广西三种甜瓜病毒分离物的分子检测与鉴定

瓜类褪绿黄化病毒Cucurbits chlorotic yellows virus(CCYV)、甜瓜黄化斑点病毒Melon yellow spot virus(MYSV)及甜瓜坏死斑点病毒Melon necrotic spot virus(MNSV)是近年来报道侵染瓜类作物的新病毒,在个别种植区大面积发生,对生产构成严重威胁。为了解3种病毒在广西的发生情况,先后到各西甜瓜种植区进行了调查,并采集疑似CCYV、MYSV及MNSV的甜瓜病叶样品,提取病叶总RNA,通过特异性引物分别进行一步法RTPCR扩增,电泳结果显示RT-PCR产物与预期大小一致的序列条带;将扩增产物分别连接到pMD19-T克隆载体上,挑选阳性克隆子进行序列测定及比对分析。结果表明:CCYV、MYSV和MNSV广西分离物的CP基因序列与其他已报道核苷酸序列一致性分别达95.1%~100%、96.5%~99%和83.7%~92.5%。     

两个马铃薯Y病毒黑龙江马铃薯分离物株系鉴定

为明确分离自黑龙江省克山县马铃薯上的2个病毒分离物KS4和KS7的分类地位,通过RTPCR扩增、克隆获得其基因组序列,利用重组分析程序包和最大似然法分别进行重组分析和系统发育分析。结果显示,分离物KS4和KS7的开放阅读框均有9 186个核苷酸,编码3 061个氨基酸,分离物KS4的核苷酸和氨基酸序列均与马铃薯Y病毒(potato virus Y,PVY)分离物Mb112一致率最高,分别为96.9%和98.4%;分离物KS7的核苷酸序列与PVY分离物12-94一致率最高,为97.4%,其氨基酸序列与PVY分离物SYR-Ⅱ-Be1一致率最高,为97.8%。重组分析表明,分离物KS4和KS7均为分离物N-605和Oz的重组体,其中KS4基因组5′-端的2 392个核苷酸来自分离物N-605,其余核苷酸来自分离物Oz;KS7基因组的第800～2 227个核苷酸和第5 637～8 950个核苷酸来自分离物N-605,其余核苷酸来自分离物Oz。系统发育分析发现,分离物KS4被聚类到N:O株系(PVY~(N:O)),分离物KS7被聚类到NTN株系(PVY~(NTN))b型。     

马铃薯Y病毒在种薯中检出率及其株系鉴定

 马铃薯是我国重要粮食和经济作物。马铃薯Y病毒(potato virus Y,PVY)是危害马铃薯生产的重要病害。种植脱毒种薯是防治PVY最有效的途径。马铃薯种薯携带PVY问题严重,但种薯中PVY株系还不清楚。本研究利用PVY特异性抗体检测了7个马铃薯品种362个种薯,发现不同品种种薯带毒率差异较大,最高达12%。通过RT-PCR方法扩增获得了7个PVY分离物编码区全序列。重组分析发现7个分离物基因组均为重组型,根据重组位点的差异可以分为PVY^NTN-NW(SYR-II型)、Rec-1、Rec-2和Rec-3等4种重组类型,后3种为新重组类型。系统进化分析发现,分离物HQH18G3-10与PVY^NTN-NW(SYR-II型)处于同一个大的分支,但与中国PVY大田分离物聚集在一起形成一个相对独立的组,命名为PVY^NTN-NW(CN型);其余6个分离物与数据库中的中国分离物聚集在PVY^N-Wi组。这暗示PVY中国分离物具有相对独立的进化过程,PVY马铃薯大田分离物和种薯分离物进化上相近。所有分离物均能在珊西烟上引起典型叶脉坏死症状,HQH18G3-10引起的坏死症状最为严重。本研究首次报道了我国种薯内PVY发生情况,对分析病毒发生发展规律和防控具有借鉴作用。     

马铃薯Y病毒在种薯中检出率及其株系鉴定

马铃薯是我国重要粮食和经济作物。马铃薯Y病毒(potato virus Y,PVY)是危害马铃薯生产的重要病害。种植脱毒种薯是防治PVY最有效的途径。马铃薯种薯携带PVY问题严重,但种薯中PVY株系还不清楚。本研究利用PVY特异性抗体检测了7个马铃薯品种362个种薯,发现不同品种种薯带毒率差异较大,最高达12%。通过RT-PCR方法扩增获得了7个PVY分离物编码区全序列。重组分析发现7个分离物基因组均为重组型,根据重组位点的差异可以分为PVY^NTN-NW(SYR-II型)、Rec-1、Rec-2和Rec-3等4种重组类型,后3种为新重组类型。系统进化分析发现,分离物HQH18G3-10与PVY^NTN-NW(SYR-II型)处于同一个大的分支,但与中国PVY大田分离物聚集在一起形成一个相对独立的组,命名为PVY^NTN-NW(CN型);其余6个分离物与数据库中的中国分离物聚集在PVY^N-Wi组。这暗示PVY中国分离物具有相对独立的进化过程,PVY马铃薯大田分离物和种薯分离物进化上相近。所有分离物均能在珊...     

Detection of potato virus Y in early-harvested potato tubers by cDNA hybridization and three modifications of ELISA1

The standard ELISA method for detection of PVY in dormant potato tubers was compared with recently developed methods: (1) a hybridization technique, using a complementary DNA-probe to PVY^N; (2) three modifications of ELISA (standard monoclonal, Flegg & Clark polyclonal or monoclonal). Sap of six Dutch cultivars secondarily infected with PVY was subjected to these methods directly after lifting and after six subsequent one-week intervals during storage at 20°C. During storage the detectability of PVY decreased, irrespective of the diagnostic method applied. However, decrease was less in susceptible than in resistant cultivars. Although the hybridization technique yielded slightly better results than the standard ELISA procedure, it was time-consuming and involved hazardous reagents. The modifications of ELISA investigated were not, in general, more sensitive than standard ELISA.     

Comparison of three methods for the detection of Potato virus Y in seed potato certification

Journal of Plant Diseases and Protection - Potato virus Y (PVY) is becoming increasingly important in potato growing regions worldwide. The main reason for this is an increase in the incidence of...     

Breakdown of potato virus X resistance gene NX: selection of a group four strain from strain group three

Isolate DX of potato virus X (PVX) caused the typical reactions of a group three strain, systemic top necrosis in presence of potato hypersensitivity gene Nx and mosaic with gene Nb. When tubers harvested from DX-infected plants of the Nx: Nb cultivars Maris Piper and Pentland Dell were, grown on, most progeny plants were healthy, but some had systemic top necrosis caused by DX while others had mosaic symptoms in upper leaves and severe necrosis in lower ones. Infective sap from the plants with mosaic and necrosis always caused similar symptoms when inoculated to Pentland Dell. Only group four PVX strains can cause systemic infection without top necrosis in Nx:Nb cultivars, so the affected plants all contained a group four strain. That the severe necrosis of their lower leaves resulted from a shock reaction rather than from isolate DX also being present was indicated because the plants partially recovered, inoculations from near to the dilution end-point failed to demonstrate any separation of strains and behaviour on back-testing to Pentland Dell was unchanged after passage twice through tubers of this cultivar. Also, although DX caused severe necrotic symptoms in tubers of both Nx: Nb cultivars, these symptoms did not develop in tubers of plants with mosaic and necrosis. When cultured serially in Nicotiana glutinosa , the group four strain eventually reverted to group three.     

The movement of potato virus Y (PVY) in the vascular system of potato plants

Potato virus Y (PVY) is responsible for major viral diseases in most potato seed areas. It is transmitted by aphids in a non-persistent manner, and it is spread in potato fields by the winged aphids flying from an infected source plant to a healthy one. Six different PVY strains groups affect potato crops: PVY^C, PVY^N, PVY^O, PVY^N:O, PVY^NTN, and PVY^N-Wi. Nowadays, PVY^NTN and PVY^N-Wi are the predominant strains in Europe and the USA. After the infection of the leaf and accumulation of the virus, the virus is translocated to the progeny tubers. It is known that PVY^N is better translocated than PVY^O, but little is known about the translocation of the other PVY strains. The translocation of PVY occurs faster in young plants than in old plants; this mature plant resistance is generally explained by a restriction of the cell-to-cell movement of the virus in the leaves. The mother tuber may play an important role in explaining mature plant resistance. PVY is able to pass from one stem to the other stems of the same plant through the vascular system of the mother tuber, but it is unknown whether this vascular link between stems is permanent during the whole life of the plant. Two greenhouse trials were set up to study the spread of PVY in the vascular system of the potato plant. The PVY-susceptible cultivar Charlotte was used for both trials. It was demonstrated that all stems growing from a PVY-infected tuber will become infected sooner or later, and that PVY^N-Wi translocates more efficiently to progeny tubers than PVY^NTN. It was also demonstrated that the progressive decay of the mother tuber in the soil reduces the possibility for virus particles to infect healthy stems through the vascular system of the mother tuber. This new element contributes to a better understanding of the mechanism of mature plant resistance.     

Properties of viruses of the potyvirus group. 1. A simple method to purify bean yellow mosaic virus,pea mosaic virus,lettuce mosaic virus and potato virus YN

Bean yellow mosaic virus, pea mosaic virus, lettuce mosaic virus, and potato virus Y^N were purified by homogenizing and clarifying infected leaves in a mixture of 0.1 M tris-thioglycollic acid buffer pH 9, carbon tetrachloride and chloroform, followed by differential centrifuging applying moderate centrifugal forces.Samenvatting Het bonescherpmozaïekvirus B25, het erwtemozaïekvirus E198, het slamozaïekvirus en het aardappelvirus Y^N konden worden gezuiverd door geïnfecteerde bladeren te vermalen in een mengsel van 0,1 M tris-thioglycolzuur pH 9, tetrachloorkoolstof en chloroform, en de geklaarde suspensie vervolgens differentiëel te centrifugeren bij matige centrifugaalkrachten.     

Distribution of Potato virus Y in potato plant organs, tissues, and cells

The distribution of Potato virus Y (PVY) in the systemically infected potato (Solanum tuberosum) plants of the highly susceptible cultivar Igor was investigated. Virus presence and accumulation was analyzed in different plant organs and tissues using real-time polymerase chain reaction and transmission electron microscopy (TEM) negative staining methods. To get a complete insight into the location of viral RNA within the tissue, in situ hybridization was developed and optimized for the detection of PVY RNA at the cellular level. PVY was shown to accumulate in all studied leaf and stem tissues, in shoot tips, roots, and tubers; however, the level of virus accumulation was specific for each organ or tissue. The highest amounts of viral RNA and viral particles were found in symptomatic leaves and stem. By observing cell ultrastructure with TEM, viral cytoplasmic inclusion bodies were localized in close vicinity to the epidermis and in trichomes. Our results show that viral RNA, viral particles, and cytoplasmic inclusion bodies colocalize within the same type of cells or in close vicinity.     

福建马铃薯S病毒的分子鉴定及发生情况

为明确福建省马铃薯S病毒(PVS)的发生与分布情况,对福建省马铃薯主要种植区的PVS进行了鉴定和普查.在利用电镜技术和传统生物学方法鉴定的基础上,克隆了PVS外壳蛋白(cp)基因,依据PVS外壳蛋白氨基酸序列建立了PVS不同分离物的系统进化树.研究表明,利用PVS 外壳蛋白氨基酸序列分析可准确鉴定PVS,同时可分析不同分离物间的分子差异.利用病毒特异性引物和DIG标记的PVS cp基因为探针,分别利用RT-PCR技术和核酸斑点杂交技术(NASH)对PVS进行了检测,并对检测技术进行了改进.调查结果表明,PVS在福建省广泛分布,发病率最高可达80%以上,当地农家自留种可能是田间PVS的主要来源.     

侵染西瓜的小西葫芦花叶病毒的生物学特性鉴定

西瓜病毒病是制约西瓜产量及品质的顽疾.据报道,该病害造成的经济损失达到30%～50%.1981～1999年皖、苏、鲁、豫西瓜的病毒病曾几次大发生.危害西瓜的病毒病有西瓜花叶病毒1号(WMV-1)、西瓜花叶病毒2号(WMV-2)、黄瓜花叶病毒(CMV)、烟草花叶病毒(TMV)、烟草环斑病毒(TRSV)、南瓜花叶病毒(SqMV)等32种[1].小西葫芦花叶病毒(ZYMV)是侵染葫芦科的主要病毒之一,1973年首次在意大利北部发现此病毒,至今已成为一个全球性的植物病毒[2].笔者对ZYMV合肥分离物的生物学特性进行了研究.     

Enzyme-linked immunosorbent assay (ELISA) for the detection of potato viruses A and Y in potato leaves and sprouts

With the enzyme-linked immunosorbent assay (ELISA) potato virus A (PVA) could be detected reliably in potato sprouts, especially when these were young and sappy. The detection of this virus in leaves of glasshouse-grown potato plants was less reliable. The tobacco veinal necrosis strain of potato virus Y (PVY^N) was readily demonstrated in foliage of glass-house-grown potato plants using an antiserum to this strain. Plants infected with the common strain (PVY^O) did not react in ELISA with this antiserum. In young sappy sprouts, using the PVY^N antiserum, PVY^N could be detected reliably when samples with PVY^O were excluded, as the reaction of samples infected with the latter virus was intermediate between PVY^N-diseased and PVY-free samples. PVY was also detected in plants inadvertently infected during the experiments.     

Some experiments and considerations on the identification of witches' broom viruses,especially in clovers,in the Netherlands and Italy

Witches' broom phenomena (witches' broom growth + antholysis) observed inTropaeolum majus and in clovers in the Netherlands and in several wild and cultivated plants in Italy were demonstrated to be due to virus infection. The virus(es) could be transmitted by grafting, through dodder and by means of leaf-hoppers (Euscelis spp. primarily). The virus isolates found in both countries did not differ appreciably in the symptoms they caused. Many of the deviations concerned have been known for a long time as teratological phenomena. In the extensive literature considerable confusion exists concerning the identity of several witches' broom virus diseases. They constitute a good example of the problem of virus variability. A continuous splitting of witches' broom viruses on the basis of slight differences in symptom expression or of vector specificity does not seem justified. Since no intrinsic properties of the virus(es) concerned are known, the only conclusion that can be drawn from the information available is that the Dutch and the Italian isolates show a great similarity to aster yellows virus as well as to tomato big bud, stolbur and other witches' broom viruses.     

Reactions on Nicotiana species to potato viruses A,X and Y and tobacco mosaic virus in relation to their taxonomy and geographical origin

When testing the response of species of the genusNicotiana to 14 isolates of potato viruses A (PVA), X (PVX) and Y (PVY) and tobacco mosaic virus (TMV), sections and section parts of the genus could be divided into five groups according to the overall reaction of their species. Species from arid regions of australia and belonging to the sectionSuaveolentes (subgenusPetunioides) were most sensitive and least resistant, whereas the sectionsPaniculatae (subgenusRustica),Tomentosae (Tabacum) andNoctiflorae (Petunioides) appeared least sensitive and most resistant. Sixty-one percent of the accessions of the latter sections proved resistant to at least two of the viruses. The most resistant species have their main geographical distribution in the central Andes (southern Peru, Bolivia and north-western Argentina), where the viruses may have originated. One other resistant species wasN. africana (Suaveolentes) indigenous to south-western Africa. The most sensitive American sections, viz.Genuinae (Tabacum) andTrigonophyllae, Alatae, Acuminatae andBigelovianae (Petunioides), were generally more sensitive than species of theSuaveolentes section from outside the arid regions of Australia. The remaining part of the genus, viz. the American sectionsThyrsiflorae andRusticae (Rustica) andUndulatae, Repandea andNudicaules (Petunioides) was intermediate between the latter group ofSuaveolentes species and the resistant group of sections with regard to sensitivity and resistance, but had a high rate of tolerance to PVA, PVX and PVY and of hypersensitivity-associated resistance to TMV. The results indicate that the viral respons in the genusNicotiana is mainly determined geographically and to a lesser extent taxonomically.Samenvatting Na inoculatie van een grote collectieNicotiana-soorten met 14 isolaten van de aardappelvirussen A (PVA), X (PVX) en Y (PVY) en tabaksmozaïekvirus (TMV), konden de secties of sectiedelen van het genus op basis van de gemiddelde reactie van hun soorten in vijf groepen worden verdeeld. Soorten uit Australische woestijngebieden en taxonomisch behorend tot de sectieSuaveolentes (subgenusPetunioides) bleken het meest gevoelig en het minst resistent, terwijl de sectiesPaniculatae (subgenusRustica),Tomentosae (Tabacum) enNoctiflorae (Petunioides) het minst gevoelig en het meest resistent waren. Van de collectienummers van de laatstgenoemde secties bleek 61% resistentie tegen minstens twee van de virussen te bezitten. De meest resistente soorten hebben hun verspreidingsgebied vooral in het centrale deel van het Andesgebergte (het zuidelijk deel van Peru, Bolivia en noord-westelijk Argentinië). Mogelijk is dit het ontstaansgebied van deze virussen. De einge andere resistente soort wasN. africana (Suaveolentes) uit zuid-westelijk Afrika. De meest gevoelige Amerikaanse secties, te wetenGenuinae (Tabacum) enTrigonophyllae, Alatae, Acuminatae enBigelovianae (Petunioides), waren gemiddeld gevoeliger danSuaveolentes-soorten die niet uit de Australische woestijnen afkomstig zijn. Het resterende deel van het genus, bestaande uit de Amerikaanse sectiesThyrsiflorae enRusticae (Rustica) enUndulatae, Repandae enNudicaules (Petunioides), stond met betrekking tot gevoeligheid en resistentie tussen laatstgenoemd deel van de sectieSuaveolentes en de groep resistente secties in, maar vertoonde in hoge mate tolerantie voor PVA, PVX en PVY en met overgevoeligheid samengaande resistentie voor TMV. Uit de resultaten blijkt dat de reacties van het genusNicotiana op virussen vooral geografisch bepaald zijn en in mindere mate taxonomisch.     

Serological identification of some soil-borne viruses causing diseases in fruit crops in the Netherlands

Samenvatting Tot voor kort was slechts weinig bekend over de identiteit van de virussen, die verantwoordelijk zijn voor de lepelbladziekte van rode bes en de Eckelraderziekte van kers. Een antiserum werd gemaakt tegen het lepelbladvirus en toetsingen werden uitgevoerd. Het lepelbladvirus bleek een stam van het Schotse raspberry ring spot-virus te zijn. Ook een virus uit Eckelraderzieke kersen bleek met het antiserum tegen lepelbladvirus te reageren. Het antiserum tegen lepelbladvirus reageerde met alle getoetste isolaties van dit virus tot een verdunning van 1/512, doch met een viertal isolaties uit Eckelraderzieke kersen tot een verdunning van slechts 1/128. Hoewel het lepelbladvirus en het virus uit Eckelraderzieke kersen dus duidelijk verwant zijn, bleek, ook uit premunitieproeven, dat er verschillen zijn. De verwantschap tussen het virus uit Eckelraderzieke kersen en het raspberry ring spot-virus is uit verschillende proefresultaten gebleken.Een derde virus, afkomstig uit aardbeiplanten van het ras Red Gauntlet, geïmporteerd uit Schotland, bleek te reageren met een antiserum tegenHarrisons beet ring spot stam van het tomato black ring-virus. Dit antiserum werd verstrekt door Dr.C. H. Cadman, Schotland.     

引进加拿大马铃薯种薯中病毒的检测

应用酶联免疫吸附法（DAS-ELISA）对2000年种植于网室和田间隔离种植地的引进加拿大马铃薯种薯3个品种进行马铃薯黄矮病毒、马铃薯A病毒（PVA）、马铃薯Y病毒N株系（PVY^n）、马铃薯黑环斑病毒、烟草脆裂病毒、马铃薯X病毒和马铃薯S病毒（PVS）的检测。对网室样品中经ELISA检测怀疑为阳性样品再用组织超薄切片、直沾法和免疫电镜法制备电镜检样品做电镜观察。结果在网室的Russet Burbank品种上检出PVA和PVS，其它两品种检出PVS；田间隔离种植中检出PVY^n和PVS。     

湖北甘薯病毒病的检测与鉴定

2013—2015年采集了湖北黄冈、鄂州、武汉、荆州以及宜昌等5个地区的甘薯病毒病样品,通过双生病毒通用引物PCR扩增、ds RNA技术和序列分析等方法,鉴定了这5个地区甘薯病毒病的病原。结果显示,甘薯羽状斑驳病毒(Sweet potato feathery mottle virus,SPFMV)、甘薯褪绿矮化病毒(Sweet potato chlorotic stunt virus,SPCSV)、黄瓜花叶病毒(Cucumber mosaic virus,CMV)和甘薯卷叶病毒(Sweet potato leaf curl Georgia virus,SPLCGV)等4种病毒被检出。其中,SPFMV SPLCGV这两种病毒在湖北皆为首次报道。     

甘薯病毒2中国分离物全基因组序列克隆及遗传进化分析

正甘薯病毒2(Sweet potato virus 2,SPV2)是马铃薯Y病毒科(Potyviridae)马铃薯Y病毒属(Potyvirus)成员。SPV2也称为甘薯脉花叶病毒(ipomoea vein mosaic virus,IVMV)和甘薯Y病毒(sweet potato virus Y,SPVY)~[1],是甘薯上常见的病毒之一。SPV2病毒粒体为线条状,长度为850 nm,在细胞质中形成风轮状或卷轴状内含体~[2]。SPV2可由桃