Establishment of Multiplex PCR for Three Virus of Potato

[Objective]The aim was to establish the multiplex PCR method for three virus of potato：PVA（potato virus A）,TMV（Tobacco mosaic virus）and PVY（potato virus Y）.[Method]According to the PVA,TMV and PVY sequences available in GenBank,pairs of primer were designed for establishing a multiplex PCR method,and constructing recombinant plasmid of target genes by PCR amplified of three viruses as reference standard simple to be used in sensitivity test;PVX（Potato virus X）,PVM（Potato virus M）,PVS（Potato virus S）,PVV（Potato virus V）and CMV（Cucumber mosaic virus）were used to carry out the specificity test and detection of 11 samples which were suspected of virus infected.[Result]The detection limit for PVA,TMV and PVY was 14,14 and 14 copies/ml,respectively.No cross-reactivity was observed with other viruses.Seven of 11 samples were infected by three viruses.[Conclusion]The multiplex PCR for PVA,TMV,PVY three viruses of potato was established successfully,which had provided basis for the detection technology of potato virus.

Aphids and their transmitted potato viruses: A continuous challenges in potato crops

Aphid is one of the most destructive insect pests on cultivated plants in temperate regions.Their piercing-sucking mouthparts and phloem feeding behavior directly damage crops and deplete plant nutrients.Potato(Solanum tuberosum L.)is one of the most important food sources on the planet,and several aphid species,e.g.,Myzus persicae(Sulzer)(green peach aphid)and Macrosiphum euphorbiae(Thomas)(potato aphid)(Hemiptera:Aphididae)colonize potato and transmit several economically important viruses.Aphid-transmitted potato viruses have been emerging all over the world as a very serious problem in potato production,inducing a wide variety of foliar and tuber symptoms,leading to severe yield reduction and loss of tuber quality.In this review,recent advances in understanding the interactions of potato viruses with their hosts,aphid vectors and the environment are described.

Monoclonal antibody-based serological detection of potato virus M in potato plants and tubers

Potato virus M(PVM) is one of the common and economically important potato viruses in potato-growing regions worldwide. To investigate and control this viral disease, efficient and specific detection techniques are needed. In this study, PVM virions were purified from infected potato plants and used as the immunogen to produce hybridomas secreting PVM-specific monoclonal antibodies(MAbs). Four highly specific and sensitive murine MAbs, i.e., 1 E1, 2 A5, 8 A1 and 17 G8 were prepared through a conventional hybridoma technology. Using these four MAbs, we have developed an antigen-coated plate(ACP)-ELISA, a dot-ELISA and a Tissue print-ELISA for detecting PVM infection in potato plants and tubers. PVM could be detected in infected potato plant tissue crude extracts diluted at 1:10 240(w/v, g mL^(–1)) by the dot-ELISA or at 1:163 840(w/v, g mL^(–1)) by the ACP-ELISA. The Tissue print-ELISA is the quickest and easiest assay among the three established serological assays and is more suitable for onsite large-scale sample detection. Detection results of the field-collected samples showed that PVM is currently widespread in the Yunnan and the Heilongjiang provinces in China. The field sample test results of the developed serological assays were supported by the results from RT-PCR and DNA sequencing. We consider that the newly established ACP-ELISA, dot-ELISA and Tissue print-ELISA can benefit PVM detection in potato plant and tuber samples and field epidemiological studies of PVM. These assays can also facilitate the production of virus-free seed potatoes and breeding for PVM-resistant potato cultivars, leading to the successful prevention of this potato viral disease.

Simulation modelling of potato virus Y spread in relation to initial inoculum and vector activity

Potato virus Y(PVY)is a non-persistent virus that is transmitted by many aphid species and causes significant damage to potato production.We constructed a spatially-explicit model simulating PVY spread in a potato field and used it to investigate possible effects of transmission efficiency,initial inoculum levels,vector behavior,vector abundance,and timing of peak vector activity on PVY incidence at the end of a simulated growing season.Lower PVY incidence in planted seed resulted in lower virus infection at the end of the season.However,when populations of efficient PVY vectors were high,significant PVY spread occurred even when initial virus inoculum was low.Non-colonizing aphids were more important for PVY spread compared to colonizing aphids,particularly at high densities.An early-season peak in the numbers of noncolonizing aphids resulted in the highest number of infected plants in the end of the season,while mid-and late-season peaks caused relatively little virus spread.Our results highlight the importance of integrating different techniques to prevent the number of PVY-infected plants from exceeding economically acceptable levels instead of trying to control aphids within potato fields.Such management plans should be implemented very early in a growing season.

Research Progress and Prospect on Prevention and Control Measures of Sweet Potato Virus Disease

Sweet potato virus disease is a serious biological threat to sweet potato,which seriously affects the development of sweet potato industry in China.This paper gives a brief introduction to the main defensive measures of sweet potato virus disease,such as cutting of virus infection source,killing of viral transmission media,cultivation and application of virus-free sweet potato,use of antiviral agents,breeding of varieties with virus resistance,and mild strain cross protection,so as to provide some reference for this field.

Three sensitive and reliable serological assays for detection of potato virus A in potato plants

Vegetative propagation of seed potato often allows passaging of viruses to seed tubers,resulting in significant yield losses and reduction of potato tuber quality.Thus,virus detection approach is crucial for effective virus management programs and the production of virus-free seed potatoes.Among the reported potato-infecting viruses,potato virus A(PVA)is considered as one of the most important viruses in potato-growing regions worldwide.This study prepared four hybridoma lines secreting PVA-specific monoclonal antibodies(MAbs)(2D4,8E11,14A6 and 16H10)using purified PVA virions as an immunogen.Western blotting results indicated that all the four MAbs reacted strongly and specifically with the putative capsid protein of PVA.Using these four MAbs,this study developed antigen-coated plate enzyme-linked immunosorbent assay(ACP-ELISA),Dot-ELISA and Tissue print-ELISA for detection of PVA infection in potato plants.The results indicated that PVA can be detected in crude tissue extracts from infected potato plants diluted up to 1:327680(w/v,g m L^(-1))by ACP-ELISA or up to1:10240 by Dot-ELISA.The Tissue print-ELISA is the quickest and easiest approach among the three serological assays,and is more suitable for onsite large-scale potato screening programs.Further analyses of field-collected potato samples showed that the sensitivities and specificities of the three serological approaches were similar to those of RT-PCR in PVA detection and confirmed that PVA is currently widespread in Yunnan and Zhejiang provinces of China.Hence,the results strongly suggest that these highly sensitive serological approaches based on PVA-specific MAbs are useful and powerful for PVA-free seed potato production programs and PVA field surveys.

烟草马铃薯X病毒(Potatoes Virus X, PVX)快速检测方法及抗性诱导物质抑制效果研究

对注射PVX后烟苗的发病症状;总RNA快速检测PVX侵染烟草叶片的方法;注射抗病毒抗性诱导物质处理后周围病斑扩散情况;摩擦法接种抗性诱导物质效果等方面进行了研究。结果表明:注射1周后观察云烟叶片,PVX在云烟中可产生严重的花叶症状,PVX在云烟中表达成功;提取RNA,利用病毒的引物检测PVX病毒的纯度,经检测RNA OD260/280 = 1.955,C = 755.89 ng/μL,说明发病叶片是由纯的PVX引起;注射1 d后用抗性诱导物质比施壮处理叶片,2 d后利用UV光照射并拍照,发现经比施壮处理后,病毒扩散斑明显小于非施用处理;在抗性诱导物质胁迫下,病毒复制效率运动扩散能力都有所降低,抗性诱导物质处理后的叶片荧光斑虽有所扩大,但病毒尚未发生明显转移。由此可见:提取侵染烟草叶片总RNA进行PVX纯度检测,可灵敏地检测出马铃薯X病毒,可作为快速评价感染马铃薯X病毒的指标。抗性诱导物质(比施壮)可抑制马铃薯X病毒的扩散。

Potato Virus Y mRNA Expression Knockdown Mediated by siRNAs in Cultured Mammalian Cell Line

RNA interference(RNAi) is a powerful tool for functional gene analysis which has been successfully used to downregulate the expression levels of target genes.The goal of this research was to provide a highly robust and concise methodology for in-vitro screening of efficient siRNAs from a bulk to be used as a tool to protect potato plants against PVY invasion.In our study,a 480bp fragment of the capsid protein gene of potato virus Y(CP-PVY) was used as a target to downregulate PVY mRNA expression in-vitro,as the CP gene interferes with viral uncoating,translation and replication.A total of six siRNAs were designed and screened through transient transfection assay and knockdown in expression of CP-PVY mRNA was calculated in CHO-k cells.CP-PVY mRNA knockdown efficiency was analyzed by RT-PCR and real-time PCR of CHO-k cells co-transfected with a CP gene construct and siRNAs.Six biological replicates were performed in this study.In our findings,one CP gene specific siRNA out of a total of six was found to be the most effective for knockdown of CP-PVY mRNA in transfected CHO-k cells by up to 80%-90%.

Screening and Identification of Tobacco Mutants Resistant to Potato Virus Y( PVY)

[ Objective] This study aimed to screen and identify tobacco mutants resistant to potato virus Y （PVY）, thus laying the foundation for obtaining PVY resistance genes. [ Method ] At seedling stage, tobacco mutant materials were inoculated with PVY virus and preliminarily screened by naked-eye observation. Enzyme-linked immunosorbent assay （ELISA）, real-time fluorescence quantitative PCR and test strip assay were performed to further identify the pre-screened PVY- resistant seedlings. [ Result ] In 2011, two highly PVY-resistant tobacco mutants （MZE2-15 and MZE2-16） and three PVY-tolerant mutants （MZE2-70, MZE2- 207 and MZE2-228） were obtained, which were further screened and identified in 2012. According to the results, tobacco mutant materials MZE2-407 and MZE2- 428 were susceptible to PVY; mutant materials MZE2-16 and MZE2-15 were resistant to PVY. [ Conclusion] This study provide theoretical basis for the control of tobacco PVY disease.

Sweet Potato Leaf Curl Virus: Coat Protein Gene Expression in <i>Escherichia coli</i>and Product Identification by Mass Spectrometry

Sweet potato is one of the first natural GMOs, genetically modified 8000 years ago by Agrobacterium rhizogenes as reported recently by Kyndt et al. A section of 10 kbp long DNA (Transferred- DNA or T-DNA) of the Ri (Root-inducing) plasmid was transferred to the plant genome by A. rhizo-genes and has been maintained in all 291 hexaploid sweet potato cultivars of the world. The maintenance in the sweet potato genome and expression of two T-DNA genes for tryptophan-2-monooxygenease (iaaM) and for indole-3-acetamide hydrolase (iaaH) are likely to be physiologically significant since these enzymes convert tryptophan to indole-3-acetic acid, a major plant growth hormone auxin. Sweet potato (Ipomoea batatas (L.) Lam) is ranked the third most important root crop after potato and cassava, and the seventh in global food crop production with more than 126 million metric tons. Although sweet potato originated in Central or South America, China currently produces over 86% of world production with 109 million metric tons. In the United States, North Carolina is the leading producer with 38.5% of the 2007 sweet potato production, followed by California, Mississippi, and Louisiana with 23%, 19%, and 15.9%, respectively. Leaf curl virus diseases have been reported in sweet potato throughout the world. One of the causal agents is Sweet potato leaf curl virus (SPLCV) belonging to the genus Begomovirus (family Geminiviridae). Although SPLCV does not cause symptoms on Beauregard, one of the most predominant sweet potato cultivars in the US, it can reduce the yield up to 26%. Serological detection of SPLCV is not currently available due to the difficulties in obtaining purified virions that can be used as antigen for antiserum production. In attempts to obtain the coat protein (CP) of SPLCV for antibody production, primers were designed to amplify the CP gene. This gene was cloned into the expression vector pMAL-c2E as a fusion protein with maltose-binding protein, and transformed into Escherichia coli strain XL1-Blue. After gene induction, a fusion protein of 72 kDa was purified by amylose affinity chromatography. The yield of the purified fusion protein was approximately 200 μg/liter of bacterial culture. Digestion with enterokinase cleaved the fusion protein into a 42.5 kDa maltosebinding protein and a 29.4 kDa protein. The latter protein was identified by mass spectrometry analysis as the coat protein of SPLCV based on the fact that the mass spectrometry elucidated the sequences corresponding to 37% of amino acid positions of the SPLCV coat protein.

Cloning and Sequencing of Kappa Light Chain Gene of a Mouse Monoclonal Antibody Directed Against Potato Virus Y

A cDNA library was constructed in λgt11 vectors, complementary to the mRNA isolated from a mouse hybridoma raised against potato virus Y(PVY). Thirty cDNA clones were selected from the cDNA library by in situ immunohybridization with goat anti-mouse kappa-chain-specific antibody conjugated to alkaline phosphatase, from which one clone, k6, having the largest insert was characterized by sequence analysis. The result shows that the immunoglobulin messenger RNA corresponding to k6 is 956 nucleotides in length excluding the poly(A) region, among which 31 bases code for the 5’ non-coding region, 57 for the leader sequence of the protein, 657 for the mature protein and 211 for the 3’ non-coding region. Comparison of deduced amino acid sequences of the protein and other kappa light chains shows that they share a 100% identity in their constant regions(CL) and 93.7% identity in their variable regions(VL). The kappa light chain encoded by k6 is considered to be specific to PVY since only one type of light chain is expressed in the hybridoma.

Correlations between delayed fluorescence of chlorophyll, metabolism and yield of plants. III. Influence of viral infection on field plants and new technology of clone selection of virus-free planting potato

At the end of potato plants vegetation virus in-fection induced both decrease in maximum amplitude of delayed fluorescence maximal amplitude and increase half time of its decrease, as well as reduction in the amount of stems, plants’ height and assimilation area surface, yield, acceleration of plants development and their early die-off. The differences of DF pa-rameters and yields between strongly and weakly infected plants increase in case of a combined virus infection. In industrial test of the selection of virus-free planting potato by the use of DF parameter, a rise in the yield and de-crease degree of viral infection of crops was obtained.

Farmers’ Knowledge of Potato Viruses and Management Strategies in the Western Highlands of Cameroon

Potato (Solanum tuberosum L.), important staple food and a source of income to small-scale farmers, is mostly cultivated in Cameroon in the Western Highlands. Production constraints are exerted on this crop by many pathogens including viruses responsible for considerable yield losses. This study aimed at assessing the perception of farmers on the virus diseases that can affect potatoes, and to identify the control methods adopted against them. A semi-structured survey was carried out among 230 farmers in 24 villages of the Western Highlands zone of Cameroon. Out of these farmers, 80.87% had never heard of potato viruses. Those having pre-knowledge about potato viruses were 19.13%. Among the latter, 16.52% had heard of potato viruses and transmission mode during capacity building workshops while 2.61% didn’t know about the means of transmission. Insect control is essentially chemical (100%). However, few farmers use biological methods such as intercropping (7.39%) and application of plant extracts (4.78%) to control insects. Twelve plant species, belonging to nine families, were mentioned for insect control. In addition to plants, farmers also use wood ash and rabbit urine for insect control. These results show the knowledge gap possessed by farmers with respect to potato viruses and their transmission mode. It is thus speculated that this spans to other crops in Cameroon settings. This finding can serve as a base and a working document for policymaking to ameliorate teaching, research and devilment related to plant viruses for better sustainable food production.

Effects of Synergism Between PVY and PVX on Virus Titer and Cell Ultrastructure in Tobacco Plants

The viruses titer and the ultrastructure of infected cells in tobacco host (Nicotiana tabacum cv. Samsun), which doubly infected with potato virus Y necrosis strain (PVYN) and potato virus X (PVX), were studied under greenhouse conditions. The results indicated that PVYN and PVX interacted synergistically. and tobacco plants which doubly infected with PVX and PVYN could greatly increase symptom severity as compared with that induced by the single virus. As determined by triple antibody sandwich enzyme-linked immunosor-bent assay (TAS-ELISA), the titer of PVX in the tobacco leaves infected with both PVYN and PVX was up to 9.10 times higher than the plants infected with PVX only. No significant differences in PVYN titer were detected between singly and doubly infected plants. The enhancement of PVX titer in doubly infected plants was evident in greenhouse and was independent of the virus strains, tested seasons, intervals between PVYN and PVX inoculation. When sections of doubly infected leaves were examined with an electron microscope, it could be frequently found that cells contained pinwheels and large masses of PVX-like particles, pinwheels and laminate inclusions, or all three structures, most of them were swollen, and their chloroplast and other organella were destructed heavily. This suggested that doubly infected cells were involved in the enhancement phenomenon, which seemed to be the result of an increase in the amount of PVX synthesized in them.

甘薯褪绿矮化病毒西非株系RT-RPA-LFD检测方法的建立与应用

【目的】利用逆转录酶重组酶聚合酶扩增(reverse transcriptase recombinase polymerase amplification,RT-RPA)结合侧向流层析(lateral flow dipstick,LFD)试纸条技术,建立甘薯褪绿矮化病毒(sweet potato chlorotic stunt virus,SPCSV)西非株系(west African strain,WA)的RT-RPA-LFD检测方法。【方法】根据SPCSV-WA外壳蛋白基因和热激蛋白基因的保守序列设计并筛选扩增效果好、特异性强的引物和探针。然后对引物和探针的浓度、扩增体系、温度、反应时间等条件进行优化,建立SPCSV-WA的RT-RPA-LFD检测方法。利用该方法对SPCSV-EA、甘薯羽状斑驳病毒(sweet potato feathery mottle virus,SPFMV)、甘薯潜隐病毒(sweet potato latent virus,SPLV)、甘薯G病毒(sweet potato virus G,SPVG)等常见的甘薯病毒进行检测,验证方法的特异性;将感染SPCSV-WA甘薯叶片样品总RNA进行10倍梯度稀释,分别采用RT-PCR和RT-RPA-LFD进行检测,比较RT-PCR和RT-RPA-LFD方法的灵敏度;对采自田间的甘薯样品和试管苗样品进行RT-RPA、RT-RPA-LFD和RT-PCR检测,验证该方法的实用性。【结果】建立了SPCSV-WA的RT-RPA-LFD检测方法,最适引物为CSV357F/R,探针CSV-CP-Probe(47 bp),引物和探针工作浓度分别为0.2和0.06μmol·L^(-1),温度为42℃,时间5 min。该方法可对SPCSV-WA进行特异性检测,与甘薯上其他常见病毒无交叉反应。RT-RPA-LFD最低可检测到总RNA稀释至10^(-4)溶液,而RT-PCR最低检测到总RNA稀释至10^(-3)溶液,RT-RPA-LFD灵敏度是RT-PCR的10倍。田间采集的22份甘薯样品经RT-PCR、RT-RPA和RT-RPA-LFD检测,均检出11份阳性样品,3种方法检测结果一致。28份试管苗样品的检测结果表明,RT-PCR和RT-RPA-LFD检测结果一致,均检出5份阳性样品。【结论】建立了SPCSV-WA的RT-RPA-LFD检测方法,该方法具有快速、简便、特异、灵敏、可视的特点,既可用于甘薯脱毒试管苗样品的病毒检测,也适用于基层单位田间甘薯病毒样品的现场快速检测。

‘藏农薯1号’不同级别种薯的病毒种类及生物学性状比较

种薯退化是制约马铃薯(Solanum tuberosum L.)产业发展的重要因素,不同级别种薯播种后在田间发病情况通常差异较大,进而影响其生物学性状及经济效益。以西藏自治区选育品种‘藏农薯1号’为试验材料,分析各级种薯(原原种、原种、一级种、二级种)在田间病毒再侵染情况,比较各级种薯植株生长发育、块茎品质和产量等生物学特性。原原种植株中仅检测到马铃薯S病毒,其他级别种薯植株中可检测到马铃薯卷叶病毒和S病毒。同时,原原种植株田间马铃薯卷叶病毒和S病毒病发病率和病情指数均较低,二级种则相反。在生长发育过程中,现蕾期叶片的叶绿素含量和光合速率最高;随着种薯级别降低,叶绿素含量和光合速率呈下降趋势,而株高差异小。不同级别种薯块茎淀粉和干物质含量差异小,还原糖含量随着种薯级别降低而降低。产量和商品薯率统计结果显示,原原种最高、二级种最低,而原种和一级种之间未见显著差异。考虑到种薯成本和经济效益,在‘藏农薯1号’马铃薯生产中,种薯应优先选用原种和一级种。

河北保定地区黄瓜花叶病毒与马铃薯Y病毒复合侵染叶用莴苣分子鉴定

利用RT-PCR方法对采集自河北保定地区的疑似叶用莴苣病毒病样品进行分子生物学鉴定,并构建系统发育进化树。结果表明,特异性引物CMV和PVY分别扩增得到大小为260、420 bp的单一条带;通过核苷酸序列Blast比对及系统进化树分析,河北保定地区叶用莴苣病毒分离物分别与印度番茄CMV分离物(MN514839.1)、印度马铃薯PVY分离物(JX945850.1)聚在同一分支上,同源性分别为95%、92%。本研究首次检测到河北保定地区叶用莴苣病毒病是由CMV与PVY复合侵染所致。

外源ABA、ZR及IAA对甘薯脱毒苗生长及结薯的影响

以甘薯品种龙薯9号的脱毒试管苗为试验材料,采用盆栽基质培养、向根部营养液加入植物激素的处理方式,设置单因素试验,分别探究了0(CK)、0.7、7.0、14.0 mg/L的ABA与IAA,以及0(CK)、0.8、8.0、16.0 mg/L ZR对脱毒苗根生长发育及结薯的影响。结果表明:0.8 mg/L ZR处理的单株结薯数和单株薯重分别较CK提高2.25个和5.93 g;0.7 mg/L ABA处理的单株结薯数和单株薯重分别较CK提高2.00个和5.43 g;14 mg/L IAA处理可显著增加脱毒苗的根长和茎长。综上所述,在脱毒薯生产过程中,根部施加0.7 mg/L ABA或0.8 mg/L ZR均可显著增加脱毒苗的单株结薯数和单株薯重,根部添加14 mg/L IAA可显著促进脱毒苗根与茎的伸长。本试验结果可为脱毒种薯种苗高效生产提供技术支撑。

马铃薯转录组数据中的病毒序列挖掘

转录组测序数据中包含寄主所感染的病毒序列信息。为分析和验证马铃薯转录组数据中存在的病毒序列,试验收集了关于马铃薯块茎发育、抗病、抗逆等研究的9个马铃薯转录组测序数据样本,过滤掉来源于马铃薯转录本的读段,组装并与病毒数据库进行本地比对,获得样本中相关病毒序列的种类和数量信息。病毒来源的读段在各样本中普遍存在,占总读段的0.12%~20.24%。这些读段组装得到10种植物病毒,包括9种RNA病毒和1种DNA病毒。其中来源于马铃薯S病毒(Potato virus S,PVS)的序列最多,组装得到28条近全长序列。进一步系统进化分析表明,这些样品中同时存在PVSA和PVSO两种株系类型。此外,通过RT-PCR对转录组数据原始样本中的马铃薯H病毒(Potato virus H,PVH)、马铃薯卷叶病毒(Potato leafroll virus,PLRV)、马铃薯M病毒(Potato virus M,PVM)、PVS、马铃薯X病毒(Potato virus X,PVX)和马铃薯Y病毒(Potato virus Y,PVY)6种病毒进行检测,结果显示RT-PCR验证结果与测序数据组装结果基本一致。该研究为马铃薯病毒多样性和进化分析提供一种高通量测序的方法。

马铃薯育种现状及发展对策

本文基于马铃薯现阶段的育种工作情况,总结分析了其有待进一步提升的环节,并提出相应的发展对策。现阶段,马铃薯育种主要依据其无性繁殖特性进行,具有简便、有效和确保薯种纯度等优点,同时做好薯种脱毒、病虫害防治和田间管理等工作;育种过程中,需进一步提升马铃薯品种资源收集、种薯生产能力、种薯生产技术、脱毒种薯质量以及马铃薯产品加工能力。提出马铃薯育种工作发展对策,包括建立良好的育种环境、科学引进马铃薯品种、创新马铃薯育种方式以及更新生产设备及生产技术,为促进马铃薯产业的规范化和合理化发展提供参考。