A蛋白碱性磷酸酯酶的标记及酶联测试

以戊二醛为交联剂,采用两步法将Ａ蛋白及碱性磷酸酯酶标记,获得的酶标记物可稀释１／２０００,酶联对香石竹斑驳病毒,马铃薯Ｘ病毒、南方菜豆花叶病毒,烟草环斑病毒的提纯制剂及病汁液检测,分别达到２０ｎｇ和稀释１０＾－２￣１０＾－５。而阴性对照维持在较低的水平。     

香石竹脉斑驳病毒的研究——Ⅰ.病毒的生物学、病理学变化及血清学

 香石竹脉斑驳病毒的寄主范围比较狭窄,只能侵染石竹科、藜科、苋科等几个科中的少数几种植物。昆诺藜和美国石竹是该病毒理想的鉴别寄主和繁殖寄主。病毒粒体为微弯曲的线条状,长790nm,宽12nm,能为国外提供的香石竹脉斑驳病毒抗血清均一包被。该病毒能被桃蚜以非持久性的方式传播,传播效率可达58%。本文还对病组织的超薄切片、病毒侵染组织中的双链RNA、病毒的体外抗性进行了研究。     

香石竹脉斑驳病毒的研究——Ⅰ.病毒的生物学、病理学变化及血清学

香石竹脉斑驳病毒的寄主范围比较狭窄,只能侵染石竹科、藜科、苋科等几个科中的少数几种植物。昆诺藜和美国石竹是该病毒理想的鉴别寄主和繁殖寄主。病毒粒体为微弯曲的线条状,长790nm,宽12nm,能为国外提供的香石竹脉斑驳病毒抗血清均一包被。该病毒能被桃蚜以非持久性的方式传播,传播效率可达58%。本文还对病组织的超薄切片、病毒侵染组织中的双链RNA、病毒的体外抗性进行了研究。     

香石竹环斑病毒的提纯及抗血清制备

选用豇豆黑种三尺作繁殖寄主,接种４周后采收病叶,采用聚乙二醇沉淀,差速离心和１０～４０％蔗糖梯度离心,得到提纯的香石竹环斑病毒,紫外吸收呈典型核蛋白吸收曲线,电镜下见到大量球状病毒粒体。采用多种途径免疫家兔获得的抗血清,琼脂双扩散测得效价为１∶１６     

香石竹环斑病毒的分子检测

试验具体研究了两类不同性质的ｃＤＮＡ探针对香石竹环斑病毒的检测效果。毒源由中国农业大学植物病毒室提供，繁殖于苋色藜Ｃｈｅｎｏｐｏｄｉｕｍａｍａｒａｎｔｉｃｏｌｏｒ上，提纯该病毒，然后抽提其核酸ＲＮＡ，将ＲＮＡ多聚腺苷化后，以Ｏｌｉｇｏ（ｄＴ）为引物，反转录合成ｃＤＮＡ，补平后与载体连接，转化大肠杆菌，获得阳性克隆，用３２Ｐ和光敏生物素分别标记ｃＤＮＡ制作探针，与ＣＲＳＶ及其ＲＮＡ进行点杂交，结果表明，两类不同性质的探针在检测ＲＮＡ水平上具有相同的灵敏度，其检测极限均达到１ｎｇ／ｍｌ，并对二类探针进行了比较。     

香石竹斑驳病毒单克隆抗体的制备及检测应用

 用香石竹斑驳病毒(CarMV)免疫的BALB/c鼠脾细胞与Sp2/0鼠骨髓瘤细胞融合,经筛选克隆,获得5株能稳定传代且分泌抗CarMV单克隆抗体(MAb)的杂交瘤细胞,并分别制备它们的单克隆抗体腹水。5株单克隆抗体腹水间接ELISA效价达10^-6,其中3G1、1B9、2A9和2F8的抗体类型及亚类均为IgG1,而2F₂为IgG3。Western-blot分析表明,5株单克隆抗体均与CarMV 38 kD的外壳蛋白亚基有特异性反应。利用2A9单抗建立的抗原包被的间接ELISA (ACP-ELISA)检测CarMV方法,病叶1:800倍稀释、提纯CarMV病毒浓度为1 ng/mL (绝对检测量为0.1 ng)时仍能检测到病毒。利用ACP-ELISA对田间香石竹样品的检测表明,CarMV在香石竹上发病很普遍。     

苹果褪绿叶斑病毒PBM1（李假痘）毒株的提纯、抗血清制备及ELISA检测

从木本植物分离的苹果褪绿叶斑病毒（ａｐｐｌｅｃｈｌｏｒｏｔｉｃｌｅａｆｓｐｏｔｖｉｒｕｓ，Ｔｒｉｃｈｏｖｉｒｕｓ组）的ＰＢＭ１毒株能引起李品种“Ｈａｕｓｚｗｅｔｓｈｅ”的假痘病，将其繁殖在昆诺藜（Ｃｈｅｎｏｐｏｄｉｕｍｑｕｉｎｏａ）上。感染ＰＢＭ１毒株的昆诺藜叶组织用两次蔗糖梯度离心提纯。每１００ｇ叶组织的病毒产量平均为１．６８ｍｇ。在电镜下可观察到弯曲状病毒颗粒上的螺纹结构。通过ＳＤＳ－聚丙烯酰胺凝胶电泳分析，病毒外壳蛋白的分子量为２１．５ｋＤａ。用提纯的病毒免疫家兔制备的抗血清和ＥＬＩＳＡ法，对感染ＰＢＭ１的昆诺藜叶片和桃花进行了灵敏的检测。     

侵染唐菖蒲的烟草环斑病毒的研究

 1987年3月,从荷兰引进的唐菖蒲球茎,在Saxany品种上,选取症状为斑驳的病株,经鉴定是烟草环斑病毒侵害的结果。通过病毒寄主范围的测定、它可侵染豆科、茄科、藜科、葫芦科、番杏科中的17种植物。摩擦接种在菜豆"Pinto"、"家雀旦";豇豆"黑种三尺","黑眼",按种叶出现局部坏死斑,系统生长点坏死。大豆"猴子毛","晋豆84"局部坏死斑,顶枯。在苋色藜及昆诺阿藜上,接种叶局部坏死斑,无系统症状。普通烟White Buriey,接种叶局部退绿环斑,系统退绿环斑和橡叶。
番杏、二青黄瓜接种叶局部退绿斑,系统花叶。
病毒的致死温度为65℃,稀释终点10-⁴,体外存活期6-7天(室温)。病毒粒体为等轴对称,直径约28nm。琼脂双扩散其沉淀线与标准的TRSV沉淀线完全相接,说明其抗原性相同。病毒外壳蛋白由一种蛋白亚基构成,分子量约58000道尔顿。     

花生矮化病毒(PSV)的研究

 1983年7月,从山东薛城的花生上分离到一个病毒分离物PS-34,以汁液摩擦接种测定了9科48种植物,PS-34可侵染6科15种植物,在苋色藜上表现系统花叶。由桃蚜、豆蚜以非持久性方式传病。体外抗性测定,失毒温度50-55℃,稀释限点10^-3-10^-4,体外存治期3-4天.提纯后经电镜观察,病毒粒体呈球形,直径±29nm.提纯病毒的抗血清和花生矮化病毒日本株系(PSV-J)的抗血清交互测定,都与PS-34有明显的沉淀线反应。故将病毒分离物PS-34鉴定为黄瓜花叶病毒组的花生矮化病毒(PSV)。因苋色藜和昆诺藜上表现为系统花叶,与在寄主反应上明显不同.因此,确定其为一种新的花生矮化病毒PSV-C株系.     

侵染蚕豆的芜菁花叶病毒的鉴定与提纯

 在江苏浙江等地的蚕豆苗上出现的红褐色环斑或白色斑驳症状.经鉴定是芜菁花叶病毒侵害的结果.在病组织中的病毒粒体为弯杆状.长度750-800nm。摩擦接种在苋色藜上,接种叶上出现大块黄色枯斑,直径约3毫米,顶叶系统感染,在克氏烟、芜菁、油菜上为系统花叶.在蚕豆苗上的症状,视品种和环境条件而异,在有限生长的品种上多为红褐色环斑.在启东蚕豆上多为白色斑驳,豆荚上也可出现变色斑。对病毒的提纯方法作了改进,用冻融的20%蔗糖溶液可代替常规的10-40%蔗糖梯度液,精提纯的效果相同。     

A new aubergine disease caused by a whitefly‐borne strain of Tomato mild mottle virus (TomMMoV)

The aims of the present study were to further characterize the causal agent of a new viral disease of aubergines in Israel, first observed in 2003 and tentatively named eggplant mild leaf mottle virus (EMLMV) in a previous work, and to identify the vector responsible for its spread. The disease could be transmitted mechanically from infected source plants to healthy aubergines or laboratory test plants. Transmission electron microscopy (TEM) analysis of purified virus preparations indicated the presence of viral particles with a flexible filamentous morphology (approximately 720 nm long). TEM analysis of ultrathin sections prepared from infected leaf tissue revealed the presence of cytoplasmic inclusion bodies with pinwheel and crystalline structures, typical of those induced by potyviral infection. The viral coat protein subunit was shown to have a molecular weight of 37·5 kDa by SDS‐PAGE analysis. The viral particles reacted positively in western blot analysis with an antiserum against Tomato mild mottle virus (TomMMoV) from Yemen, described as a potyvirus, vectored by the aphid Myzus persicae. The current study describes some biological properties of EMLMV and presents evidence for its transmission by the whitefly Bemisia tabaci, but not by three aphid species. The taxonomic relationship between EMLMV and TomMMoV is discussed based on their biological characteristics and sequence analysis of their genomes. It is suggested that the Israeli EMLMV should be considered a distant strain of TomMMoV, designated TomMMoV‐IL, according to the present rules of Potyviridae molecular taxonomy.     

Occurrence of a variant of cucumber green mottle mosaic virus in ISRAEL

A virus was isolated from naturally infected melons and squirting cucumber(Ecballium elaterium (L.) Rich) plants collected in the northern part of Israel. Some data are presented on the biological, serological and physical properties of the virus, which was identified as a variant of cucumber green mottle mosaic virus.     

Characterization and detection ofCucumber green mottle mosaic virus in Greece

The host range specificity of Greek isolates ofCucumber green mottle mosaic virus (CGMMV) was determined and the ability of the virus to retain its infectivity in naturally contaminated soil, after storage at 4°C for at least 10 months, was established. An immunocapture RT-PCR protocol was developed for the detection of the virus and proved to be 10⁵ times more sensitive than DAS-ELISA and 10² times more sensitive than F(ab’)₂-ELISA using F(ab’)₂ fragments that were prepared from an antiserum, which was raised against a local virus isolate. In order to clarify dissimilarities that were revelaed between the coat protein (CP) genes of the Greek isolates after restriction mapping of their respective PCR products, the CP genes of three isolates were sequenced and found to be very similar but not identical to the CP gene of CGMMV-SH. http://www.phytoparasitica.org posting Aug. 27, 2002.     

Characterization of a carlavirus from dandelion (Taraxacum officinale)

A carlavirus was isolated from leaves of a dandelion plant raised in the experimental garden of the Hugo de Vries Laboratory in Amsterdam. The virus was readily sap-transmissible and infected 24 out of the 52 plant species and cultivars tested, with visible symptoms in 18 of them.Myzus persicae andCuscuta subinclusa (dodder) did not transmit the virus. In addition the virus was not seed-transmitted in dandelion. Dilution end-point was 10^–5, thermal inactivation occurred at between 80–85°C and longevity in vitro was approximately 24h. The virus had a sedimentation coefficient of 136 S. Polyacrylamide gel electrophoresis of the coat protein gave two bands, consisting of proteins with molecular masses ranging from 37 000 to 34 300 Da (band I) and from 34 000 to 32 800 Da (band II). The molecular mass of the RNA was 2.84 x 10⁶ Da. The average buoyant density of the virus was 1.306 gcm^–3 and the average A₂₆₀/A₂₈₀ ratio 1.16. The virus particles had a normal length of 668 nm. with the light microscope, large mainly vacuolate inclusions were observed in the epidermal cells of infectedNicotiana cleavelandii leaves. In ultra-thin sections of systemically infected leaves ofN. clevelandii, bundles of aggregated virus particles were detected, whereas in infected dandelion leaves there were fewer aggregates and more scattered virus particles. There was a close serological relationship to dandelion latent virus, chrysanthemum virus B and potato virus S and a more distant one to carnation latent virus, elderberry carlavirus,Helenium virus S and potato virus M. The occurrence of the virus was found to be restricted to dandelion plants in the experimental garden in Amsterdam. On the basis of large differences in host range, symptomatology and lack of transmission byM. persicae it was decided that the virus could not be considered a strain of either dandelion latent virus, chrysanthemum virus B or potato virus S. We therefore propose that it be called dandelion carlavirus.Samenvatting Een carlavirus werd geïsoleerd uit een paardebloemplant, die opgekweekt was in de proeftuin van het Hugo de Vries-Laboratorium in Amsterdam. Het virus kon gemakkelijk met sap worden overgebracht en was in staat 24 van de 52 getoetste plantesoorten en-cultivars te infecteren, waarbij op 18 van deze symptomen zichtbaar werden.Myzus persicae en warkruid (Cuscuta subinclusa) konden het virus niet overbrengen. Evenmin kon het virus met zaad van geïnfecteerde planten van paardebloem overgaan. De verdunningsgrens was 10^–5, de inactiveringstemperatuur 80–80°C en de houdbaarheid in vitro ongeveer 24 uur. Het virus had een sedimentatiecoëfficiënt van 136 S. Polyacrylamide-gelelektroforese van het manteleiwit resulteerde in twee banden, bestaande uit eiwitten met molecuulmassa's die varieerden van 37000 tot 34 3000 Da (band I) en van 34 000 tot 32 800 Da (band II). De molecuulmassa van het RNA was 2,84×10⁶Da. De gemiddelde zweefdichtheid van het virus bedroeg 1,306g cm^–3 en de gemiddelde A₂₆₀/A₂₈₀ verhouding was 1,16. Het virus had een normale lengte van 668 nm. In de epidermiscellen van geïnfecteerde bladeren vanNicotiana clevelandii werden met de lichtmicroscoop insluitsels met draderige en vacuoleachtige structuren waargenomen. In ultradunne coupes van systemisch geïnfecteerde bladeren vanN. clevelandii waren bundels geaggregeerde virusdeeltjes zichtbaar. In geïnfecteerde bladeren van paardebloem werden daarentegen meer verspreid voorkomende virusdeeltjes gevonden en minder aggregaten. Het virus vertoonde een sterke serologische verwantschap met het dandelion latent virus, chrysantevirus B en aardappelvirus S; er was een geringe verwantschap met het latente anjervirus, het carlavirus van vlier, Helenium virus S en het aardappelvirus M. Het vóórkomen van het virus bleek beperkt te zijn tot paardebloemen in de proeftuin in Amsterdam. Gezien de grote verschillen in waardplantenreeks, symptomatologie en overdracht metM. persicae hebben we gemeend, dat het virus niet slechts als een stam kon worden beschouwd van hetzij het dandelion latent virus, hetzij het chrysantevirus B en het aardappelvirus S. We stellen voor de naam carlavirus van paardebloem aan dit virus te geven.     

A Cucumber mosaic virus isolated from Momordica charantia L.

Momordica charantia L. plants systemically infected with Cucumber mosaic virus (CMV) were found in Oita Prefecture. The virus isolated from the host plant was characterized by biological, serological, and molecular biological methods. The purified virus was used to mechanically inoculate the host and produced green mottle, green mosaic, and/or chlorotic spots in the noninoculated upper leaves of the host. The virus was identified as an isolate of CMV containing genomic RNA3 derived from subgroup IA by several lines of evidence based on electron microscopy, serological detection, host range, symptoms, and the entire nucleotide sequence of RNA3.     

Patterns in disease progress and the influence of single and multiple viral infections on pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) growth

The patterns and progress of disease caused by multiple infections of Cucumber mosaic virus (CMV), Pepper mild mottle virus (PMMoV) and Pepper mottle virus (PepMoV) and their effects on growth of pepper plants (Capsicum annuum L.) were investigated in this study. Each virus induced distinct symptoms, but more severe symptoms, including reduced growth rates, were observed when pepper plants were simultaneously infected by more than one virus. When CMV was included in multiple viral inoculations, co-inoculations and sequential inoculations, PepMoV and PMMoV symptoms were observed but the symptoms characteristic of CMV were not masked, even though CMV titre did not increase greatly. In multiple viral infections, PepMoV titre and CMV did not increase significantly, but PMMoV titre gradually increased in most cases. Growth rates of pepper plants were greatly reduced during the 30 to 40-day post-inoculation period under both single-infection and multiple-infection conditions, but multiple viral infections of CMV pre-inoculated peppers were affected to a greater extent. A significant reduction in fruit size and fruit number was observed in single and multiple viral inoculations, and fruit malformation rates were high in CMV single-infection and multiple viral infections with CMV.     

Broad bean mottle virus in Morocco; curculionid vectors,and natural occurrence in food legumes other than faba bean (Vicia faba)

Broad bean mottle virus (BBMV) was transmitted from infected to healthy faba-bean plants by the curculionid weevilsApion radiolus Kirby,Hypera variabilis Herbst,Pachytychius strumarius Gyll,Smicronyx cyaneus Gyll, andSitona lineatus L. The latter appeared to be an efficient vector: acquisition and inoculation occurred at the first bite, the rate of transmission was c. 41%, and virus retention lasted for at least seven days.S. lineatus transmitted the virus from faba bean to lentil and pea, but not to the three genotypes of chickpea tested. This is the first report on the generaHypera, Pachytychius, andSmicronyx as virus vectors, and onA. radiolus, H. variabilis, P. strumarius, andS. cyaneus as vectors of BBMV.Out of 351 samples of food legumes with symptoms suggestive of virus infection, 16, 11, 19, and 17% of the samples of chickpea, lentil, pea, and common bean, respectively, were found infected when tested for BBMV in DAS-ELISA. This is the first report on the natural occurrence of BBMV in chickpea, lentil, pea, and common bean. The virus should be regarded as a food-legume virus rather than a faba-bean virus solely, and is considered an actual threat to food legume improvement programmes.     

Impact of <Emphasis Type="Italic">Erysiphe alphitoides</Emphasis> on transpiration and photosynthesis in <Emphasis Type="Italic">Quercus robur</Emphasis> leaves

Oak powdery mildew, (Erysiphe alphitoides) causes one of the most common diseases of oaks. We assessed the impact of this pathogen on photosynthesis and water relations of infected leaves using greenhouse-grown oak seedlings. Transpiration of seedlings infected by oak powdery mildew was also investigated. Altogether, E. alphitoides had a low impact on host gas exchange whether at the leaf or whole plant scale. Maximal stomatal conductance of infected leaves was reduced by 20–30% compared to healthy controls. Severely infected seedlings did not experience any detectable change of whole plant transpiration. The reduction in net CO₂ assimilation, A_n, was less than proportional to the fraction of leaf area infected. Powdery mildew reduced both the maximal light-driven electron flux (J_max) and the apparent maximal carboxylation velocity (Vc_max) although Vc_max was slightly more impacted than J_max. No compensation for the infection occurred in healthy leaves of partly infected seedlings as the reduced photosynthesis in the infected leaves was not paralleled by increased A_n levels in the healthy leaves of the seedlings. However, E. alphitoides had a strong impact on the leaf life-span of infected leaves. It is concluded that the moderate effect of E. alphitoides on oak might be related to the small impact on net CO₂ assimilation rates and on tree transpiration; nevertheless, the severe reduction in leaf life-span of heavily infected leaves may lead to decreased carbon uptake over the growth season.