基因芯片在百合无症病毒检测中的应用

设计百合无症病毒、植物18SrRNA基因的特异扩增引物和各种对照的探针，将探针固定在醛基化玻璃片基上，完成植物基因芯片制备。提取百合种球总RNA，经RT-PCR扩增相应区段并用Cy3dCTP进行标记，用标记的PCR产物与芯片杂交，扫描仪对杂交结果进行扫描，GenePixProd．0软件对杂交图像进行分析。结果从荷兰进口的百合种球中检测出百合无症病毒，证明了该实验研制的植物病毒基因芯片的准确性和灵敏性。     

Control of the spread of tulip breaking virus in tulips with mineral-oil sprays

Several mineral-oil sprays used to curtail the spread of stylet-borne tulip breaking virus (TBV) in tulips ‘Elmus’ were tested. The similarly concentrated sprays prepared with summer oil, winter oil, Albolineum, and Asepthion oil, decreased the spread of TBV considerably. Control was improved by the more concentrated Albolineum sprays (2.5, 5, 10%), and spread was reduced more effectively, when variable quantities of emulsions providing good leaf coverage were used (2.5, 5%). The weight ratios of the bulb yields of plots given a 2.5% spray in all years and a 5% spray in 1972 and 1973 fluctuated closely (0–6%) around the value for the untreated plots, which was taken as 100. These ratios dropped by 11–19% after more concentrated sprays were used in variable quantities in 1971. Spraying was slightly more effective at weekly than at fortnightly intervals, but the weight ratios scarcely differed. Better control of TBV spread was obtained when spraying was started at the beginning of May; when started in June, the sprays were not effective. The weight ratios were not clearly influenced differentially except when spraying was begun in the first week of May. The efficacy of mineral-oil sprays is discussed in relation to tulips and lilies, with reference to comparable experiments. The application of mineral-oil sprays for curtailing TBV spread in commercial tulip culture is discussed.     

北京地区百合无症病毒CP基因的克隆与分析

以感染百合无症病毒(LSV)的百合叶片为材料,提取总RNA为模板,通过反转录聚合酶链式反应(RT-PCR)扩增出856bp大小的LSV CP基因片段.经Blast比对发现,该基因片段与Genbank上发表的ISV CP基因序列(DQ294655.1)同源性为97.31%.由该序列推导出的氨基酸序列与其相似度为98.6%,仅存在某些氨基酸的差异.经过聚类分析表明,该CP基因的氨基酸序列与厦门和兰州分离的病毒遗传距离较近,而与海宁和广州分离的病毒遗传距离较远.     

百合无症病毒对百合光合生理、酶活性及叶绿体超微结构的影响

 以东方百合“西伯利亚”为试验材料,研究百合无症病毒(LSV)侵染百合对其叶片生理生化以及叶绿体超微结构的影响。检测结果表明:叶片中叶绿素a、b以及总叶绿素含量与健康对照相比分别下降了28.6%、33.3%和23.5%,净光合速率、气孔导度及胞间CO₂浓度分别下降33.3%、25%和13.8%;超氧化物歧化酶(SOD)、过氧化物酶(POD)、多酚氧化酶(PPO)和苯丙氨酸解氨酶(PAL)与健康对照相比,分别增加了16.6%、29.4%、16.7%和22.2%。电镜观察发现:感病植株叶绿体膨胀变形,基质片层散乱,叶绿体内淀粉粒肿大且数目增多,从而证明LSV侵染破坏叶绿体结构,影响植株的光合作用。     

Mechanical transmission of a strain of tulip breaking virus fromlilium longiflorum to chenopodium spp

A virus, mechanically transmitted fromLilium longiflorum toChenopodium spp., was identified in morphological, serological and ultrastructural studies as tulip breaking virus (TBV). Data presented indicate that the isolate differs from other TBV strains in host range and ultrastructural pathology.     

Detection of tobacco rattle virus in different parts of tulip by ELISA and cDNA hybridisation assays

Different parts of tulips cv. Apeldoorn were assayed for the presence of tobacco rattle virus (TRV) by means of ELISA, cDNA hybridisation and immuno-electron microscopy. Assays were periodically performed during the growing season and upon storage of the bulbs, During the growing season in the field the relative TRV concentrations detected by ELISA and cDNA were highest mainly in the basal stem-parts and basal leaf-parts, respectively. When, during storage, infected bulbs were divided into a number of sections, TRV could be detected only in some of the sections, irrespective of the test used. However, nearly all sprouts of infected bulbs, stored at 5°C for 7 months, appeared to contain detectable amounts of TRV upon testing with ELISA and cDNA. Thus, testing of sprouts may offer a possibility to develop a routine test for TRV in tulip bulbs in due course.Samenvatting Verschillende delen van tulp cv. Apeldoorn werden getoetst op de aanwezigheid van tabaksratelvirus (TRV) met behulp van ELISA, cDNA-hybridisatie en immuno-elektronemicroscopie. Tijdens het groeiseizoen en de bewaring van de bollen werden regelmatig toetsingen uitgevoerd. Gedurende het groeiseizoen op het veld werden de relatief hoogste TRV concentraties voornamelijk gevonden in het basale deel van de stengel en het okselgedeelte van het blad met respectievelijk ELISA en cDNA-hybridisatie. TRV bleek gelokaliseerd aanwezig te zijn in een of meer stukjes van een gedeelde bol, onafhankelijk van de gebruikte toetsmethode. Bijna alle spruiten van geïnfecteerde bollen die gedurende 7 maanden bij 5°C bewaard waren, bleken bij het toetsen met behulp van ELISA en cDNA aantoonbare hoeveelheden van TRV te bevatten. Het toetsen van spruiten biedt de mogelijkheid te zijner tijd een routinetoets voor TRV in tulpebollen te ontwikkelen.     

郁金香和百合主要病害、病原及鉴定方法

郁金香和百合是最重要的百合科球根花卉。病害是制约郁金香和百合产业健康发展的重要因素。本文介绍了国内为害郁金香和百合的主要病害和病原。国内为害郁金香和百合的主要病原真菌有镰刀菌、灰霉和炭疽菌;主要病原细菌有萎蔫短小杆菌、果胶杆菌属和迪基氏属软腐细菌;主要病毒有郁金香碎色病毒、百合斑驳病毒、百合无症病毒和黄瓜花叶病毒等RNA病毒。主要植物病原线虫有短体线虫、茎线虫和滑刃线虫。线虫取食伤害郁金香和百合的根、鳞茎和芽,促进病原真菌和细菌对植物的复合侵染;毛刺属和拟毛刺属线虫除侵害郁金香和百合,还作为介体向植物传播烟草脆裂病毒。为害郁金香和百合的线虫,南芥菜花叶病毒、草莓潜隐环斑病毒和番茄环斑病毒等RNA病毒是与郁金香和百合相关的主要进境检疫性有害生物。本文也介绍了鉴定这些病原的常规方法和新方法,为建立郁金香和百合主要病原的检测检疫体系提供技术理论支持。     

检测兰花病毒的斑点酶联法的建立

本文建立了齿兰环斑病毒（Odntoglossum ringspot virus,ORSV）和建兰花叶病毒（Cymbidium mosaic virus,CyMV）两种兰花主要病毒的斑点酶联法检测技术（Dot-ELISA）。用Dot-ELISA对提纯病毒和田间病叶进行检测,结果表明;检测ORSV和CyMV提纯病毒的灵敏度分别为0.82μg/mL和0.244μg/mL;病叶汁液的稀释倍数分别为1280倍和2650倍。该方法具有操作简便、成本低等优点,适合于兰花生产中的病毒检测。     

Evolutionary Characterization of Two Lily Isolates of Cucumber mosaic virus Isolated in Japan and Korea

We analyzed the evolutionary histories of two lily strains of Cucumber mosaic virus (CMV) isolated in Japan and Korea (HL- and Ly2-CMVs). They share common biological characteristics in that their host ranges are very restricted perhaps from a unique adaptation to lily plants. Although HL and Ly2 were isolated independently from different lily species in separate countries, their RNA3 sequences had a very high sequence similarity (97%). The evolutionary relationships between the two isolates were characterized by comparing their phylogenetic trees for the 3a and CP genes. The two lily CMVs always formed a distinct cluster within subgroup IB in 3a, but within IA in CP. Together, the phylogenetic tree topology and the sequence identity between the two lily CMVs suggest that they evolved from a common progenitor. Received 5 November 2001/ Accepted in revised form 11 January 2002     

南方菜豆花叶病毒酶联试剂盒的测评

南方菜豆花叶病毒酶联检测试剂盒,采用直接双抗体夹心法。主要由已包被特异性的酶联板、碱性／磷酸酯酶标记的抗体、对硝基苯磷酸盐底物以及浓缩清洗液等几部分组成。该试剂盒的检测灵敏度可达８ｎｇ／ｍｌ提纯病毒。检测大豆病叶可达１：１０００。检测大豆病种子可达１：１００,与黄瓜花叶病毒,马铃薯Ｙ病毒,烟草环斑病毒,烟草花叶病毒呈阴性反应,同时选用大柬告示５和阴性对组,ＯＤ值读数均较低,背景反应好,未出现假阳性     

Purification and some properties of pea leafroll virus

Pea leafroll virus (PeLRV) was purified from infected pea plants using polyethylene glycol precipitation of crude extracts followed by clarification with chloroform-butanol, differential centrifugation and density-gradient centrifugation. Higher yields of virus were obtained from roots than from shoots. The A260/A280 was 1.83. Particles were isometric with a diameter of 27 nm in preparations stained with uranyl acetate.An antiserum with a homologous titre of 1/512 in gel-diffusion tests was prepared. PeLRV was precipitated by antisera to soybean dwarf virus and beet western yellows virus but did not react with antiserum to a New Zealand strain of barley yellow dwarf virus.Polyacrylamide gel electrophoresis of disrupted virions revealed a single RNA component with a relative molecular mass of about 2.4×10⁶ and a single polypeptide with a relative molecular mass of 30–35×10³.Samenvatting Erwtetopvergelingsvirus (PeLRV) werd gezuiverd uit geïnfecteerde erwteplanten met behulp van polyethyleenglycolprecipitatie uit ruwe extracten, gevolgd door klaren met chloroform en butanol, differentiëel centrifugeren en scheiden op dichtheidsgradiënten. Uit wortels werden hogere virusopbrengsten verkregen dan uit scheuten (Fig. 1). Het virus bereikte een evenwicht in één band bij evenwichtscentrifugering in Cs₂SO₄ (Fig. 2). De zweefdichtheid bedroeg 1,32×10³ kg/m³. De A260/A280 was 1,83 (Fig. 3). In met uranylacetaat gekleurde preparaten waren de deeltjes isometrisch met een diameter van 27 nm (Fig. 4).Er werd een antiserum bereid dat een homologe titer van 1/512 in de geldiffusietoets had. PeLRV reageerde zowel in de immune density-gradient-toets (Fig. 5) als in de gel-diffusietoets met antisera tegen soybean dwarf virus en slavergelingsvirus (beet western yellows virus), maar het reageerde niet met een Nieuwzeelandse stam van het gerstevergelingsvirus.Polyacrylamide-gelelektroforese van gedissocieerd virus toonde de aanwezigheid aan van één RNA-component met een relatieve moleculaire massa van 2,4×10⁶ en één eiwitcomponent met een relatieve moleculaire massa van 30–35×10³ (Fig. 6).Guest research worker, Plant Diseases Division, DSIR, Private Bag, Christchurch, New Zealand. With financial support of the International Agricultural Centre, Wageningen, and the Ministry of Education and Science, The Hague.     

Brown ring formation and streak mottle,two distinct syndromes in lilies associated with complex infections of lily symptomless virus and tulip breaking virus

Brown ring formation in bulbs of lilies, particularly of Mid-century hybrids, is described as a newly recognized disease. Symptoms of streak mottle in cultivars ofLilium speciosum Thunb., not associated with abnormalities of bulbs, are briefly described with reference to the literature. Sometimes the two syndromes occur in the same crop such as in the Mid-century hybrid Enchantment, showing brown ring formation in bulbs and an indistinct mottling in field plants. Severe leaf mottling appears in Midcentury hybrids andL. speciosum when plants are forced under glass. In both diseases tulip breaking virus (TBV) was always found to occur in complex with lily symptomless virus (LSV), which was consistently detected in apparently healthy plants of the Mid-century hybrid Enchantment and in severalL. speciosum cultivars.The part of TBV involved in the complex diseases described has been demonstrated by serological, electron-microscopical, and inoculation studies with lilies and tulips. LSV was sap-transmitted from lily to tulip but it could not be detected in several randomly taken samples of a dozen field-grown tulip cultivars. Suppression of TBV in plants of Enchantment, grown from bulbs with brown ring formation under field conditions, is discussed. TBV was serologically and electron-microscopically detectable only in plants grown under glass. A similar phenomenon was observed inL. speciosum cultivars under both conditions.Samenvatting Bruinkringerigheid in bollen van Midcentury-hybriden wordt beschreven als een nieuw onderkende virusziekte (Fig. 1, 2 en 3). Streperige vlekkerigheid op bladeren vanL. speciosum cultivars (Fig. 4), niet tegelijkertijd optredend met bolafwijkingen, wordt in het kort beschreven, en deed zich voor zoals in de literatuur wordt vermeld.Soms treden de twee syndromen op in hetzelfde gewas zoals in de Midcentury-hybride Enchantment, die bruinkringerigheid in bollen laat zien en een onduidelijke vlekkerigheid in veldplanten. Ernstige vlekkerigheid op de bladeren komt naar voren in Midcentury-hybriden enL. speciosum wanneer de planten in de kas worden gebroeid.Bij de beschreven ziekten was het complex van tulpemozaïekvirus (syn.: tulpebloembrekingsvirus; TBV) en symptoomloos lelievirus (LSV) steeds aanwezig. Het laatstgenoemde virus werd steeds aangetoond in ogenschijnlijk gezonde planten van de Midcentury-hybride Enchantment en van verscheidene cultivars vanL. speciosum.Het aandeel van het TBV in de beschreven complexe ziekten bleek uit serologisch en elektronenmicroscopisch onderzoek en uit inoculatieproeven met lelies en tulpen (Tabel 1 en 2). LSV werd met sap overgebracht van lelie naar tulp (Fig. 5 en 6). LSV kon niet worden aangetoond in verscheidene willekeurig genomen monsters uit planen met mozaïeksymptomen op de bladeren van een twaalftal te velde geteelde tulpecultivars. In planten met bruinkringerigheid te velde werd een teruglopen van de concentratie van het TBV waargenomen. TBV was serologisch en elektronenmicroscopisch lechts aantoonbaar in lelie-planten die in de kas groeiden. Een overeenkomstig verschijnsel werd waargenomen inL. speciosum-cultivars.     

Purification and some properties of Papaya meleira virus, a novel virus infecting papayas in Brazil

'Meleira', or 'sticky disease', is currently the most damaging papaya disease in the mid-eastern Brazilian growing regions. Consistent disease transmission via latex injection, presence of similar isometric particles in the laticiferous vessels of diseased plants, and detection of double-stranded DNA in naturally and experimentally infected papaya trees suggest that a virus is the causal agent. Conclusive evidence for viral aetiology was previously lacking, mostly because every attempt to purify the putative virus from infected papayas had failed. Following the successful purification and partial characterization of the meleira virus, healthy papaya seedlings injected with purified virus particles later developed typical symptoms of the disease. Negatively stained, isometric, full and 'empty' purified virus particles measured 42 and 38 nm, respectively. The viral genome was a single dsRNA molecule of about 12 kbp. Several capsid proteins, ranging in size from 14·4 to 45 kDa, were consistently revealed by PAGE. Papaya meleira virus (PMeV) appears to represent a novel group of viruses, with no known similar counterpart among known plant-, vertebrate-, invertebrate- or prokaryote-infecting viruses.     

苜蓿花叶病毒ELISA检测方法的建立及其在大豆病毒调查和抗性资源鉴定中的应用

苜蓿花叶病毒(alfalfa mosaic virus, AMV)是一种世界性分布、宿主范围广、具有严重危害性的植物病毒,能引起大豆的严重病害。本研究利用原核表达的AMV CP蛋白制备的抗血清,建立了高效、准确的AMV间接ELISA检测方法,并应用于病害调查和抗性鉴定,结果表明制备的3份抗血清对重组蛋白和AMV感染的大豆植物粗提液的效价均达到256 000倍,血清特异性分析结果显示3份抗血清仅识别感染AMV的大豆叶片,不识别感染大豆花叶病毒(soybean mosaic virus, SMV)的大豆叶片。通过建立的AMV间接ELISA与常规RT-PCR同时对采集的50份疑似感染AMV的大豆样品进行检测,有46份样品检测结果一致,符合率达92%。利用建立的AMV ELISA方法和课题组已建立的SMV ELISA方法对吉林省大豆主产区的大豆样品进行病毒检测的结果表明,病毒检出率为38.30%,SMV的检出率达30.85%,AMV的检出率达17.06%,复合侵染率为9.61%。对接种AMV的40个大豆品种进行抗性鉴定,结果显示40份大豆全部感染AMV,但是病毒载量存在差异,部分品种表现出AM...     

Detection of tobacco rattle virus by ELISA and test plants in main sprouts of tulip bulbs during storage at different temperatures

The detectability of tobacco rattle virus (TRV) in the main sprouts of primarily and secondarily infected tulip bulbs of cv. Apeldoorn stored at different temperatures from the lifting in July up to February is described. Detection by ELISA was not affected by the size of the main sprout, nor by the size of the bulbs. The rates of TRV-infected bulbs found by ELISA were highest during storage at 13, 9 or 5°C continuously, and when temperatures were lowered from 20 or 17°C to 5°C in October. The percentages detected via test plants, but undetectable by ELISA were also lowest at these temperatures. The unfavourable effect of continuous storage at 20, 17, or 2°C as expressed in low ELISA absorbances not significantly different from the mean value of healthy bulbs, was largely overcome during long storage by the change of temperature down to 5°C from 20 and 17°C or upwards from 2°C. The reverse from 5°C upwards to 17 and 20°C affected the detectability by ELISA unfavourably. The rate of detection via test plants in the main sprout and in the small sprouts from different positions in bulbs was only possible at low percentages.The effect of some factors, like different temperatures during storage, detectability of different TRV serotypes, interference of irregular occurrence of TRV in the removed scale and basal-plate tissue with the main sprout, and variable recurrence of TRV in progeny bulbs, is discussed in view of its impact on routine testing of bulbs during storage.     

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     

Control of air-borne field spread of tulip breaking virus,lily symptomless virus and lily virus X in lilies by mineral oils,synthetic pyrethroids,and a nematicide in the Netherlands

The inhibitory effect on the spread of viruses in lilies viz., tulip breaking virus (TBV; nonpersistently aphid-borne, potyvirus,) lily symptomless virus (LSV; non-persistently alphidborne, carlavirus), and lily virus X (LVX; potexvirus of unknown etiology), was studied of brands of mineral oil (Luxan oil H and Duphar-7E oil) and synthetic pyrethroid insecticides (l-cyhalothrin and deltamethrin), and a nematicide (aldicarb) in crops in which virus-infected plants were present as virus sources. The spread of TBV and LSV were controlled by sprays of mineral oil and insecticide, while that of LSV was also limited by the soil-applied nematicide. The spread of LVX was reduced by the insecticides and, not effectively by the mineral-oil spraying, by which data the mode of transmission may be presumed to be by an insect in the persistent or semi-persistent manner.Mixtures of mineral oil and pyrethroid were more effective in the reduction of spread of TBV and LSV than either components tested alone. The mineral oil was the most effective component in the mixtures in which pyrethroid added a slight extra effect. The addition of pyrethroid did not mask either the lower efficacy of the oil brand Duphar-7E oil, or the diminished inhibitory effect of low dosages of oil. The normal rate of mineral oil gave similar control to that of a mixture of mineral oil at half rate plus the pyrethroid at full dosage. Low rates of oil, or even synthetic pyrethroids alone may be used on cultivars which suffer of the loss of bulb weight by the use of normal or decreased rates of oil. Weekly sprays were more effective than fortnightly sprays. The rate of control by the weekly sprays ranged between 90 and 95% for Luxan oil H at half dosage plus the full rate of pyrethroid. Weekly sprayed synthetic pyrethroids alone onto the virus sources and the plants to be infected gave 60–70% control. The weight ratios tended to be slightly reduced if the half dosage of the efficient Luxan oil H was used. Factors which affect the control of the air-borne field spread of viruses by mineral oils and synthetic pyrethroid insecticides in lilies are discussed.     

Plum pox virus detection in dormant plum trees by PCR and ELISA

An immunocapture polymerase chain reaction (IC-PCR) protocol and ELISA were compared for their effectiveness in detecting plum pox virus (PPV) in dormant plum material. Although the IC-PCR was about one thousand times more sensitive than ELISA, PPV was detected by ELISA in 71–80% of bark samples collected in December, January and March 1996/97 from pot-grown rootstock trees inoculated with PPV the previous March, compared with 85–86% detection in the same samples by IC-PCR. In similar samples from one-year-old shoots taken from infected branches of orchard trees, 66–81% were positive by ELISA compared with 81–87% by IC-PCR. With bulked samples taken from the fibrous roots of the pot-grown trees, PPV was detected in 92–100% of samples by IC-PCR in winter compared with only 38–65% by ELISA. These results were confirmed in samples from the roots and shoots of the same trees in 1997/98. Three samples per shoot would have been sufficient to detect PPV by ELISA in 87 of the 88 infected shoots tested during the two winters. However, infected shoots are irregularly distributed in diseased trees and PCR assays of root samples offer the potential for improving the reliability of identifying trees infected with PPV.