tabaci could transmit TYLCV-Mld from cucumber into tomato and jimsonweed plants. However, all infected tomato plants remained symptomless. In addition, results of semi-quantitative PCR (sqPCR) analysis showed that the relative amount of TYLCV-Mld DNA acquired by B. tabaci from cucumber plants was less than that acquired from tomato plants.     

Development of simplified and rapid nucleic acid release protocol for PCR based detection of <Emphasis Type="Italic">Potato viruses</Emphasis>

A simple, cost-effective and rapid viral nucleic acid release (NAR) buffer suitable for RT-PCR based diagnostic assay was developed for the detection of potato viruses. The NAR buffer and commercially available RNA isolation kit were compared for RT-PCR based assay, where an amplicon of expected size (~380 bp) targeting PVY was observed in both isolations indicating that it can be used in RT-PCR based diagnostic assays. The same was further validated for its repeatability by running across more than hundred suspected potato leaf samples collected from different sources where, it showed consistent results for the presence of PVY indicating its reliability. The NAR buffer assay was examined for its sensitivity in comparison with the kit based isolation where both the assays were able to detect even up to 10^?5 dilution without affecting the sensitivity. NAR buffer was found stable up to 28 days at -20 °C and for 14 days at 4 °C without losing PCR sensitivity. The assay was also found effective to release the nucleic acid from potato leaves, thrips and aphids for PCR and RT-PCR based detection of DNA viruses like ToLCNDV-potato and other RNA viruses. The developed protocol is simple, less laborious, time-saving (10-15 min) and economical (1/100th of kit) as compared to kit based protocol. The assay can be adopted in diagnostic laboratories for detection of RNA/DNA viruses from potato plants and in thrips as well.     

美人蕉黄斑驳病毒巢式PCR检测方法的建立

本文以我国台湾进境美人蕉病株为材料,根据已报道的美人蕉黄斑驳病毒Canna yellow mottle virus(CaYMV)基因序列设计2对特异性引物(外侧引物1对、内侧引物1对),建立了巢式PCR快速检测CaYMV的方法,并对进境的50份美人蕉样品进行了检测。结果显示,该方法特异性强,且灵敏度高于常规PCR,是常规PCR的1 000倍,表明该方法能够实现对CaYMV的快速、准确、灵敏检测,适用于口岸快速检测CaYMV。