截形叶螨对阿维菌素的抗性遗传方式研究

采用室内盆栽试验研究了截形叶螨(Tetranychus truncates Ehara)抗性品系(RR)对杀菌剂阿维菌素的遗传方式。结果表明,截形叶螨抗性种群的杂交F1代(SS♀×RR♂和RR♀×SS♂)的显性度D值分别为0.479和0.636,均在0 D1范围内,表明抗性由不完全显性基因控制;两个D值(DSR和DRS)95%置信限有重叠,并且经t检验,两个D值不存在显著差异(P0.05),证明截形叶螨对阿维菌素的抗性遗传受母体或核外效应的影响不大,其抗性基因在常染色体上;回交F2代两个种群的(BC1SR和BC1RS)的实际死亡率和期望值经χ2检验,均差异显著,证实由多基因控制。     

褐飞虱对环氧虫啶的抗性选育和遗传方式

为明确褐飞虱Nilaparvata lugens对环氧虫啶的抗性遗传方式, 进而指导田间合理用药。经过34代选育获得抗环氧虫啶褐飞虱品系(CYC-R), 其抗性水平相较于敏感品系(CYC-S)提高了102.42倍。抗性遗传方式分析结果表明, 环氧虫啶对CYC-R和CYC-S的正、反交后代(F1RS和F1SR)的毒力无显著性差异, 且正、反交后代显性度分别为0.33和0.34, 表明褐飞虱对环氧虫啶的抗性为常染色体、不完全显性遗传。自交和回交的后代(F2RS、F2SR和F2BC)经环氧虫啶处理后的实际死亡率与期望值经卡方检验, 均差异显著, 表明褐飞虱对环氧虫啶的抗性由多基因控制。因此, 一旦褐飞虱田间种群对环氧虫啶产生抗性, 环氧虫啶的使用寿命将会缩短, 抗性治理难度较大, 建议采用预防性抗性治理策略。     

褐飞虱对甲胺磷的抗性遗传

室内抗性筛选表明，褐飞虱对甲胺磷抗性的发展呈不规则的“S”形，第5代前抗性上升很慢，第5～15代间迅速上升，第15代后上升再次变慢。利用选育的抗甲胺磷褐飞虱品系和敏感品系，研究了褐飞虱对甲胺磷的抗性遗传形式。结果显示，正交和反交子代的显性度D值分别为0．5130和0．4673，表明抗性主效基因是不完全显性遗传的。利用剂量对数-死亡机率值曲线分析法，初步判断抗性遗传不是单基因控制的；卡方(χ^2)测验进一步证实，抗性遗传可能是两个或两个以上的基因控制。抗性筛选全过程和F5～F13的现实遗传力(h^2)分别为0．2190和0．2457，说明褐飞虱对甲胺磷抗性产生的速率较快，出现高抗性的风险较大。     

土耳其斯坦叶螨对杀螨剂的抗性选育及解毒酶活力变化

为探索土耳其斯坦叶螨的抗药性及其生化机理,在室内对敏感系土耳其斯坦叶螨分别用螺螨酯、甲氰菊酯和阿维菌素逐代处理,选育出抗性种群。结果表明,选育至15代,土耳其斯坦叶螨对螺螨酯、甲氰菊酯和阿维菌素的抗性指数分别达到268.63、37.98和112.68倍。分别测定敏感品系(SS)、抗螺螨酯(RS)、抗甲氰菊酯(RF)、抗阿维菌素(RA)品系的解毒酶活性显示,3种不同抗性品系相对SS品系的羧酸酯酶(CarE)、谷胱甘肽S-转移酶(GSTs)和多功能氧化酶(MFO)的比活力均有不同程度的提高,差异均达到显著水平(P0.05)。其中,RF品系的MFO比活力上升最快,是SS品系的12.7倍;RA品系的MFO比活力次之,是SS品系的5.76倍;RS品系的3种解毒酶比活力均增长较慢,其中CarE比活力上升最慢,是SS品系的1.31倍。由此表明,CarE、GSTs、MFO的活性增大可促进土耳其斯坦叶螨对3种杀虫剂的抗性形成;螺螨酯的抗性增强可能与CarE关系甚微;MFO活性的增加可能与甲氰菊酯抗性升高密切相关;GSTs、MFO的活性升高可能是土耳其斯坦叶螨对阿维菌素产生抗性的主要原因。     

二斑叶螨抗甲氰菊酯、四螨嗪、螺虫乙酯、哒螨灵品系的微卫星DNA分析

采用微卫星标记法,对二斑叶螨（Tetranychus urticae Koch）敏感品系（S）和室内筛选的4个抗性品系（抗甲氰菊酯品系、抗四螨嗪品系、抗螺虫乙酯品系、抗哒螨灵品系）进行微卫星DNA多态性分析。结果表明,二斑叶螨4个抗性品系显示出微卫星DNA 多态性。引物Tu11在相似系数0.80处将5个二斑叶螨品系区分开。引物Tu1在相似系数0.70处将5个二斑叶螨品系区分开。微卫星DNA多态性虽不能定位抗性基因,但可结合聚类分析软件,为二斑叶螨对甲氰菊酯、四螨嗪、螺虫乙酯、哒螨灵抗性快速分子检测技术提供依据,指导二斑叶螨田间抗药性监测及抗药性治理。     

二斑叶螨对螺螨酯抗药性及对18种杀螨剂交互抗性

采自甘肃兰州兴隆山公园的二斑叶螨(Tetranychus urticae Koch),用雌雄单系培养敏感品系(S),用螺螨酯处理二斑叶螨种群培养抗性品系(SP R),用室内生测法对二斑叶螨S和SP R品系进行室内毒力测定。结果表明,二斑叶螨对螺螨酯抗性发展初期较慢,中期稳定,后期较快,选育至26代抗性指数(RI)达58.83。SP R对甲氰菊酯、氯氰菊酯有一定的交互抗性,RI分别为11.54和10.03;对苯丁锡、四螨嗪、苦皮藤生物碱、阿维菌素、氯氟氰菊酯、哒螨&#8226;四螨嗪、哒&#8226;水胺硫磷、三唑锡、三氯杀螨醇、哒螨灵、氧化乐果无交互抗性(1＜RI<5.00);对浏阳霉素、毒死蜱、噻螨酮、柴油、哒螨灵、唑螨酯可能存在负交互抗性(RI<1)。     

吡螨胺和哒螨灵亚致死剂量对截形叶螨的亚致死效应

通过吡螨胺（tebufenpyrad）和哒螨灵（pyridaben）的亚致死剂量处理截形叶螨（Tetranychustruncatus Ehara）成螨和螨卵,研究了两药剂对实验种群的亚致死效应。结果表明：吡螨胺和哒螨灵处理后当代成螨的每雌日均产卵量均有不同程度的降低,日平均产卵量、卵孵化率均低于对照; F1的卵期、幼螨期、若螨期延长显著,净增殖率低于对照、内禀增长率和周限增长率与对照差异不大。两药剂处理卵后,卵期、幼螨期、若螨期延长,净增殖率低于对照。吡螨胺和哒螨灵的亚致死剂量处理截形叶螨成螨和螨卵都无刺激增殖的作用,能延缓种群的发育速度。     

河南和宁夏朱砂叶螨、截形叶螨对3种药剂的抗性检测

以玻片浸渍法检测河南和宁夏两地区朱砂叶螨(Tetranychus cinnabarinus)和截形叶螨T.truncatus对3种药剂的抗性。结果表明,河南的朱砂叶螨和截形叶螨对三氯杀螨醇、久效磷表现抗性;对氧化乐果仍处在敏感水平。宁夏的两种叶螨对3种药剂都表现敏感。同时,截形叶螨对3种药剂的LC₅₀均高于朱砂叶螨。大田防治时应减少三氯杀螨醇的使用次数。截形叶螨为优势种时,禁止使用久效磷防治。提倡氧化乐果在防治棉花叶螨和兼治棉蚜时应用。     

二斑叶螨抗阿维菌素品系选育及其解毒酶系活力变化

采用室内生物测定和生化分析方法, 以采自甘肃兰州国家级森林公园兴隆山的二斑叶螨为敏感品系(SS), 研究二斑叶螨对阿维菌素的抗药性及抗性生化机理。结果表明：在室内用阿维菌素强化筛选24代, 获得了二斑叶螨抗阿维菌素品系(Ab-R24), 抗性指数(resistance index, RI)为321.5。对SS和Ab-R24解毒酶活性的分析表明, Ab-R24品系体内羧酸酯酶(CarE)、乙酰胆碱酯酶(AchE)、酸性磷酸酯酶(ACP)、碱性磷酸酯酶(ALP)、谷胱甘肽S-转移酶(GSTs)和多功能氧化酶(MFO)活性分别是SS品系的1.43、1.18、1.56、1.48、1.55倍和4.02倍, 差异达到显著水平(P < 0.05), 其中MFO的活性上升最为显著。对SS和Ab-R-24解毒酶动力学常数的分析表明, Ab-R-24品系体内AchE、GSTs和MFO的 Km分别是SS品系的1.14、2.31倍和2.58倍; Vmax分别是SS品系的1.19、2.34倍和1.76倍, 差异均达到显著水平(P <0.05)。说明二斑叶螨对阿维菌素抗性增高与MFO活性快速升高有关, AchE和GSTs也参与阿维菌素抗性的形成。     

桔全爪螨对哒螨灵抗性的选育及其生化机理

模拟田间药剂的选择压力 ,用哒螨灵对桔全爪螨 (Panonychus citri Mc Gregor)逐代处理 ,以选育其抗药性。结果表明 :选育 12代 ,抗性增长到 35.0倍 ;哒螨灵抗性品系对氧乐果、双甲脒、氯氟氰菊酯、水胺硫磷和炔螨特有交互抗性。通过增效剂和离体酶活性测定证明 :桔全爪螨对哒螨灵的抗性主要与谷胱甘肽 S-转移酶、多功能氧化酶和乙酰胆碱酯酶活性的提高有关。     

广东省番茄黄化曲叶病毒的分子鉴定及序列分析

番茄黄化曲叶病毒病是世界番茄生产上一种毁灭性病害,番茄黄化曲叶病毒Tomato yellow leaf curl virus(TYLCV)是引起该病害的主要病原病毒之一。本文采用滚环扩增及基因克隆方法,获得了侵染广东佛山和肇庆番茄的TYLCV 4个分离物全基因组;它们均为2 781 nt,编码6个ORF,其中病毒链上编码AV1和AV2,互补链上编码AC1、AC2、AC3和AC4。同源性比较结果表明,4个广东分离物基因组序列两两间同源性为99%以上;与已报道的TYLCV各分离物同源性在90%以上,而与来自中国不同地区的TYLCV分离物的同源率均在98%以上。系统进化分析显示,广东分离物与来自中国不同地区的TYLCV分离物亲缘关系较近,并聚类在一个分支。因此,侵染引起广东佛山和肇庆番茄黄化曲叶病的病毒应来自国内其他地区。本研究是对TYLCV广东分离物分子特征的首次报道。     

侵染广西番茄的番茄黄化曲叶病毒的分子鉴定

番茄黄化曲叶病毒病是番茄生产上的毁灭性病害,严重影响番茄产量及品质。2019年从广西百色市采集疑似番茄黄化曲叶病叶片样品,采用滚环扩增(RCA)及基因克隆等方法,获得了4个烟粉虱传双生病毒的全基因序列,4个分离物的全长序列分别为2 776、2 781、2 752、2 781 bp,均编码6个开放阅读框;核苷酸相似性比较发现,4个分离物彼此间的相似性均在90%以上,与已报道的番茄黄化曲叶病毒Tomato yellow leaf curl virus (TYLCV)各分离物间的相似性也在91%以上;系统进化分析表明,TYLCV广西分离物BS-2和BS-4与TYLCV-Hunan、BS-1和BS-3与TYLCV-YN6553等分离物处于独立的小分支,说明TYLCV广西分离物与TYLCV-Hunan、TYLCV-YN6553具有较近的亲缘关系。本研究首次报道TYLCV在广西发生。     

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。     

陕西省番茄黄化曲叶病毒基因间隔区缺失突变体的鉴定与不同分离物间群体进化研究

为明确番茄黄化曲叶病毒(tomato yellow leaf curl virus, TYLCV)基因组的变异情况, 分别从陕西省杨凌区?泾阳县?大荔县和阎良区采集了表现为番茄黄化曲叶病症状的抗病品种番茄样品20份以及感病品种番茄样品4份?经TYLCV特异性引物检测, 24个样品均为阳性?通过全长扩增测序获得了15个TYLCV分离物的基因组序列?序列分析显示, 其中7个分离物基因组大小为正常的2 781 bp, 而有8个TYLCV分离物基因组大小为2 768 bp?与正常基因组相比, 这8个分离物在基因间隔区(intergenic region, IR)的TATA-box与保守9核苷酸序列之间发生了13个核苷酸的缺失?对我国不同地区TYLCV分离物全基因组序列进行变异分析, 发现IR区域是发生核苷酸变异最大的区域?系统进化分析表明, 本研究分离到的15个TYLCV分离物均属于TYLCV-Israel进化分支下的中国亚群, 其中8个TYLCV IR缺失突变体与国内已报道的IR缺失突变体BJ04和BJ06亲缘关系较远, 而与本研究中分离得到的正常TYLCV分离物亲缘关系最近, 疑似为陕西地区出现的一类新TYLCV IR缺失突变体?本研究首次对我国不同地区多个TYLCV分离物出现IR区缺失现象进行了报道?     

北京地区番茄黄化曲叶病病毒分离物测定及株系的初步鉴定

 番茄黄化曲叶病毒病是番茄生产中的一种毁灭性病毒病害,2009年传入北京。利用烟粉虱传双生病毒简并引物PA/PB对2010年~2011年采集自北京市5个区县的53个番茄样品进行检测,30个表现典型黄化曲叶病症状的样品均扩增得到约500 bp的特异条带,测定了其中7个样品的部分序列,经序列比对分析表明其为番茄黄化曲叶病毒（Tomato yellow leaf curl virus, TYLCV）。利用TYLCV特异引物TJ-F/TJ-R、TY-F/TY-R对样品BJDXXY、BJFS02、BJFS03、BJMY2231进行TYLCV基因组克隆和序列测定,经分析4个样品携带的TYLCV基因组长度均为2 781碱基,编码6个蛋白。基因组序列比较发现,这4个分离物与TYLCV-Israel株系同源性达到98%以上;通过建立系统发育树,发现BJDXXY、BJFS02、BJFS03与河北分离物（HBLF4）、山东分离物（SDSG）亲缘关系较近,BJMY2231与上海分离物（TYLCV-Israel）、江苏分离物（JSNJ1）亲缘关系较近。     

南疆温室番茄黄化曲叶病病毒种类的分子鉴定

为明确南疆温室番茄黄化曲叶病的病毒种类,利用双生病毒的兼并引物通过PCR扩增,对采集的20个番茄病株进行了分子检测.从20个病株中均扩增到约500 bp的目标片段,对其中4株进行克隆和测序,其相互间序列同源性为97.1％ ～99.3％,与番茄黄化曲叶病毒(Tomato yellow leaf curl virus,TYLCV)的同源性较高,为98.6％ ～ 99.5％.随机选取莎车分离物KS2-5进行全基因组的克隆和测序,KS2-5 DNA全长为2781 nt(序列号:JQ807735),具有典型的双生病毒基因组特征,与TYLCV其它分离物同源性达到98.9％～99.5％,而与其它粉虱传双生病毒的序列同源性较低,为68.3％ ～75.5％,表明危害南疆温室番茄的病毒种类为番茄黄化曲叶病毒TYLCV.     

Pepper (Capsicum annuum) Is a Dead-End Host for Tomato yellow leaf curl virus

ABSTRACT Tomato yellow leaf curl (TYLC) is one of the most devastating pathogens affecting tomato (Lycopersicon esculentum) worldwide. The disease is caused by a complex of begomovirus species, two of which, Tomato yellow leaf curl Sardinia virus (TYLCSV) and Tomato yellow leaf curl virus (TYLCV), are responsible for epidemics in Southern Spain. TYLCV also has been reported to cause severe damage to common bean (Phaseolus vulgaris) crops. Pepper (Capsicum annuum) plants collected from commercial crops were found to be infected by isolates of two TYLCV strains: TYLCV-Mld[ES01/99], an isolate of the mild strain similar to other TYLCVs isolated from tomato crops in Spain, and TYLCV-[Alm], an isolate of the more virulent TYLCV type strain, not previously reported in the Iberian Peninsula. In this work, pepper, Nicotiana benthamiana, common bean, and tomato were tested for susceptibility to TYLCV-Mld[ES01/99]and TYLCV-[Alm] by Agrobacterium tumefaciens infiltration, biolistic bombardment, or Bemisia tabaci inoculation. Results indicate that both strains are able to infect plants of these species, including pepper. This is the first time that infection of pepper plants with TYLCV clones has been shown. Implications of pepper infection for the epidemiology of TYLCV are discussed.     

应用纳米磁珠实时荧光定量PCR方法检测番茄黄化曲叶病毒

In order to develop a rapid, sensitive and specific qPCR assay for detection and quantification of Tomato yellow leaf curl virus (TYLCV), a pair of primers and TaqMan probe were designed according to the conserved sequence of known TYLCV isolates. Combining with MNP technique, a novel MNP-qPCR detection method was established and verified based on specificity, sensitivity and reproducibility tests. The results indicated that the Ct value of plotted standard curve showed good linear relationship(R² =0.9994)with the log of copy number of template. The established method showed a high specificity for TYLCV detection without crossing reaction with Tomato severe leaf curl virus and Tomato yellow leaf curl Sadinia virus, and was 10-fold more sensitive than routine PCR. Both coefficients of variation were less than 2%, indicating a good reproducibility. We have provided a novel method for detection of TYLCV in plant samples rapidly and quantitatively.     

Detection and typing of tomato yellow leaf curl viruses

The denomination Tomato yellow leaf curl virus (TYLCV) comprises several viruses that cause severe damage to tomato crops in warm and temperate regions worldwide. TYLCV viruses are widespread in the Mediterranean Basin, in which two species have been reported: Tomato yellow leaf curl Sardinia virus (TYLCSV) and Tomato yellow leaf curl virus (TYLCV, previously TYLCV-Is). The availability of methods convenient for the diagnosis of these viruses is essential. We have investigated several alternatives for reliable detection and differentiation of TYLCSV and TYLCV. Triple-antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) proved to be very useful for large-scale diagnosis in field situations, but lacked discriminating capacity and sensitivity in the stages of infection in which low virus titre is present. The DNA-based methods are suited to laboratory operations and plant disease clinics, where accuracy of detection and discrimination of viruses is required. Polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) was the most reliable method to discriminate between TYLCSV and TYLCV, but is not suited to high sample turnover. For large-scale testing, tissue print hybridization assay provides a reliable and sensitive alternative to PCR.     

Tomato yellow leaf curl virus in the Dominican Republic: Characterization of an Infectious Clone, Virus Monitoring in Whiteflies, and Identification of Reservoir Hosts

ABSTRACT Epidemics of tomato yellow leaf curl disease (TYLCD) in the Dominican Republic in the early to mid-1990s resulted in catastrophic losses to processing tomato production. As part of an integrated management approach to TYLCD, the complete nucleotide sequence of a full-length infectious clone of an isolate of Tomato yellow leaf curl virus (TYLCV) from the Dominican Republic (TYLCV-[DO]) was determined. The TYLCV-[DO] genome was nearly identical in sequence (>97%) and genome organization to TYLCV isolates from Israel and Cuba. This established that TYLCV-[DO] is a bonafide TYLCV isolate (rather than a recombinant virus, such as isolates from Israel [Mild], Portugal, Japan, and Iran), and provided further evidence for the introduction of the virus from the eastern Mediterranean. A reduction in the incidence of TYLCV in the northern and southern processing tomato production areas of the Dominican Republic has been associated with the implementation of a mandatory 3-month whitefly host-free period (including tomato, common bean, cucurbits, eggplant, and pepper). Monitoring TYLCV levels in whiteflies, by polymerase chain reaction with TYLCV-specific primers, established that the incidence of TYLCV decreased markedly during the host-free period, and then gradually increased during the tomato-growing season. In contrast, TYLCV persisted in whiteflies and tomato plants in an area in which the host-free period was not implemented. Surveys for TYLCV reservoir hosts, conducted to identify where TYLCV persists during the host-free period, revealed symptomless infections in a number of weed species. The implications of these findings for TYLCV management in the Dominican Republic are discussed.