斜纹夜蛾核型多角体病毒的ELISA定量检测分析

本研究采用ELISA间接法检测试验,对纯化的斜纹夜蛾核型多角体病毒和五龄、六龄幼虫、蛹及卵块内所含的病毒进行了定量检测。当检测纯化病毒样品时,吸光值(OD_(450))与浓度值对敷值(LnC)有显著的直线相关关系,标准曲线的直线方程为:Y=8.1367X+0.7608(相关系数,r=0.9928)。其结果与生物测定方法比较无显著差异(P<0.01)。本试验所能测出的最低浓度值为:1630PIBs/ml,即27.38ng。而普遍采用的血球计数法所能测出的最低浓度值比这个结果高出近1000倍。当检测感染病毒的五龄、六龄幼虫样品,幼虫体内所检出的病毒含量与血球计数法基本相同(0.01相似文献   

用ELISA检测人血清中茶毛虫核型多角体病毒

用间接ELISA法,以109份人血清作为抗原及抗体,与茶毛虫核型多角体病毒及其抗血清进行检测,以确定茶毛虫核型多角体病毒对人群的安全性,结果均呈阴性反应。试验说明,茶园应用茶毛虫核型多角体病毒杀虫剂对人群无害。     

甘蓝夜蛾核型多角体病毒杀虫剂防治水稻害虫效果及增产作用

昆虫病毒杀虫剂核型多角体病毒在生物农药杀虫剂中占有重要地位。在我国农业部推荐使用的杀虫剂名录中,核型多角体病毒杀虫剂品种占有很大份额。经过多年的推广使用,核型多角体病毒杀虫剂已经越来越为广大农户所接受和喜爱。甘蓝夜蛾核型多角体病毒杀虫剂为中国科学院武汉病毒研究所张忠信研究员所发现分离的毒株,由江西省新龙生物科技有限公司进行批量生产。为研究甘蓝夜蛾核型多角体病毒悬浮剂（康邦TM）在水稻上的杀虫增产效果,     

核型多角体病毒的杀虫作用及其田间应用方法

核型多角体病毒杀虫剂具有特异性强、不易产生抗药性、安全、无害、后效作用明显等优点,然而,在田间实际应用中也存在杀虫谱窄、杀虫速度慢、受自然因素影响较大等限制因素.结合国内、外已开展的核型多角体病毒生物特性和田间应用研究情况,对核型多角体病毒杀虫剂的杀虫特点和应用技术进行了总结与阐述,并提出了田间应用技术措施.     

亚洲玉米螟核型多角体病毒分离株鉴定及其对草地贪夜蛾的室内毒力测定

本文对从湖北省恩施州亚洲玉米螟幼虫上新分离的一种广谱病毒进行了形态学和分子特征鉴定,并以该病毒与另一种广谱杆状病毒对草地贪夜蛾幼虫进行了生物活性测定。结果表明新分离的核型多角体病毒与薄荷灰夜蛾核型多角体病毒和芹菜夜蛾核型多角体病毒具有较高的同源性,但遗传距离分析不支持它与这两种病毒为同一个种。生物活性测定表明,分离的病毒对草地贪夜蛾3龄幼虫的半数致死剂量是甘蓝夜蛾核型多角体病毒的3.86倍。本研究的结果为开发防治草地贪夜蛾的高效病毒杀虫剂提供了依据。     

棉铃虫核型多角体病毒分子生物学和基因工程研究进展

棉铃虫核型多角体病毒(HaSNPV)是棉铃虫专一性病原物,隶属于杆状病毒科核型多角体病毒属.对其分子生物学和基因工程的研究主要包括以下几个方面:测定了HaSNPV G4株和C1株基因组核苷酸全序列,并与其它病毒进行了同源性比较;研究了HaSNPV部分基因的结构、转录、表达及其功能.构建了HaSNPV Bac to Bac杆状病毒表达系统;重组病毒杀虫剂的研究为HaSNPV大面积防治棉铃虫展示了广阔的前景.随着棉铃虫核型多角体病毒分子生物学和基因工程研究的不断深入,重组病毒杀虫剂将在棉铃虫综合防治中发挥更为重要的作用.     

PCR法证实棉铃虫核型多角体病毒对棉铃虫经卵和蛹的垂直传播

根据棉铃虫核型多角体病毒的多角体蛋白基因设计5’引物和3’引物，通过PCR技术成功扩增389bp的目的片段，并对该片段进行了克隆和测序。结果表明，扩增片段为棉铃虫核型多角体病毒多角体蛋白基因序列。该检测方法的灵敏度为10fg，应用此技术从带毒的虫卵和蛹的总DNA中也检测到该病毒的存在。该方法从分子水平上证明了病毒的垂直传递。     

基于流式细胞术的黄瓜霜霉病菌孢子囊计数研究

 为病原菌时空流行动态监测及病害预测模型构建提供病原菌快速计数方法,以卵菌中代表——葫芦科霜霉病病原菌古巴假霜霉菌(Pseudoperonospora cubensis)为例,研究了孢子囊悬浮液的荧光染色方法和基于流式细胞术计数方法。研究表明EDTA和SYBR Green Ⅰ 的联合作用能够对孢子囊进行荧光标记。对SYBR Green Ⅰ 染色剂的最佳染色条件进行研究,结果表明在染色时间相同时,荧光染色剂浓度越高,染色效果越好;同一浓度荧光染色剂,随着染色时间的增加,孢子囊染色率增加。使用流式细胞术对荧光染色的孢子囊进行计数,并与显微镜血球计数板计数结果进行比较,结果显示:流式细胞仪计数结果与显微镜血球计数板计数结果得到的孢子囊浓度没有显著差异(P<0.01),对两种方法获得的孢子囊浓度取对数后进行相关性分析,显示流式细胞仪测定的孢子囊浓度与显微镜血球计数板计数得到的孢子囊浓度高度相关,相关系数为 0.993 4,表明流式细胞术可以应用于古巴假霜霉菌孢子囊的计数。     

昆虫病毒与苏云金杆菌混用研究应用进展

利用昆虫病毒防治农林害虫是生物防治的一种重要手段,用于害虫生物防治的昆虫病毒主要是杆状病毒科的核型多角体病毒(NPV)、质型多角体病毒(CPV)、颗粒体病毒(GV)和痘病毒科的昆虫痘病毒(EPV).     

复合型苜蓿银纹夜蛾核型多角体病毒制剂

复合型苜蓿银纹夜蛾核型多角体病毒制剂主要由苜蓿银纹夜蛾核型多角体病毒和斜纹夜蛾核型多角体病毒两株病毒毒株和苏云金杆菌按一定比例复合而成,经单一型,复合型病毒制剂毒力测定比较,获得增效比值为５．１－９．５的最佳配比,并以甜菜夜蛾为宿扩大增殖形成雏型产品。     

花椒凤蝶核型多角体病毒的研究

从花椒凤蝶(Papilio xuthus Linnaeus)幼虫中分离到一株核型多角体病毒,其多角体平面图象为四、五边形或近圆形,直径1.6—4.0微米,病毒粒子杆状,微弯,两端圆滑,约360×45毫微米。室内和野外在自然条件下试验结果表明,此株病毒对花椒凤蝶幼虫有较强的致病力,对三龄幼虫的LC_(50)为1.4×10~(4.2)多角体/毫升,y=2.48+0.61x。野外防治效果可达85％以上,好于常用化学农药乐果和马拉硫磷。此株病毒对家蚕、柞蚕和瓢虫无致病作用。     

Insects and Mites

Abstract

During August 1986 when there was a build up of Parasa lepida Cramer on coconut in the study area in Kerala, India, many dead larvae were found on the underside of the leaves. A few of these cadavers which were suspected to be infected by a virus were collected. Pathogenicity tests proved positive. PIBs were negative to stain by Giemsa, and section of the polyhedra shows that the virions are rod shaped. The pathogen is a multiple embedded baculovirus.     

The Extraction and Diagnosis of Corynebacterium sepedonicum from Diseased Potato Tubers1

Corynebacterium sepedonicum causes a very severe disease in potato and it is therefore necessary for the method of its diagnosis to be specific and for the extraction technique to ensure that even small amounts of bacteria will be found. By using indirect immunofluorescence staining it was possible to get a reliable diagnosis. For this purpose a specific serum was produced. To find the best technique for extracting the bacteria from potato tubers, two methods were compared. One was based on maceration of the plant material in a Waring blender followed by centrifugation. Immunofluorescence staining was done on the resuspended sediment. The other method was carried out by cutting the potatoes into small pieces (about 1 cm3) and transfering them to a bottle containing sterile water. The bottle was shaken for 3 or 16 h and samples for immunofluorescence tests were taken from the fluid. Centrifugation could be used if necessary before immunofluorescence staining. The results show that the most reliable technique for extraction was to shake the plant material for 16 h followed by centrifugation. If there are heavy symptoms, dilution of the extracted material may be necessary.     

Electron microscope observations of nuclear polyhedra fromMalacosoma neustria (Lepidoptera: Lasiocampidae

The nuclear polyhedral bodies fromMalacosoma neustria are enclosed within a membrane. The diameter of the nuclear polyhedra varies from 0.9 to 2.8 with an average of 1.8 . In the nuclear polyhedra the rod-like virus particles occur both singly and in bundles. The single virus rods are enclosed within two membranes, namely the intimate membrane and the developmental membrane. The virus rods which occur in bundles have an intimate membrane just like the single virus rods, whereas the developmental membrane encloses the whole bundle. The virus rods are closely packed by the intimate membrane and between the intimate and the developmental membrane is a space. The diameter of the virus rods without membranes, determined from sectioned polyhedra, is about 25 m and the length 250 m.     

Weakening and Delayed Mortality of Fusarium oxysporum by Heat Treatment: Flow Cytometry and Growth Studies

ABSTRACT Survival of Fusarium oxysporum f. sp. niveum following heat treatments was studied using flow cytometric, physiological, and microscopic assays. We exposed germinating conidia to sublethal temperatures from 36 to 42 degrees C for 60 min, followed by rhodamine 123 staining and flow cytometry, and found increasing levels of fluorescence that reflect a change in mitochondrial membrane potential, indicating a weakening induced by stress. Viability of conidia or germinating conidia of the fungus exposed to heat decreased with increasing temperature, as assessed by fluorescent staining. However, viability was higher than that assessed with the 5-day-long plate count method and was further reduced 13 and 24 h after treatment, suggesting delayed mortality of the heat-treated germinating conidia. Delayed mortality was substantiated by observing these conidia with light and fluorescent scanning electron microscopy and by subculturing single germinating conidia that had been previously heated. Programmed cell death was not observed in heat-treated conidia or germinating conidia of F. oxysporum based on the detection of plasma membrane phosphatidylserine translocation, cell-cycle measurements, detection of DNA fragmentation, or microscopic observation of apoptotic bodies. We hypothesize that propagules, which survived the heating and apparently are alive, may undergo further irreversible detrimental processes, eventually leading to their death by yet unidentified mechanisms. These findings suggest that pathogen propagules also might be affected under lower temperatures, possibly facilitating pathogen control by heating.     

Ralstonia solanacearum preferential colonization in the shoot apical meristem explains its pathogenicity pattern in tomato seedlings

Ralstonia solanacearum causes a lethal bacterial wilt disease in many plants by colonizing the vascular tissues of the hosts. Upon inoculation of tomato seedlings through either leaf or root, the wilting symptoms occur first at the apical region and then proceed downward along the shoot. The systemic order of the disease initiation and progression in the host, independent of the site of pathogen inoculation, is yet to be investigated. To understand the disease progression more clearly, we have carried out a systematic study of the pathogen localization by GUS staining of inoculated tomato seedlings, at 24-hour intervals from 0 days post-inoculation (dpi) to 5 dpi. In both inoculation methods, pathogen colonization was observed at 1 dpi at the apical meristem as well as the cotyledon leaves, where the disease initiates. As the disease progressed, colonization by the pathogen towards the lower region of the shoot was observed. Disease consistency and pathogenicity magnitude were observed to be higher using the leaf inoculation method than the root inoculation method. Several R. solanacearum transposon-induced mutants that were reduced in virulence by root inoculation but virulent by leaf inoculation were obtained. Using GUS staining, it was observed that these mutants were unable to localize in the shoot region when inoculated in the root. Our study indicates that the apical meristem and the cotyledon leaves are the first regions to be colonized in inoculated tomato seedlings, which might explain the disease initiation from this region.     

Sensitivity of different methods for the detection of Ralstonia solanacearum in potato tuber extracts

The sensitivities of various methods for the detection of Ralstonia solanacearum following dilution in healthy potato tuber tissue macerate were compared. Estimated pathogen populations in undiluted macerates, from samples of 200 heel-end vascular cores each containing a single diseased and 199 healthy tubers, ranged from 1.2 × 10⁶–7.4 × 10⁷ colony-forming units per ml. Following concentration by high-speed centrifugation and resuspension in phosphate buffer, the pathogen was detected by all methods studied, including culture on semi-selective media, enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescent-antibody staining (IFAS) of fixed cells, immunofluorescent colony staining (IFCS), detection of specific DNA sequences following amplification by the polymerase chain reaction (PCR) and bioassay in tomato seedlings. Both ELISA and PCR methods were improved by pre-enrichment of samples in semi-selective broth prior to testing. A nested PCR method was evaluated which could detect fewer than 10 cells per ml in the potato extracts. Of the other methods only dilution plating on semi-selective medium and tomato bioassay could detect fewer than 10⁴ cells per ml. In order to combine ease and speed of use with sensitive detection, it was recommended that a series of methods be used for routine screening of potato tuber stocks for infection by R. solanacearum.     

甜菜夜蛾核型多角体病毒青岛分离株SeMNPV-QD的毒力测定

为评价一株新的甜菜夜蛾核型多角体病毒（青岛分离株SeMNPV-QD）在害虫生物防治中的应用潜力,本文以甜菜夜蛾核型多角体病毒青岛株SeMNPV-QD与美国株SeMNPV-US1为材料,分别测定了病毒对甜菜夜蛾细胞系的感染、对甜菜夜蛾幼虫的室内生物活性以及田间防治效果。研究结果表明,SeMNPV-QD与SeMNPV-US1对甜菜夜蛾细胞系Se-3的感染率分别为92.34%和93.65%,平均每个细胞的病毒多角体产量分别为23.97和24.10 PIB,差异均不显著;SeMNPV-QD与SeMNPV-US1对初孵甜菜夜蛾幼虫的LC₅₀分别为4.43×10⁴和4.35×10⁴ PIB/mL,LT₅₀分别为4.12和4.02 d;SeMNPV-QD与SeMNPV-US1对4龄甜菜夜蛾幼虫的LC₅₀分别为9.25×10⁵和4.44×10⁵ PIB/mL,LT₅₀分别为6.20和5.50 d;SeMNPV-QD和SeMNPV-US1对大葱田甜菜夜蛾幼虫具有较好的防治作用,7 d的校正防效分别达到74.99%和79.04%。研究结果将为新病毒株的深入研究开发以及甜菜夜蛾的生物防治提供理论依据。     

斑点免疫金和免疫金/银染色法检测烟草环斑病毒

在硝酸纤维素膜上进行的斑点免疫胶体金检测烟草环斑病毒技术已经建立.免疫金染色检测提纯病毒的灵敏度为25ng,检测病汁液可稀释10倍.使用免疫金/银染色则灵敏度更进一步提高.