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二重RT-PCR快速检测马铃薯病毒的方法 总被引：6，自引：0，他引：6
本研究采用传统的蛋白酶K法和病毒RNA简易浸提法,从马铃薯块茎、茎干、叶梗、叶片中提取马铃薯X病毒,马铃薯Y病毒,马铃薯A病毒及马铃薯卷叶病毒RNA,并设计了4种马铃薯病毒引物,优化了二重RT-PCR反应条件,可以同步扩增出上述4种病毒,扩增产生的靶带分别为562bp(PVX)、480bp(PVY)、336bp(PLRV)、255bp(PVA).应用病毒RNA简易制样技术和优化的二重RT-PCR反应条件,可以同步快速检测田间自然感染的马铃薯病毒,此研究还可适合于检测马铃薯脱毒种薯及试管苗,对马铃薯病毒病早期监测有一定的作用. 相似文献
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引进加拿大马铃薯种薯中病毒的检测 总被引：1，自引：1，他引：1
应用酶联免疫吸附法（DAS-ELISA）对2000年种植于网室和田间隔离种植地的引进加拿大马铃薯种薯3个品种进行马铃薯黄矮病毒、马铃薯A病毒（PVA）、马铃薯Y病毒N株系（PVY^n）、马铃薯黑环斑病毒、烟草脆裂病毒、马铃薯X病毒和马铃薯S病毒（PVS）的检测。对网室样品中经ELISA检测怀疑为阳性样品再用组织超薄切片、直沾法和免疫电镜法制备电镜检样品做电镜观察。结果在网室的Russet Burbank品种上检出PVA和PVS,其它两品种检出PVS;田间隔离种植中检出PVY^n和PVS。 相似文献
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病毒病是限制我国马铃薯生产的重要因子之一。本研究通过引物设计和体系优化建立了一套多重RT-PCR检测方法,可以在一个反应体系中同时检测6种马铃薯病毒和1种马铃薯类病毒以及1个马铃薯内参基因,包括马铃薯Y病毒(potato virus Y, PVY)、马铃薯M病毒(potato virus M, PVM)、马铃薯S病毒(potato virus S, PVS)、马铃薯X病毒(potato virus X, PVX)、马铃薯A病毒(potato virus A, PVA)、马铃薯卷叶病毒(potato leaf curl virus, PLRV)、马铃薯纺锤块茎类病毒(potato spindle tuber viroid, PSTVd)和细胞色素c氧化酶亚基Ⅰ(cytochrome c oxidase subunitⅠ,CoxⅠ)基因的特异片段,产物大小依次为181、226、275、565、630、681、359、500 bp,符合理论预期。采用建立的方法以相应病毒和类病毒的质粒作模板进行检测,其检测灵敏度在1.6×10-3～1.8×10-1 ... 相似文献
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基于马铃薯病毒ssRNA的快速制备方法,根据马铃薯病毒CP基因序列设计PVX、PVS、PVY和PLRV特异性引物对、P1基因序列设计PVA特异性引物对,建立了多重RT PCR快速检测体系,可以同步检出复合侵染马铃薯种苗主要病毒,灵敏度比传统的ELISA至少高100倍。结合生物活性稳定剂研制的固相化检测试剂盒,已用于四川和重庆等15个县市田间与苗圃248个马铃薯显症或无症样品的实际检测,表明四川和重庆地区2~3种马铃薯病毒往往复合侵染(PVX、PVA和PVS三种病毒复合侵染或PLRV和PVY二种病毒复合侵染)。 相似文献
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我国马铃薯病毒主要有马铃薯Y病毒(PVY)、马铃薯X病毒(PVX)、马铃薯S病毒(PVS)、马铃薯卷叶病毒(PLRV),常发生复合侵染。根据GenBank中4种马铃薯病毒的外壳蛋白(coat protein,CP)基因全长设计引物,通过RT-PCR扩增得到4种病毒CP基因全长片段,测序结果显示序列同源性96%以上;针对4种病毒CP基因的保守序列分别设计引物,在一个PCR体系中同步对4种病毒进行扩增,得到421、202、516、330bp的特异性条带,优化建立了能同步检测PVY、PVX、PVS和PLRV的多重RT-PCR检测体系。检测结果证明优化后的多重RT-PCR体系能在田间样品中快速、高效地检测出4种病毒。 相似文献
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选用显斑驳症状的兼云香、赤线金钩和叶片黄化的泉乡万圣和泉乡冲天等共4个品种的葡萄母株,龙茎脱毒获得的茎组培苗用生物学和血清学方法检测葡萄母株和组培苗的带毒情况。鉴定结果表明,兼云香母株（1A）带有葡萄B病毒,其组培苗未能脱去该病毒。赤线金钩母株（2A）带有番茄不孕病毒（TAV）和葡萄B病毒（CVB）复合侵染,其组培苗脱去了TAV病毒还带有CVB病毒。万乡泉圣母株（3A）和泉乡冲天母株（4A）带有菊花B病毒,其组培苗均脱去了此病毒,是真正的脱毒组培苗。 相似文献
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基于小RNA深度测序和RT-PCR检测侵染广东省冬种马铃薯的病毒 总被引：1，自引：1，他引：1
为明确侵染广东省冬种马铃薯的病毒种类及优势病毒,结合小RNA深度测序技术及RTPCR检测方法,对采集于广东省冬种马铃薯7个主产区的189份疑似病样进行检测分析。结果表明,经小RNA深度测序技术检测马铃薯病毒病混合样,发现存在马铃薯Y病毒(Potato virus Y,PVY)、马铃薯S病毒(Potato virus S,PVS)和马铃薯卷叶病毒(Potato leaf-roll virus,PLRV)3种病毒。进一步设计3种病毒的特异性引物并利用国内已报道的其它5种马铃薯病毒的特异性引物进行RT-PCR检测,发现189份马铃薯病毒病疑似病样中仅检测到PVY、PVS和PLRV这3种病毒,检出率依次为75.13%、10.05%和4.76%,且3种病毒在马铃薯上还存在复合侵染,复合侵染率为14.19%,其中PVY在各马铃薯产区均可检测到。表明侵染广东省冬种马铃薯的病毒为PVY、PVS和PLRV,其中PVY是优势病毒。 相似文献
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M.P.M. Nagtzaam A.J. Termorshuizen G.J. Bollen 《European journal of plant pathology / European Foundation for Plant Pathology》1997,103(7):597-605
Using potato, eggplant and thorn apple as test plants, the relationship between soil inoculum density and plant infection was studied as a basis for the development of a quantitative bioassay of Verticillium dahliae. A linear relationship was demonstrated (P < 0.05) between soil inoculum density and population density on roots for all three test plants and for soil inoculum density and population density in sap extracted from stems for eggplant. Correlation coefficients were higher with densities on or in roots (R2 varying from 0.45 to 0.99) than with densities in stems (R2 varying from 0.04 to 0.26). With eggplant, population densities on/in root and in sap extracted from stems were significantly correlated at 20 and 25°C with Pearson's correlation coefficients of 0.41 and 0.53, respectively. For potato, root colonization was higher at 15 than at 20°C, whereas the reverse applied to eggplant. Stems of potato were less colonized than stems of eggplant. The pathozone sensu Gilligan (1985) was calculated to be <300 µm, indicating that infection was caused by microsclerotia which were located close to the roots. To assess the density of V. dahliae in plant tissue pipetting infested plant sap on solidified ethanol agar medium without salts yielded higher densities than using pectate medium or mixing sap with molten agar. A bioassay for determining effects of (a)biotic factors on development of V. dahliae in the plant is recommended with eggplants as a test plant, grown in soil infested with 300 single, viable microsclerotia g-1 soil at a matric potential of –6.2 kPa, and incubated at 20°C for 8 weeks. 相似文献
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A reliable, rapid and low-cost screening bioassay, based on measuring lesion sizes, rotten tissue weight and tissue colonization 1 week after the injection of a conidial suspension into the stem scars of mature green tomato fruits, was developed to assess the aggressiveness of Colletotrichum coccodes isolates. This protocol was compared with inoculation of either potato plantlets from in vitro multiplication, or ripe tomato fruits, using C. coccodes isolates from potato and from tomato. Aggressiveness to mature green tomato fruits was scored by measuring lesion size and weight at the scar end of the fruit, while colonization was measured in samples taken from the blossom end. Values of all three disease parameters were significantly higher ( ca . 3 times) with aggressive isolates of the pathogen than with less aggressive ones. High correlation levels among these three parameters were obtained. Also, the aggressiveness to mature green tomatoes was highly correlated with the aggressiveness to potato tissue culture plantlets. It was concluded that lesion size in inoculated tomatoes can be used as a sole measure for estimating the aggressiveness of C. coccodes isolates to either tomato or potato. 相似文献
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Reactions of three Polish potato cultivars to potato virus S (PVS) were investigated at 22°C. Cultivars Tajfun and Tonacja exhibited partial resistance with systemic infection detected in some inoculated plants; cultivar Bryza was susceptible to PVS with systemic infection detected in all inoculated plants. The virus was not detectable by ELISA at 23 days postinoculation (dpi) but was detected after 40 dpi. Infection rate and viral accumulation were significantly lower in Tonacja and Tajfun than in Bryza, but no statistically significant difference between Tajfun and Tonacja was detected. Both susceptible and resistant genotypes displayed various, either common or cultivar-specific, symptoms. Delayed systemic infection at 56 dpi was observed in some cases in Tonacja and Tajfun. Resistance-related alteration of a set of miRNAs and mRNA targets in the tested cultivars in response to PVS at 22°C exhibited inter- and intracultivar variability. The majority of tested genes were altered only in the partially resistant Tajfun and Tonacja but not in the susceptible Bryza. Enhanced expression of AGO1-2, DCL1, stu-miR482 and its target Gpa2 was observed in Tonacja and plants of Tajfun in which PVS was detected, with the highest induction of Gpa2 in Tajfun (30.2-fold). However, their expression remained unchanged or decreased in plants of Tajfun in which PVS was undetected. Increased expression of stu-miR168a and stu-miR172e was observed in Tonacja and the PVS-undetected plants of Tajfun, respectively, but not in the PVS-detected plants of Tajfun. This is the first report on cultivar-specific alteration of miRNA in a potato–PVS resistance interaction. 相似文献
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Endogenous levels of free and conjugated salicylic (SA) and gentisic (GA) acids, both putative signal molecules in plant defence, were analysed in order to investigate their involvement in the resistance of four potato ( Solanum tuberosum ) genotypes with different susceptibilities to Potato virus YNTN (PVYNTN ) infection: the highly susceptible cv. Igor and its extremely resistant transgenic line, the extremely resistant cv. Sante and the tolerant cv. Pentland Squire. The lowest levels of free and conjugated SA were observed in the extremely resistant cv. Sante, while free GA, which was detected in all the other varieties, was absent. The extremely resistant transgenic cv. Igor contained the highest basal total SA level and the lowest level of total GA of all four cultivars. In susceptible cv. Igor, but not in resistant transgenic cv. Igor, a systemic increase of free SA was measured 1 day postinfection (dpi). Even more significant increases of free and conjugated SA and GA were detected 11 dpi when systemic symptoms appeared. In inoculated but not in upper noninoculated leaves of resistant transgenic cv. Igor, significant increase of SA conjugates occurred, but not before 11 dpi. The increase of SA and GA in susceptible cv. Igor could contribute to the general elevated levels of phenolic compounds as a response to stress caused by virus infection. It appears that basal levels of SA and GA do not correlate with resistance to PVYNTN in potato plants. 相似文献
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Joaquim A. F. Vicente Maria S. Santos Anibal E. Vercesi Vítor M. C. Madeira 《Pest management science》1998,54(1):43-51
The herbicide dinitro-o-cresol (DNOC) was evaluated for its effects on bioenergetic activities of potato tuber mitochondria to elucidate its mechanism of action and to compare its toxicological properties with those of the chemically related uncoupler dinitrophenol (DNP). DNOC acts as a typical uncoupler, similarly to the classical uncouplers DNP and carbonylcyanide p-trifluoromethoxyphenyl-hydrazone (FCCP). Low concentrations of DNOC (<100 μM ) maximally stimulate succinate-supported respiration of plant mitochondria, with simultaneous collapse of trans-membrane electrical potential, more efficiently than DNP. The herbicide makes the plant mitochondrial membrane more permeable to protons, acting as a protonophore even in non-energized mitochondria. High concentrations of DNOC (>100 μM ) act also more efficiently than DNP simultaneously as a protonophore and inhibitor of respiration, especially when respiration is supported by substrates that are transported to the matrix. The efficiency of DNOC is decreased with increase of mitochondrial protein, BSA and exogenous orthophosphate. Although similar effects were observed for animal and plant mitochondria, rat-liver mitochondrial respiration was more sensitive to DNOC than plant mitochondria. Furthermore, in the presence of DNOC, liver mitochondria exhibited a higher state 3 respiratory coupling level than potato tuber mitochondria, as a result of a considerable stimulation (60%) of state 3 respiration. In conclusion, DNOC is a more potent mitochondrial uncoupler and respiratory chain inhibitor than DNP, although their chemical structures are very similar. Apparently, the additional methyl group of DNOC increases its efficiency as an uncoupler and as an inhibitor, as compared to DNP. Plant mitochondria were shown to be as useful as animal mitochondria in evaluating the toxicity of these xenobiotics. © 1998 Society of Chemical Industry. 相似文献
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D. Andrivon J. Avendaño‐Córcoles S. F. Carnegie L. R. Cooke R. Corbière D. Detourné L. J. Dowley D. Evans K. Forisekova D. G. Griffin A. Hannukkala A. K. Lees R. Lebecka F. Niepold Z. Polgar D. S. Shaw J. Thompson B. Trognitz H. M. G. van Raaij E. Zimnoch‐Guzowska 《Plant pathology》2011,60(3):556-565
Determining virulence towards race‐specific resistance genes is a prerequisite to understanding the response of pathogen populations to resistant cultivars, and therefore to assess the durability of these resistance genes and the performance of resistance management strategies. In Phytophthora infestans, virulence testing began shortly after the introduction of R‐genes from Solanum demissum into S. tuberosum cultivars. However, the characteristics of R‐gene expression, the sensitivity of the phenotype to environmental and physiological parameters, and the diversity of experimental protocols make the comparison of data from different studies problematic. This prompted European teams working on P. infestans diversity to: (i) design a joint protocol, using detached leaflets from greenhouse‐grown plants of a shared set of differential cultivars inoculated with standardized suspensions of inoculum, and (ii) assess the performance of this protocol in a blind ring test involving 12 laboratories and 10 European isolates of the pathogen. A high level of consensus in the determination of virulence/avirulence to R1, R3, R4, R7, R8, R10 and R11 was achieved among the collaborators, showing that the protocol could be robustly applied across a range of laboratories. However, virulence to R2, R5 or R9 was detected more frequently in some laboratories, essentially from northern Europe; these genes are known to be highly sensitive to host and environmental conditions. The consensus determination was often markedly different from the original virulence phenotype of the isolates, suggesting virulence instability in stored P. infestans isolates. This indicates that creating reliable core collections of pathogen isolates with known virulences could be difficult. 相似文献
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根据5种病毒小西葫芦黄花叶病毒(Zucchini yellow mosaic virus,ZYMV)、西瓜花叶病毒(Watermelon mosaic virus,WMV)、烟草花叶病毒(Tobacco mosaic virus,TMV)、南瓜花叶病毒(Squash mosaic virus,SqMV)和黄瓜花叶病毒(Cucumber mosaic virus,CMV)的核苷酸保守区序列,设计特异性引物对,从影响多重RT-PCR (mRT-PCR)扩增的引物浓度、Mg2+浓度、Taq DNA聚合酶浓度、dNTPs浓度、退火温度等方面进行反应体系的优化,建立了一种能够同时检测ZYMV、WMV、TMV、SqMV和CMV的多重RT-PCR技术体系,并进行了实际应用。在一个体系中对上述5种病毒复合侵染的西瓜材料进行多重RT-PCR扩增,得到与试验设计相符的5条特异性条带,依次是542、485、410、354和293bp。该体系实现了对侵染西瓜的5种病毒的同时检测,极大地提高了检测效率,降低了检测成本,体现了多重RT-PCR的优越性。 相似文献
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为研究新疆葡萄中沙地葡萄茎痘伴随病毒(Grapevine rupestris stem pitting associated virus,GRSPa V)、葡萄斑点病毒(Grapevine fleck virus,GFk V)及葡萄病毒A(Grapevine virus A,GVA)的发生情况和新疆分离株系统进化关系,分别克隆3种病毒新疆分离株部分基因区域,应用RT-PCR对新疆64份葡萄样品中上述3种病毒进行检测,并进行系统进化分析。结果显示,GRSPa V、GFk V和GVA的检出率分别为31.3%、62.5%和25.0%。新疆GRSPa V分离株(KJ801847)与美国GRSPa V分离株(AY368590)同源性达96.59%;新疆GFk V分离株(KJ801846)与日本GFk V分离株(AB222861)及中国辽宁GFk V分离株(JF927942)的同源性分别为91.70%和91.03%;新疆GVA分离株(KJ801845)与波兰GVA分离株(JN860997)同源性为93.88%,与中国四川GVA分离株(HQ671655)及辽宁GVA分离株(FJ445220)的同源性分别为92.92%和89.53%。表明3种葡萄病毒在新疆发生比较普遍,且新疆分离株与国内其它地方的分离株存在较大差异。 相似文献