Biotinylated DNA probes for detecting virus Y and aucuba mosaic virus in leaves and dormant tubers of potato

Summary Detection of potato virus Y (PVY) in dormant potato tubers using the enzyme-linked immunosorbent assay (ELISA) has been reported not to be accurate and reliable and it requires breaking of the dormancy of tubers prior to testing. We describe a simple, practical and highly sensitive hybridization method based on the use of biotinylated DNA probes for detecting PVY and potato aucuba mosaic virus (PAMV) in dilutions made of crude extracts of infected potato leaves and dormant tubers. As little as 50 fg of RNA can be detected by this method, and the probes are highly specific for their targets, even in crude plant extracts. The presence of one virus did not interfere with the detection of the other.     

Improved efficiency of detection of potato mop-top furovirus in potato tubers and in the roots and leaves of soil-bait plants

Summary A reverse trancription-polymerase chain reaction (RT-PCR) assay was devised and shown to be sensitive and reliable for the detection of potato mop-top virus (PMTV) RNA sequences in the flesh of virus infected potato tubers, and in the roots and leaves of soil-bait plants. This assay was compared with an enzyme-linked immunosorbent assay incorporating PMTV specific monoclonal antibodies (TAS-ELISA). The tests were devised to improve the efficiency of detection of viruliferousSpongospora subterranea in agricultural soils, and PMTV in potato tubers. RT-PCR detected PMTV RNA sequences in the roots and leaves of bait plants after three weeks growth in viruliferous soil, three weeks before the bait plants themselves developed symptoms, and two weeks before the virus was detected by TAS-ELISA. Both RT-PCR and TAS-ELISA detected PMTV in the tubers of primary-infected potatoes. RT-PCR and TAS-ELISA were shown to be more sensitive and reliable than conventional baittests and sap inoculation methods for the detection and diagnosis of PMTV.     

马铃薯病毒外壳蛋白融合基因转化马铃薯及其抗病性分析

将多种病毒的有效核酸片断拼接成融合基因转入马铃薯可获得多抗马铃薯材料。针对马铃薯生产中分布广泛、危害严重并经常混合感染的马铃薯X病毒(PVX)、马铃薯Y病毒(PVY)、马铃薯卷叶病毒(PLRV)和马铃薯S病毒(PVS),开展了利用基因工程方法获得兼抗4种马铃薯病毒转基因马铃薯材料的研究。试验在前期获得含4种马铃薯病毒外壳蛋白基因片段的质粒pART27-XSYV-rh的基础上,通过根癌农杆菌(Agrobacterium tumefaciens)介导转化马铃薯(Solanum tuberosum)品种‘陇薯3号’,PCR扩增和PCR-Southern杂交证明,4价融合基因已整合到马铃薯基因组中。qRT-PCR分析表明,该融合基因在转基因植株中能正常表达。3株转基因植株的抗病性鉴定结果表明,2株对4种病毒同时具有抗性;1株对PLRV侵染表现阳性,对另外3种病毒同时具有抗性。     

马铃薯脱毒种薯的快速繁殖技术

马铃薯退化现象是由于感染一种或多种过滤性病毒引起的,它是影响高产稳产的主要因素。病毒不仅赖于植物体内部生存,并且参加和改变植物体内许多代谢途径的生理活动。当被不同病毒侵染的马铃薯出现各式各样症状,就是病毒在植物体内部干扰机体代谢作用的结果,所以只有从内部清除病毒,获得脱毒健康种薯,才是治本的办法。     

PVYCP基因导入加工型马铃薯甘农薯1号的研究

利用整合有马铃薯Y病毒外壳蛋白基 (PVYCP基因 )的改建质粒PEY3,以土壤农杆菌为载体转化加工型马铃薯新品种甘农薯 1号叶盘 ,细菌侵染 14d后用 10 0ml/LKM在愈伤组织水平上选择 ,转化率最高可达 2 1 3%。转化效率与细菌生理状态、NAA浓度、叶盘预培养天数有关。转化体分化 2株再生植株。冠瘿碱分析证明转化体整合了PVYCP基因。     

Detection of potato virus X in tubers with the enzymelinked immunosorbent assay (ELISA)

Summary Tubers of field-grown plants of ten Dutch potato cultivars, secondarily infected with potato virus X (PVX) or free from this virus were submitted to ELISA after storage at 4°C. About 40 weeks after lifting PVX could be detected reliably. Extinction values with the apical parts of the tubers were slightly higher than those with the basal parts.     

湖南省马铃薯主产区马铃薯病毒种类及流行分析

马铃薯是世界第四大粮食作物,其病毒病危害严重。2010年对湖南马铃薯主产区采集的66个病毒标样进行了RT-PCR检测,结果表明,检测出的马铃薯病毒有马铃薯Y病毒(PVY)、马铃薯卷叶病毒(PLRV)、马铃薯X病毒(PVX)、马铃薯S病毒(PVS)、马铃薯A病毒(PVA)和马铃薯纺锤块茎类病毒(PSTVd)。其中PVS的检出率最高,为54.5%,其次是PVX,检出率为45.5%,PVY的检出率为39.4%,PSTVd和PVA的检出率均为21.2%,PLRV的检出率为18.2%。2~4种病毒的复合侵染现象较为普遍。PVY中重组型PVY占85.7%。     

A simple assay system incorporating monoclonal antibodies for the detection of potato virus T

Summary Two monoclonal antibodies (McAbs) which react specifically with potato virus T (PVT) were produced and tested for cross reactivity with potato viruses (A, S, X, Y^o, Y^N, leaf roll), apple stem grooving virus (ASGV) and against healthy sap. They were found to be highly specific for PVT. These two McAbs form the basis of an enzyme-linked immunosorbent assay (ELISA) for the detection of PVT in infected plant material which is of key significance for potato quarantine.     

侵染湖南甘薯种质资源的曲叶病毒和杆状病毒的检测及其亲缘关系分析

采用PCR技术对2015—2016年从湖南省多个市县采集的180份甘薯种质资源进行两种DNA病毒（sweet potato leaf curl virus,SPLCV和sweet potato badnavirus,SPBV）检测,并以PCR扩增产物序列为基础,对这两种病毒的亲缘关系进行分析。结果表明：（1）180份甘薯种质资源中,22份检测出SPLCV,占总数的12.2%;32份检测出SPBV,占总数的17.8%;8份同时检出SPLCV和SPBV这两种病毒,占总数的3.9%。（2）检出SPLCV的市县有长沙、永州、怀化、邵阳、郴州、常德、娄底、益阳、岳阳和株洲;检出SPBV的市县有长沙、永州、怀化、衡阳和邵阳;检出两病毒复合侵染的市县有怀化、长沙、永州和邵阳。（3）基于病毒全基因组序列分析显示,全球已报道的36个SPLCV分离物可分为3组,本研究获得的12个湖南分离物可分别归于3个组中,其中6个归于组Ⅰ,1个归于组Ⅱ,5个归于组Ⅲ,表明湖南SPLCV具有高度多样性。（4）基于病毒运动蛋白和外壳蛋白编码基因部分序列分析显示,全球已报道的36个SPBV分离物可分为4组,绝大部分分离物属于组Ⅰ,我国1个安徽分离物单独成组（组Ⅱ）,我国2个山东分离物和1个海南分离物构成1个组（组Ⅲ）,本研究获得的2个湖南分离物其中1个归于组Ⅰ,而另1个与已报道各分离物的核苷酸序列均存在显著差异,相似性仅为74.92%,单独成组（组Ⅳ）,表明我国SPBV具有高度的变异性,存在多个变异类型。本研究结果可为湖南地区这两种病毒病防控提供依据。     

Use of jet injection to determine potato cultivar susceptibility to potato viruses A and Yc

Summary The use of a high pressure jet injector to inoculate virus rapidly was compared with graft inoculation to determine cultivar susceptibility to potato virus A (PVA) and potato virus Y^c (PVY^c). Tubers were injected with virus infected potato sap, plant reactions recorded, and virus recovered using a bioassay test and enzyme linked immuno-sorbent assay. The injection method compared well with a traditional grafting procedure for PVY^c susceptibility tests but was unsuitable for testing susceptibility to PVA.     

Diploid genotype DW.84-1457, highly resistant to potato leafroll virus (PLRV)

Summary The diploid clone DW.84-1457 which has outstanding resistance to potato leafroll virus (PLRV), has been selected at the Mlochów Centre of the Institute for Potato Research. It has in its pedigree PLRV-resistant clones from the Max Planck Institute nos. MPI 44.1016/10, MPI 44.335/130 and MPI 49.540/2. Its behaviour in the field and response to aphid inoculation indicate high resistance to infection, and the low concentration of the virus in graft-inoculated plants indicates high resistance to multiplication. This combination within one genotype of two aspects of resistance is not connected with hypersensitivity, and is heritable. Clone DW.84-1457 has other desirable characters such as extreme resistance to potato virus X (PVX), high resistance to potato virus M (PVM) and good table and processing quality. It is being utilized in the development of parental lines, both at the diploid and tetraploid level.     

Detection of potato leaf roll virus and potato viruses M,S, X and Y by dot immunobinding on plain paper

Summary Potato leafroll virus and potato viruses M, S, X and Y in green leaves were detected by dot immunobinding (DIB) on plain paper and on nitrocellulose membranes. On both materials, DIB could detect the presence of very small amounts of virus, e.g. 30 pg of purified PVX. The sensitivity of the DIB test on plain paper and on nitrocellulose was compared to Double Antibody Sandwich ELISA (DAS-ELISA) by serial dilutions of infected plant sap made in healthy plant sap. Detection of potato viruses by DIB on plain paper and nitrocellulose was found to be equally sensitive whereas DAS-ELISA was 2 to 8 times more sensitive. Possible simplifications of the DIB procedure to suit the requirements of a routine method were examined. The use of the DIB method for routine testing of potato viruses is discussed.     

An enhanced multiplication of potato virus X is not related to a synergistic reaction between the potato viruses X and Y

Summary Greenhouse experiments demonstrated a differential interaction between potato viruses X and Y in two potato cultivars. Enhancement of PVX synthesis in doubly infected plants occurred only in the PVY-susceptible potato cv. Ulla, whereas PVX multiplication was almost completely inhibited in the doubly infected PVY-resistant cv. Franzi. However, a synergistic effect was also evident in the latter cultivar in the form of a growth inhibition of the plants. An increased multiplication of one or both of the viruses is, therefore, not related to a synergistic reaction.     

Purification of potato virus A and its detection in potato by enzyme-linked immunosorbent assay (ELISA)

Potato virus A (PVA) was purified fromNicandra physaloides by a simple method that omitted organic solvent clarification and consisted of differential centrifugation followed by equilibrium centrifugation in CsCl. An antiserum was produced that specifically detected PVA in potato leaf sap using either the SDS-agar test or enzyme-linked immunosorbent assay (ELISA). No heterlogous reaction of the antiserum with potato virus Y was detected. Purified PVA was highly infectious; it had an A 258/280 nm absorbance ratio of 1.28. The particles had a normal intact appearance in the electron microscope. Detection of PVA in potato sprouts and foliage by ELISA was compared with the local lesion assay onPhysalis angulata L. plants. ELISA was superior over an indicator plant test when sprout tissue was used. PVA antiserum reacted similarly with mild and crinkle isolates.     

Territoriale Schwerpunkte des Virusbefalls von Kartoffeln im Osten Deutschlands

Zusammenfassung Auf der Grundlage von Ergebnissen der Pflanzgutkontrolle der Jahre 1979 bis 1988 konnten ca. 77% des Gebietes der ehemaligen Deutschen Demokratischen Republik in Befallsdruckzonen der ?konomisch relevanten Virusgruppen potato leafroll virus und schwere Mosaikviren (potao virus A, potato virus M, potato virus Y) eingeteilt werden. Damit wurden die Aussagen von Schick (1952) und Pfeffer (1956) zur Verteilung der Gesundheits- und Abbaulagen best?tigt.     

Enzyme-linked immunosorbent assay with single or combined antisera for viruses S and X in potato tubers and plants

Enzyme-linked immunosorbent assay (ELISA), with single or combined antisera was effective for diagnosing potato virus S (PVS), potato virus X (PVX) or both viruses in plants grown in the greenhouse or field. In dormant tubers, stolon, middle and apical end composite sampling with or without eyes and sprouted tubers produced reliable positive assays for PVX. Only tuber pieces with sprouts resulted in consistently reliable assays for PVS. Composite sampling of potato foliage was effective for detecting one PVX infected plant in a total of 100 Kennebec, Norland, or Russet Burbank plants. There were some false negative results and greater variability in composite PVS assays, but on average, one PVS infected plant can be detected in composites of 10 Kennebec, Norland, or Russet Burbank plants. Sodium diethyldithiocarbamate (0.01M NaDIECA) in phosphate buffered saline + 0.5% Tween (PBS-T) added to plant extracts enhanced specific reactions for either virus. Onceor twiceused enzyme conjugate was effective in ELISA of either virus from potato foliage.     

Solanum microdontum (PI 558098): A diagnostic host plant for potato virus A

Solanum microdontum (PI 558098) develops diagnostic symptoms when inoculated with plant sap containing potato virus A (PVA). The symptoms consist of local lesions (2–4 mm), followed by systemic necrosis of leaf veins, bronzing of leaf surface, and leaf drop. Symptoms develop in a temperature range of 20–29 C with 4 to 8 Klux light intensity of 14 h daylength. Local lesions are visible within 1 wk of inoculation and can be caused by PVA containing sap dilutions of up to 1:100. Plants multiplied by shoot cuttings as well as those produced from true potato seed are equally sensitive. True potato seed production occurs under normal greenhouse conditions in cross-pollinated plants.     

马铃薯病毒分离提纯的方法

马铃薯已被我国定为第四大粮食作物,国内马铃薯需求量也越来越大,种植面积逐年上升,同时国际种薯市场也在逐渐扩大,需要大量高质量的合格种薯,所以迫切需要用于检测马铃薯病毒的病毒检测试剂盒。本文主要介绍马铃薯病毒提纯的环节及注意事项,以便提纯出高质量的病毒,用于制备病毒检测试剂盒检测病毒。