Evaluation of Tip Culture, Thermotherapy and Chemotherapy for Elimination of Peanut Mottle Virus from Arachis hypogaea

Peanut (Arachis hypogaea L.) plants (cvs Florunner and Pronto) were inoculated at the two-leaf stage with peanut mottle virus (PMV) to obtain PMV-infected plants. Shoot-tips from plants grown in the glasshouse (27°C) or from plants maintained at 35°C were used for tip culture. In some experiments ribavirin was added to the culture medium at 5 mg/1, 10 mg/1, 15 mg/1 or 20 mg/1. Plants regenerated from meristems or shoot-tips taken from virus-infected plants were not virus-free. After 45 days at 35°C, foliar tissue of 93 % of Florunner and 95 % of Pronto plants tested negative for PMV by enzyme-linked immunosorbent assay (ELISA). When shoot-tips from the plant that tested negative by ELISA were used for tip culture, no virus-free plants were obtained. No virus-free plants were obtained from tips cultured on medium supplemnted with ribavirin. However, when tip culture, thermotherapy and chemotherapy were combined; 80 % of Florunner and 100 % of Pronto plants were found negative for PMV.     

Distribution and accumulation of PVM and PVY,PVX in infected potato plants

The ability of PVM, PVY and PVX viruses and their progeny to distribute themselves and accumulate in primary infected potato plants by mono‐ and mixed infections is analysed. It is shown, that the transport and accumulation of virus in inoculated potato plants depends on variety resistance, combination of viruses and sequence of their application.     

Interactions of the Solanum spp. of the Etuberosa group and nine potato-infecting viruses and a viroid

All 26 accessions of Solanum brevidens, one accession of S. etuberosum and one accession of S. fernandezianum tested were all extremely resistant to potato leafroll virus (PLRV) and potato viruses Y (PVY) and A (PVA). S. brevidens and S. etuberosum were also resistant to Andean potato mottle virus (APMV) and moderately resistant to potato virus X (PVX), whereas S. fernandezianum was susceptible to these viruses. Additionally, S. brevidens was resistant to sap-inoculated potato viruses M (PVM) and S (PVS). All the Etuberosa accessions were susceptible by graft-inoculation to PVM, PVS, potato virus T (PVT) and Andean potato latent virus (APLV). Infections by the above mentioned viruses were symptomless in all of the Etuberosa spp. S. etuberosum and S. fernandezianum were infected by mechanical inoculation with potato spindle tuber viroid, S. etuberosum developing severe stunting and leaf-curl symptoms, but S. brevidens was infected only by graft-inoculation. The genes conferring resistance to PVY and PVX in S. brevidens and S. etuberosum appeared to be different from those currently utilised by plant breeders.     

Experimental and natural weed host-virus relations

Weeds, as alternative hosts of plant viruses and nutrient plants of virus vectors play important role in virus ecology and epidemiology. The aim of our study was to discover new weed-virus relations. Therefore some weed species were mechanically inoculated with 28 viruses (strains or isolates) maintained in our glasshouse. Different weed species with and without visible symptoms were collected from agro-, water ecosystems and wastelands of Hungary between 1997 and 2003. Virus infections were evaluated by biotests, DAS ELISA serological methods, electronmicroscopy and immunosorbent electronmicroscopy (ISEM). Under glasshouse conditions Ambrosia artemisifolia was considered as a virophob species, showing resistance to all viruses listed above. A series of new artificial (Chenopodium album--SoMV (LH+SH)*, AMV (LH+SH); C. berlandieri--PVY(NTN) (LH), AMV (LH+SH), CMV (LH), SoMV (LH+SH), ObPV (LH+SH), ZYMV-10 (LH): C. ugandae--ObPV (LH), SoMV (L); C. glaucum--ObPV (LH), SoMV (L); Echinocystis lobata--PVX (L), ZYMV (LH+SH); Solanum nigrum--MYFV (LH+SH), PVY(N) (L), PVY(NTN) (LH+SH), SoMV (LH), TMV (SH), CMV (SH); S. dulcamara--CMV-U/246 (SH), PVY(NTN) (LH), SoMV-H (L), TMV-O (L); S. luteum--PVY(N) (SH), PVY(NTN) (LH+L), TMV(SH).) and natural (Asclepias syriaca--TMV, AMV, TSWV; Alisma plantago-aquatica--PVY, SoMV; Ambrosia artemisiifolia--CMV; Chenopodium album--CMV, PVS, PLRV; C. hybridum--CMV; Cirsium canum--CMV, PVM; Carex vulpina--CMV; Comium maculatum--PVY; Datura stramonium--PVA, PVX, PVS, PVM, CMV, TMV; Lysimachia vulgaris--ArMV, BNYVV, CMV, TMV; Lythrum salicaria--ArMV; Malva neglecta--CMV; Mercurialis annua--SoMV; Solanum nigrum--CMV, PVY, PVY(N); Solidago gigantea--CMV, RpRSV, BNYVV; Stenactis annua--PVM, PVA) weed--virus relations were detected. The epidemiological role of perennial hosts (A. syriaca, A. planlago aquatica, C. canurm, L. vulgaris, L. salicaria, S. gigantea) is especially high, because they can serve as infection sources as well as overwintering hosts of different plant viruses.     

Improved Dot-Blot Hybridization Assay for Large-Scale Detection of Potato Viruses in Crude Potato Tuber Extracts

Abstract A rapid and simple technique has been developed to enhance the sensitivity of virus detection in dot-blot hybridization assay by up to 1000 fold. The procedure generally follows that of B aulcombe et al. (1984) but includes moderate heating of the nitrocellulose filter in 10XSSC, 0.5% SDS solution at 55°C after sample application. Using this procedure, four potato viruses (PVX, PVS, PVM and PVY) were detected with cloned virus-specific ³²P-cDNA probes in 2 μl spots containing 0.2–2 pg of purufied virus (0.1–1 ng/ml). The procedure was also successfully applied for the detection of PVX, PVS, PVM and PVY in crude potato tuber extracts.     

Incidence and altitudinal distribution of 13 virus cultures in Solanum tuberosum (Solanaceae) from Costa Rica

A survey was conducted in 30 fields located at three different altitudes in Cartago, Costa Rica's main potato producing area. Twenty plants were sampled per farm, for a total of 600 samples with 200 samples per altitude. ELISA was used with commercial reagents to independently test for PVX, PVY, PVM, PVA, PVS, PLRV, PMTV, PAMV, PVV, PVT, APLV, APMoV and TRSV. The presence of the following viruses was determined: PVX (77 %), PAMV (62 %), PLRV (42 %), TRSV (42 %), PVT (39 %), PVV (37 %), PMTV (31%), PVY (30 %), PVS (19 %), PVM (13 %), PVA (8 %), and APMoV (8%). APLV was not detected in any sample. This is the first report in Costa Rica of the presence of the viruses PMTV, PAMV, PVV, PVT and APMoV. A high viral incidence in the tuber seed production area as well as a high rate of mixed infections is reported.     

Occurrence and distribution of viruses infecting potato in Russia

Potato viral disease has been a major problem in potato production worldwide including Russia. Here, we detected Potato Virus M (PVM), P (PVP), S (PVS), Y (PVY), and X (PVX) and Potato Leaf Roll Virus (PLRV) by RT-PCR on potato leaves and tubers from the Northwestern (NW), Volga (VF), and Far Eastern (FE) federal districts of Russia. Each sample was co-infected with up to five viruses. RT-PCR disclosed all six viruses in NW, three in VF, and five in FE. Phylogenetic analyses of PVM and PVS strains resolved all PVM isolates in Group O (ordinary) and all PVS isolates in Group O. Seven PVY strains were detected, and they included only recombinants. PVY recombinants were thus the dominant potato virus strains in Russia, although they widely varied among the regions. Our research provides insights into the geographical distribution and genetic variability of potato viruses in Russia.     

用IgG指示试纸同时检测多种马铃薯病毒病

用硝基纤维素膜(NCM)为载体制备的抗体IgG指示试纸,可同时检测马铃薯X病毒(PVX)、马铃薯Y病毒(PVY)、马铃薯S病毒(PVS)和马铃薯卷叶病毒(PLRV)的感染。以pH10.0的1％甘氨酸溶液为PVX、PVY和PVS的研磨缓冲液,比已报道的缓冲液效果好。在提取病毒抗原时加入4％纤维素酶,可提高病毒得率及所制备的抗血清灵敏度。     

百合不定芽培养脱毒种球生产的研究

百合不定芽培养再生植株的病毒脱毒效果因百合品种和病毒种类而有所差异;‘卡萨布兰卡’百合潜在病毒（LSV）容易脱去,脱毒率达76%,‘魅丽’黄瓜花叶病毒（CMV）能全部脱去。‘卡萨布兰卡’不定芽培养在固体培养基和液体培养基都能很好形成子球;在含有抗病毒剂（10 mg/L DHT）的液体培养基中子球增大显著,LSV脱毒效果理想。无病毒子球移到有防虫网的户外栽培,2年后部分植株被检测出病毒,再感染率因品种而不同,‘卡萨布兰卡’高达73%,麝香百合‘乔治亚’仅17%;无病毒植株‘乔治亚’,香华丽百合和‘卡萨布兰卡’的生长高度要比有病毒植株明显增高。     

Elimination of Apple Mosaic Virus and Raspberry Bushy Dwarf Virus from Infected Red Raspberry (Rubus idaeus L.) by Tissue Culture

Experiments with ApMV infected ‘Malling Landmark’ and RBDV infected ‘Schamp;önemannamp;’ and ‘Trentamp;’ plants were carried out to evaluate a) the dependence of virus eradication on explant size and mass propagation. b) the reliability of results of ELISA tests on in vitro plantlets. With ApMV a correlation between virus elimination and explant size was observed, whereas with RBDV even plantlets from the smallest established explants were still infected. With ApMV, in vitro multiplication for three subsequent subcultures did not lead to further virus elimination, with RBDV this was observed in two cases. ELISA test results for both viruses, ApMV and RBDV, were identical when small in vitro plantlets, long-term stored plants, or potted plants from the same origin were tested, indicating that virus tests are possible with very young plant material and can be used to select virus-free plants in vitro. Tissue culture permits long-term storage of plant viruses. It is also suitable tor plant virus propagation and could be a useful aid in plant virus purification. For commercial multiplication only virus-indexed plant material should be used for establishment and further propagation in vitro.     

采用液质联用方法检测黄秋葵荚果黄酮类物质含量

该研究为了培育兼抗4种病毒的马铃薯品种,采用RT￣PCR技术对PVX、PVS、PVY和PLRV的外壳蛋白( CP )基因进行克隆与分析,获得了大小分别为670、800、700、600 bp的CP基因序列,将获得的CP基因序列与NCBI中已报道的序列进行比对分析,其同源性都在96％以上。根据所克隆的CP 基因对靶标片段进行筛选,获得了大小约300 bp的靶标片段PVX￣rh、PVS￣rh、PVY￣rh和PLRV￣rh,同时利用 Overlap￣PCR技术将4种病毒的靶标片段进行拼接,得到了长度约为1200 bp的融合片段XSYV￣rh,与预期目标片段XSYV￣yxz的相似性达100％。利用DNA重组技术将融合片段XSYV￣rh克隆到pGM￣T载体上构建成克隆载体pGM￣T￣XSYV￣rh,用SpeⅠ和SacⅠ对克隆载体pGM￣T￣XSYV￣rh和植物表达载体pART27进行同步双酶切,用T4 DNA连接酶将XSYV￣rh片段连接到载体pART27上,成功构建了同时含4种病毒CP 基因片段的植物表达载体pART27￣XSYV￣rh。采用直接转化法将植物表达载体导入根癌农杆菌LBA4404中,并利用农杆菌介导法对烟草品种T12试管苗进行遗传转化,转化后的烟草植株经PCR检测,有40株转化植株可扩增出目的条带,表明XSYV￣rh融合基因已成功转入烟草基因组中。     

Accumulation of potato virus Y is enhanced in Solatium brevidens also infected with tobacco mosaic virus or potato spindle tuber viroid

The accumulation of potato virus Y?(PVY?) and potato leaf roll virus (PLRV) was studied in plants of Solanum brevidens co-infected with each of six viruses or a viroid. Virus could not be detected by ELISA in plants of S. brevidens infected solely with PVY. However, accumulation of PVY was increased c. 1000-fold in plants doubly infected with tobacco mosaic virus or potato spindle tuber viroid (PSTVd). PVY titres in doubly infected plants of S. brevidens were between 1% and 0.1% of those found in the PVY-susceptible interspecific Solanum hybrid DTO-33. Double infections of 5. brevidens by PVY and alfalfa mosaic virus or potato viruses M, S, T or X did not significantly enhance PVY accumulation. Accumulation of PLRV was not enhanced in plants co-infected with any of the six viruses or PSTVd.     

Virus infections reduce in vitro multiplication of ‘Malling Landmark’ raspberry

Summary Virus-infected plants are often symptomless and may be inadvertently used as explant sources in tissue culture research. Our objective was to determine the effect of virus infection on micropropagation. We studied the effects of single and multiple infections of three common raspberry viruses on the in vitro culture of ‘Malling Landmark’ red raspberry (Rubus idaeus L.). Virus-infected reaspberry plants were produced by leaf-graft inoculation from known-infected plants onto virus-free ‘Malling Landmark’. Single-virus source plants were infected with either tobacco streak ilarvirus (TSV), tomato ringspot nepovirus (TomRSV), or raspberry bushy dwarf idaeovirus (RBDV) and were free of other viruses as determined by enzyme-linked immunosorbent assay (ELISA) and bioassay. Virus-free, single, and multiple virus-infected ‘malling Landmark’ explants were initiated into culture and multiplied on Anderson's medium with 8.9 μM N⁶-benzyladenine (BA). At the end of the multiplication tests, ELISA reconfirmed virus infections. In vitro multiplication of ‘Malling Landmark’ was significantly reduced by multiple infections, and multiplication of plants infected with all three viruses (RBDV+TomRSV+TSV) was less than half that of virus free cultures. Shoot height and morphology of in vitro cultures were not influenced by virus infection. The greenhouse stock plant with the three-virus infection was stunted and yellow compared to the control and the other infected plants. Part of a thesis submitted by C.-W.V.T. in partial fulfilment of the requirements for the MS degree. The use of trade names in this publication does not imply endorsement by the U.S. Department of Agriculture or Oregon State University.     

Molecular characterisation of Onion yellow dwarf virus (OYDV) infecting garlic (Allium sativum L.) in Israel: Thermotherapy inhibits virus elimination by meristem tip culture

Garlic (cv. Shani) was tested using single step RT‐PCR and digoxygenin (DIG) labelled dot‐blot for a number of viruses. Following sequence analysis it was shown that at least three different polymorphs of the potyvirus Onion yellow dwarf virus (OYDV) infect the same plant simultaneously, together with the potyvirus Leek yellow stripe virus (LYSV), the carlavirus Garlic common latent virus (GCLV) and a multitude of allexiviruses (Shallot virus X (ShVX) related viruses]. Several garlic plants free of all the viruses tested were obtained through meristem‐tip culture. Plants infected with single viruses or with different combinations of viruses were similarly obtained. Meristem‐tip culture was confirmed as a satisfactory method of virus eradication, while thermotherapy treatment given to mother plantlets before meristem excision was found to specifically antagonise OYDV eradication. This work uses molecular methods for the first time to examine the effectiveness of meristem‐tip culture for the eradication of multiple viruses from garlic.     

Construction of Plant Expression Vectors and Identification of Transgenic Potato Plants

Potato virus X (PVX), potato virus Y (PVY) and potato leaf roll virus (PLRV) infection in potato may result in the loss of centrification of seed potatoes and affect the quality and yield of potatoes in agricultural production. The authors cloned coat protein (cp) genes of PVX, PVY and PLRV and constructed two kinds of plant expression vector which contain PVX and PVY or PVY and PLRV cp genes. Three major commercial cultivars of potato and one cultivar of tobacco were transformed via Agrobacterium tumefaciens mediated procedure. Transgenic plants were confirmed by PCR analysis. Transgenic tobacco plants containing both PVX and PVY cp genes were significantly resistant to PVX and PVY infection via mechanical inoculation.     

双价外壳蛋白基因植物表达载体的构建及马铃薯转基因植株的鉴定

在克隆了马铃薯Ｘ病毒（ＰＶＸ）、马铃薯Ｙ 病毒（ＰＶＹ）和马铃薯卷叶病毒（ＰＬＲＶ）的外壳蛋白基因的基础上,构建同时包含ＰＶＸ和ＰＶＹ 与ＰＶＹ 和ＰＬＲＶ 两个外壳蛋白基因植物表达框架的表达载体,通过农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ ｔｕｍｅｆａｃｉｅｎｓ）介导转化烟草（Ｎｉｃｏｔｉａｎａｔａｂａｃｕｍ ）和生产上常用的几个马铃薯（Ｓｏｌａｎｕｍ ｔｕｂｅｒｏｓｕｍ ）优良品种：“Ｆａｖｏｒｉｔａ”、“虎头”、“克４”。经ＰＣＲ检测证明外源基因已整合到植物的染色体上,得到批量转基因植株。在转ＰＶＸ＋ＰＶＹ 外壳蛋白基因的烟草上接种ＰＶＸ （５ μｇ／ｍ Ｌ）、ＰＶＹ（２０ μｇ／ｍ Ｌ）病毒,得到有一定抗性的植株