Chemotherapeutical Elimination of Potato Virus X from Potato Stem Cuttings

Potato virus X was completely eliminated from all infected potato stem cuttings grown in nutrient media containing 0.02 or 0.03 % 2,4-dioxohexahydro-1,3,5-triazine (DHT). When DHT was added to the media in concentrations of 0.01 and 0.005% the efficiency by which the virus was eliminated differed between the varieties tested. The method is less time-consuming than the generally used meristem (axillary-) tip culture in combination with chemo- or thermotherapy.     

Expression of Potato Virus Y Coat Protein Gene in Transgenic Potato

Potato virus Y (PVY) N coat protein (CP) coding sequence was cloned into a plant expression vector pMON316 under the CaMV 35S promoter. Leaf discs of potato (Solanum tuberosum) were used to Agrobacterium-mediated gene transfer. A large number of regenerated putative transgenic plants were obtained based on kanamycin resistance. Using total DNA purified from transgenic plants as templates and two oligonucleotides synthesized from 5' and 3' of the PVY coat protein gene as primers, the authors carried out polymerase chain reaction (PCR) to check the presence of this gene and obtained a 0. 8 kb specific DNA fragment after 35 cycles of amplification. Southern blot indicated that the PCR product was indeed PVY CP gene which had been integrated into the potato genome. Enzyme-linked immunosorbent assay (ELISA) of our transgenic plants showed that CP gene was expressed in at least some transgenic potato plants.     

马铃薯卷叶病毒基因间隔区转化的马铃薯抗病性研究

将本室合成、克隆的马铃薯卷叶病毒（ＰｏｔａｔｏＬｅａｆｒｏｌＶｉｒｕｓ,ＰＬＲＶ）中国分离株的基因间隔区（ｉｎｔｅｒｇｅｎｉｃｓｅｑｕｅｎｃｅ,ＩＳ）双链ｃＤＮＡ以正、反向两种方式分别构建于转化载体ｐＲＯＫ２中,通过致瘤农杆菌介导,以马铃薯叶圆片为转化材料,转化马铃薯栽培品种Ｄｅｓｉｒｅ,获得了转基因植株。卡那霉素抗性分析和ＰＣＲ检测目的基因,证明ＰＬＲＶＩＳ双链ｃＤＮＡ已经整合到转基因马铃薯的染色体基因组中。将转基因植株移栽网棚用蚜虫接种ＰＬＲＶ,观察症状并用酶联免疫吸附测定（ＥＬＩＳＡ）检测转基因植株中ＰＬＲＶ含量。结果表明,表达ＰＬＲＶＩＳ正意和反意ＲＮＡ的转基因植株,接种病毒后表现无症状或症状轻微,ＰＬＲＶ平均滴度均较未转基因对照植株低。表达正意ＲＮＡ的转基因植株ＰＬＲＶ滴度降低４３％～７２％,表达反意ＲＮＡ的转基因植株ＰＬＲＶ滴度降低７２％～８６％,由此可见,表达ＰＬＲＶＩＳ反意ＲＮＡ的转基因马铃薯对ＰＬＲＶ抗性较强。     

马铃薯卷叶病毒基因间隔区转化的马铃薯抗病性研究

将本室合成、克隆的马铃薯卷叶病毒(Potato Leafroll Virus, PLRV)中国分离株的基因间隔区(intergenic sequence, IS)双链cDNA以正、反向两种方式分别构建于转化载体pROK2中,通过致瘤农杆菌介导,以马铃薯叶圆片为转化材料,转化马铃薯栽培品种Desiree,获得了转基因植株.卡那霉素抗性分析和PCR检测目的基因,证明PLRV IS双链cDNA已经整合到转基因马铃薯的染色体基因组中.将转基因植株移栽网棚用蚜虫接种PLRV,观察症状并用酶联免疫吸附测定(ELISA)检测转基因植株中PLRV含量.结果表明,表达PLRV IS正意和反意RNA的转基因植株,接种病毒后表现无症状或症状轻微,PLRV平均滴度均较未转基因对照植株低.表达正意RNA的转基因植株PLRV滴度降低43%～72%,表达反意RNA的转基因植株PLRV滴度降低72%～86%,由此可见,表达PLRV IS反意RNA的转基因马铃薯对PLRV抗性较强.     

Chimeric Virus as a Source of the Potato Leafroll Virus Antigen

Large quantities of potato leafroll virus (PLRV) antigen are difficult to obtain because this virus accumulates in plants at a low titer. To overcome this problem, we constructed a binary vector containing chimeric cDNA, in which the coat protein (CP) gene of the crucifer infecting tobacco mosaic virus (crTMV) was substituted for the coat protein gene of PLRV. The PLRV movement protein (MP) gene, which overlaps completely with the CP gene, was doubly mutated to eliminate priming of the PLRV MP translation from ATG codons with no changes to the amino acid sequence of the CP. The untranslated long intergenic region located upstream of the CP gene was removed from the construct. Transcribed powerful tobamovirus polymerase of the produced vector synthesized PLRV CP gene that was, in turn, translated into the protein. CP PLRV packed RNAs from the helical crTMV in spherical virions. Morphology, size and antigenic specificities of the wild-type and chimeric virus were similar. The yield of isolated chimera was about three orders higher than the yield of native PLRV. The genetic manipulations facilitated the generation of antibodies against the chimeric virus, which recognize the wild-type PLRV.     

Survival and Transmission of Potato Virus Y,Pepino Mosaic Virus,and Potato Spindle Tuber Viroid in Water

Hydroponic systems and intensive irrigation are used widely in horticulture and thus have the potential for rapid spread of water-transmissible plant pathogens. Numerous plant viruses have been reported to occur in aqueous environments, although information on their survival and transmission is minimal, due mainly to the lack of effective detection methods and to the complexity of the required transmission experiments. We have assessed the role of water as a source of plant infection using three mechanically transmissible plant pathogens that constitute a serious threat to tomato and potato production: pepino mosaic virus (PepMV), potato virus Y (PVY), and potato spindle tuber viroid (PSTVd). PepMV remains infectious in water at 20 ± 4°C for up to 3 weeks, PVY (NTN strain) for up to 1 week, and PSTVd for up to 7 weeks. Experiments using a hydroponic system show that PepMV (Ch2 genotype) and PVY (NTN strain) can be released from plant roots into the nutrient solution and can infect healthy plants through their roots, ultimately spreading to the green parts, where they can be detected after a few months. In addition, tubers developed on plants grown in substrate watered with PSTVd-infested water were confirmed to be the source of viroid infection. Our data indicate that although well-known pathways of virus spread are more rapid than water-mediated infection, like insect or mechanical transmission through leaves, water is a route that provides a significant bridge for rapid virus/viroid spread. Consequently, water should be taken into account in future epidemiology and risk assessment studies.     

Functionally Homologous Host Components Recognize Potato Virus X in Gomphrena globosa and Potato

All known isolates of potato virus X (PVX), with the exception of a South American isolate PVXHB, induce an extreme resistance response on potato carrying the Rx gene and elicit the production of necrotic lesions on Gomphrena globosa: PVXHB establishes systemic infection on Rx genotypes of potato and infects the inoculated leaf of G. globosa without lesion formation. Previously, we have shown that the Rx-mediated resistance is affected by a feature of the coat protein that depends on the presence of a threonine residue at position 121 in the coat protein of PVXCP4 and that the resistance is an induced response expressed in protoplasts of potato with the Rx genotype. In this study, we provide evidence, based on the analysis of PVXCP4/PVXHB hybrids, that the elicitation of lesions on G. globosa also requires the presence of a threonine residue at position 121 of the viral coat protein. The lesion-forming phenotype was not associated with the ability of the viral isolate to accumulate in the infected plant. We therefore propose that there is a homologous component of both potato carrying Rx and G. globosa that interacts with a feature of the PVX coat protein and, following the interaction, activates an induced response in the plant cell.     

Effects of Sweet Potato Feathery Mottle Virus and Sweet Potato Chlorotic Stunt Virus on the Yield of SweetPotato in Uganda

Sweet potato feathery mottle virus (SPFMV) and Sweet potato chlorotic stunt virus (SPCSV) are the most common viruses infecting sweetpotato in Uganda. Field plots planted with graft inoculated plants of virus‐free cultivars Beauregard, Dimbuka, Ejumula, Kabode and NASPOT 1 were used to assess the effect of SPFMV and SPCSV on yield and quality of sweetpotatoes in two agro‐ecologies. SPFMV spreads rapidly to control plots at Makerere University Agricultural Research Institute Kabanyolo (MUARIK), and these plots had similar yields to those singly infected with SPFMV but at the National Semi Arid Resource Research Institute (NaSARRI) where SPFMV spreads slowly, plots infected with SPFMV yielded 40% less than the control. Recovery from SPFMV appeared to be more frequent at NaSARRI than at MUARIK. Infection by SPCSV alone resulted in yield losses of 14–52%, while mixed infections of SPFMV+SPCSV resulted in yield losses in both locations of 60–95% depending on the cultivar. SPCSV and mixed infections of SPFMV+SPCSV also reduced the number of roots formed as well as the diameter of the roots, resulting in a greater length to diameter ratio compared to the healthy control. This study, therefore, confirms that both SPFMV and SPCSV, both singly and when mixed, can reduce yield, the extent depending on the cultivar. To mitigate the effect of these viruses, farmers should use clean planting materials of resistant varieties.     

21. Purification of Potato Leaf Roll Virus

ABSTRACT

Potato leaf roll virus was purified by a combination of Takanami and Kubo's method and the use of the extraction and thin layer rotors and finally by centrifugation in tubes filled with beads.     

马铃薯卷叶病毒单克隆抗体的制备

采用杂交瘤技术,以马铃薯卷叶病毒(Potato Leafroll Virns,PLRV)为抗原,用直接将病毒注入脾脏和随后尾静脉注射的方法,免疫BALB/C小鼠。将免疫小鼠的脾细胞与小鼠骨髓瘤细胞SP2/0融合。用Dot-ELISA和间接血凝试验筛选分泌抗马铃薯卷叶病毒抗体的阳性克隆,建立了分泌抗PLRV单克隆抗体的杂交瘤细胞株。用微量玻片双扩散法测定单克隆抗体亚类为IgG_1和IgG2a,轻链为λ。注射杂交瘤细胞株A_1、A_3、C_3和D_3于小鼠腹腔,制备出含高效价单克隆抗体的腹水。用获得的四种单克隆抗体对马铃薯卷叶病毒15个分离物进行了鉴定。     

Potato Gene Xi Confers Inoculum Dependent Resistance to Potato Virus X Replication in Protoplasts

Protoplasts were obtained from genetically related Argentinepotato (Solanum tuberosum tuberosum) cultivars: Serrana INTAand Huinkul MAG and from an European cultivar: Spunta. SerranaINTA is resistant to potato virus X (PVX) infection becauseit carries the major reaction gene Xⁱ (believed to be the sameas Rx_ac1) while Huinkul MAG and Spunta are susceptible. Protoplastswere inoculated with a purified preparation of PVXcp (a SouthAmerican isolate) and then assayed for PVX concentration atdifferent times postinfection by enzyme-linked immunosorbentassay (ELISA), immunofluorescence and nucleic acid hybridization.PVX multiplication rates in Serrana INTA were about fifteentimes slower than in susceptible genotypes when cell-bound viruslevels just after infection were in the range of 0.01 to 0.1ng PVX per viable protoplast. However, when inoculum levelswere raised to 1 ng PVX per viable protoplast, PVX multiplicationwas about the same in all three genotypes. To rule out geneticbackground effects in this behaviour, protoplasts of an ArgentineS. acaule clone (PI: 320277) likely carrying the same resistancegene, were infected with PVX in similar conditions, reproducingthose results obtained with Serrana INTA. The comparison ofPVX replication in protoplasts and whole plants indicate thatalthough Xⁱ gene confers resistance at the cell level it necessitatesof tissue structure to fully express immunity. (Received January 16, 1990; Accepted April 23, 1990)     

Inhibited Long-Distance Movement of Potato Leafroll Virus to Tubers in Potato Genotypes Expressing Combined Resistance to Infection, Virus Multiplication and Accumulation

Plants of two potato clones which, in preliminary greenhouse assessments, showed resistance to multiplication and accumulation of potato leafroll virus (PLRV) were graft or aphid inoculated with the virus and grown in the greenhouse; plants of a moderately susceptible cultivar were used for comparison in all experiments. A high concentration of aphid‐borne inoculum was used to ensure strong infection pressure. Clone M62759 appeared to be highly resistant to PLRV infection, whereas clone PS1706 was more susceptible. Both clones expressed a high level of resistance to virus multiplication, when primary or secondary infection was assayed by enzyme‐linked immunosorbent assay. Moreover, PLRV was detected in only few or none of the progeny plants of clone M62759, which thus strongly inhibited virus transport to tubers. The study on PLRV translocation from aphid‐inoculated shoots to uninoculated shoots sprouted from the same tubers showed that no specific mechanisms are likely to impair PLRV movement through the tubers of the resistant genotypes. These results indicate that three valuable components of the resistance to PLRV are probably closely linked in the genotype, a combination that seems to occur rather rarely in potato clones. Nevertheless, selecting potato genotypes for the complex resistance to PLRV may prove to be a worthwhile part of breeding programmes, provided that the genetic mechanisms governing particular types of resistance are better recognized.     

甘薯羽状斑驳病毒分离提纯及血清学特性

从感染有甘薯羽状驳病毒的牵牛（I．Nil）叶片,通过超速离心,Cscl密度梯度离心提纯SPFMV粒子,每千克叶片可得病毒13－15毫克。电镜观察病毒粒子长度范围在820－860nm,也可见到900nm以上的特长粒子。病毒提纯物的紫外吸收曲线呈典型核蛋白吸收曲线,OD260／OD280＝1．21,免疫电镜检查,该病毒与SPFMV抗血清起阳性反应。