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.

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.

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.

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.

利用农杆菌介导法将PVA-CP基因导入马铃薯品种东农303的研究

以马铃薯早熟品种东农303的微型薯为受体材料,对其再生和农杆菌介导的遗传转化体系进行了研究,成功地将PVA-CP基因导入马铃薯中。结果表明:在加入5mg·L-1ZT和1mg·L-1IAA的培养基上微型薯再生频率最高达95%;农杆菌侵染薯块时,用浓度为OD600=0.5的菌液,侵染5min,共培养2d转化频率最高;在诱导芽阶段加入75mg·L-1的卡那霉素,采用延迟筛选的方法。得到的抗性芽有22株在含100mg·L-1卡那霉素的培养基上生根。经PCR和PCR-Southern杂交检测,15株为阳性植株。初步证明了PVA-CP基因已导入并整合到马铃薯品种东农303的基因组中。

马铃薯A病毒(PVA)运动相关蛋白质的研究进展

综述了马铃薯A病毒(PVA)运动相关蛋白的结构、功能方面的研究进展。

马铃薯试管苗保存毒源PVA技术的研究

报道了含PVA毒源的马铃薯试管苗保存技术及效果。在无菌条件下切取毒源马铃薯茎尖于盛MS培养基的试管中培养,当毒源马铃薯长至12 cm时单节切段进一步扩繁培养。利用免疫电镜法、电镜负染法筛选出3管纯毒源材料。进一步试验表明:免疫电镜法鉴定证明毒源马铃薯试管苗接种3周后病毒含量明显提高;毒源马铃薯试管苗移至花盆中栽培表现出典型的PVA症;用毒源马铃薯的汁液接种指示植物表现典型PVA状,说明毒源马铃薯试管苗的PVA具有侵染力。用马铃薯试管苗保存PVA毒源,克服了传统保存中的不足,提高了保存效果。

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。

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.

Effect of SPVD on Sweet Potato Yield Formation

This study was to investigate the influence of SPVD on the growth devel- opment and yield formation of sweet potato, The virus seeding, landrace, virus-free seedlings of high starch sweet potato XichengO07 were inoculated with SPVD for revealing the interaction mechanism, The results showed that SPVD could result in clustering and dwarfing of sweet potato plant type, smaller leaves and lower effec- tive of leaf area, reduced chlorophyll content, and smaller source, lowered assimila- tive ability and photosynthetic capacity. The flow became smaller, further finally led to the reduced biological yield, and the desorption of SPVD could increase leaf ＂source＂ and the chlorophyll content, improve photosynthetic and flow ability, thus raising the output of production. SPVD could reduce the activities of SOD, POD and CAT in sweet potato plant, increase the content of MDA, decrease antioxidant activity and production, damage the cell membrane. However, virus-free treatment could increase the activities of SOD, POD and CAT in plants, which was helpful to reduce the harm of MDA. After the desorption of SPVD, the production of above- ground was increased by 3.4% and the production of fresh sweet potato was up by 2.9%, while SPVD dissemination could result in the reduction of the aboveground and fresh tubers by 69.9% and 49.1%, respectively.

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。