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NCM-ELISA检测马铃薯Y病毒(PVY)技术的研究及应用 总被引：2，自引：0，他引：2
血清学方法是病毒检测的主要手段。本试验通过对马铃薯Y病毒(PVY)的提纯,免疫家兔制备PVY抗血清,并提取PYV免疫球蛋白IgG作为NCM-ELISA反应为一抗,以市售羊抗兔抗血清为二抗,在硝酸纤维素膜(NCM)上进行NCM-ELISA反应检测PVY,建立PVY NCM-ELISA检测反应体系。试验结果显示,NCM-ELISA具有反应特异性强,灵敏度高的优点,检测植物叶片样品的最高稀释度可达到1:250。通过对田间40份样品的NCM-ELISA和DAS-ELISA检测比较,其检测结果吻合率达到100%。由于NCM-ELISA方法可以将样品点在硝酸纤维素膜上,并且可贮存几个星期或将膜送到其他实验室进行检测,因此具有操作简单,使用方便,检测成本低等优点。 相似文献
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用马铃薯环腐菌菌株NCPPB2140和HEC_1免疫家兔得到效价1:2048的抗血清。通过硫酸铵沉淀法和DE52柱层析提取免疫球蛋白(IgG)。应用间接ELISA检测了马铃薯环腐菌及其供试样品。结果表明:免疫球蛋白最适浓度为5～10μg/ml,酶标记羊抗兔结合物的最适稀释度为1:75～100,检测环腐菌的灵敏度为2×10~5CFμ/ml,用NCPPB2140和HEC_1的IgG同马铃薯黑胫病菌、软腐病菌和青枯病菌等不发生反应,用间接ELISA可以检出轻度感病或受其它细菌二次感染腐烂的环腐病薯块。 相似文献
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应用PCR技术快速检测马铃薯环腐病菌 总被引：1，自引：0，他引：1
根据马铃薯环腐病菌16SrDNA基因片段核苷酸序列设计引物(引物1∶5-′TGTACTCGGCCAT-GACGTTGG-3′和引物2∶5-′TACTGGGTCATGTTGGT-3)′,进行马铃薯环腐病菌PCR特异性扩增试验。合成的引物能从马铃薯环腐病菌总基因组DNA和细菌纯培养,以及人工接种和自然发病的马铃薯块茎中特异性扩增环腐病菌16SrDNA基因片段1046bp。该试验结果为马铃薯环腐病的鉴定、检测及病害流行学研究提供了新的技术和方法。 相似文献
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传统的马铃薯环腐病菌鉴定方法主要是革兰氏染色法、茄苗接种鉴定法以及根据菌体的各种特征特性进行鉴定;应用血清学鉴定检测马铃薯环腐病菌方法主要是乳胶致敏试验(AG)、凝胶双扩散试验(DD)、间接荧光抗体染色试验(IFAS)以及酶联免疫吸附测定试验;分子生物学方法检测马铃薯环腐病菌技术主要是应用RFLP分子标记、基因探针技术、核酸斑点杂交方法以及ITS-PCR方法。对于马铃薯环腐菌的鉴定检测不能单单依靠一种方法,因为任何一种方法都有其缺点和局限性,为得到真实、可靠的判定结果,应将多种方法取得的结果结合起来进行最终判定,因此针对马铃薯环腐菌鉴定检测建立一套完整的技术体系十分必要。 相似文献
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马铃薯环腐病菌鉴定检测技术研究进展 总被引：2，自引：2，他引：2
传统的马铃薯环腐病菌鉴定方法主要是革兰氏染色法、茄苗接种鉴定法以及根据菌体的各种特征特性进行鉴定应用血清学鉴定检测马铃薯环腐病菌方法主要是乳胶致敏试验(AG)、凝胶双扩散试验(DD)、间接荧光抗体染色试验(IFAS)以及酶联免疫吸附测定试验;分子生物学方法检测马铃薯环腐病菌技术主要是应用RFLP分子标记、基因探针技术、核酸斑点杂交方法以及ITS-PCR方法.对于马铃薯环腐菌的鉴定检测不能单单依靠一种方法,因为任何一种方法都有其缺点和局限性,为得到真实、可靠的判定结果,应将多种方法取得的结果结合起来进行最终判定,因此针对马铃薯环腐菌鉴定检测建立一套完整的技术体系十分必要. 相似文献
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马铃薯种薯中存在环腐病菌潜伏侵染,这种潜伏侵染逐代累积、逐渐表现症状,这是马铃薯环腐病无法根除的根本原因。本研究应用国外成功报道的根据存在于pCS1质粒和环腐菌染色体中一个1.3 kb的插入因子IS1121、高度重复的DNA片段设计的环腐病菌亚种特异性引物序列合成出引物对CMSIF1-CMSIR1和引物对CMSIF2-CMSIR2。以环腐标准菌株、黑龙江省环腐菌株以及马铃薯上其它主要的细菌病原菌(青枯病、软腐病、黑胫病)为试验材料进行直接PCR和嵌套式PCR扩增,结果只有环腐标准菌株和黑龙江省环腐菌株出现特异性片段(直接PCR扩增出1046 bp的片段,嵌套式PCR扩增出864 bp的片段)。将环腐菌纯菌种菌悬液稀释成浓度梯度并与马铃薯组织液混合进行直接PCR和嵌套式PCR检测灵敏度比较,结果表明嵌套式PCR检测灵敏度比直接PCR检测灵敏度提高了100￣1000倍。以明显感病症状的块茎、无明显感病症状的块茎和健康块茎为试验材料进行直接PCR和嵌套式PCR,结果除明显感病症状块茎外,所有无明显感病症状的块茎也均被检测出带有环腐病菌。 相似文献
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实时定量荧光PCR法检测马铃薯黑胫病菌 总被引：1，自引：0，他引：1
马铃薯黑胫病是国内外马铃薯产区发生比较普遍的一种细菌性病害,严重地影响了马铃薯的产量和质量。本研究根据Potato Black Leg序列,设计出马铃薯黑胫病菌特异性的引物,对10种供试菌株进行了实时荧光PCR检测。结果表明,该方法的特异性好,可以将马铃薯黑胫病菌和其它马铃薯常见病害相区分;灵敏度较高,可检测出最低的黑胫病菌浓度为3.6~3.9 cfu.mL-1;而且检测时间仅用4 h,大大缩短了检测周期。该方法可有效地应用于马铃薯黑胫病菌的检测和监控。 相似文献
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１ 前 言马铃薯环腐病（ＣｌａｖｉｂａｃｔｅｒｍｉｃｈｉｇａｎｅｎｓｉｓＳｕｂｓｐ．ｓｅｐｅｄｏｎｉｃｕｍ）是一种严重危害马铃薯输导系统的细菌性病害,各国都把它列为重要植物检疫对象。它主要以带病种薯传播蔓延;因此,寻找一种简易、快速、准确的鉴定和检验方法,确保核心种薯不带病菌,是目前国内外都在研究解决的问题。多年来,国内外有关专家学者尝试建立各种检测马铃薯环腐病的方法,如茄苗接种鉴定技术、免疫荧光抗体技术、酶联免疫吸附试验、ＤＮＡ核酸杂交、ＰＣＲ检测环腐病技术等等。由于以上检测手段有的需特定仪器… 相似文献
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Corynebacterium sepedonicum was detected in symptomless potato stems and tubers with immunofluorescence using monoclonal antibodies specific for the bacterial ring rot pathogen. The concentration of bacterial cells in potato tissue preparations ranged from >500 cells/microscope field to 1 cell per preparation. Symptomless tubers containing ring rot bacteria planted in field plots yielded plants with ring rot symptoms, plants with latent ring rot infections, or plants with no detectable levels of ring rot bacteria. Tubers with the greatest number of bacteria were most likely to develop plants expressing ring rot symptoms, but even some seed tubers with a low number of bacteria developed into plants with symptoms. Some seed tubers with high levels of ring rot bacteria produced plants with only low numbers ofC. sepedonicum. 相似文献
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S. H. De Boer 《American Journal of Potato Research》1991,68(2):107-113
The bacterial ring rot disease of potato (Corynebacterium sepedonicum) can be detected in seed potato lots by serological tests with greater accuracy than by visual field inspections. Indexing tests can make use of highly specific immunofiuorescence probes, enzyme-linked immunosorbent assays, and perhaps other procedures. Sensitivity and specificity of these procedures need to be determined in order to estimate the confidence with which ring rot can be detected in a seed lot. In addition to the laboratory tests, the sampling strategy determines the efficacy and sensitivity of the indexing procedure. Samples might consist of stems, leaf petioles, or tubers in single or composite units. Disease incidence and number of plants sampled also determine the confidence level with which ring rot can be detected in a potato seed lot. Although research has been conducted on many aspects of ring rot testing procedures, further work needs to be done. Nevertheless, it is anticipated that indexing potato seed lots for the presence of ring rot will be a significant factor in control and eradication of the disease. 相似文献
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J. I. S. Perminow I. L. W. Akselsen E. Borowski ?. Ruden W. Gr?n?s 《Potato Research》2012,55(3-4):241-247
Potato ring rot, caused by the bacterium Clavibacter michiganensis subspecies sepedonicus, is considered to be one of the internationally most important seed potato diseases (Smith et al.; Eur J Plant Pathol 107:739–748, 2001) and has been a problem in Norwegian potato production since its first detection in 1964. Since 1965, Norway has had its own national legislation for the control of the disease. In recent years, this legislation has evolved to be more similar to the EU Commission Directive 2006/56/EC. In 1999, the Norwegian Food Safety Authority initiated an eradication program for potato ring rot with the aim of selling potatoes to other European countries. During the project period (1999–2008), efficient systems for sampling, analysis and eradication measures were built. From 1999 to 2002, the occurrence of potato ring rot in commercial potato production was monitored in all counties. Sampling was carried out according to the instructions of the Norwegian Food Safety Authority, while testing of samples was (from 2000 onwards) carried out by Bioforsk laboratories with modern serological and molecular detection methods. When ring rot was detected in a potato lot, the grower had to implement strict eradication measures. The survey was followed up with two monitoring periods, 2003–2004 and 2005–2008. During the project period, 328 cases of potato ring rot were found. The counties Hedmark, Nordland, Troms, and Tr?ndelag accounted for most of these. The main objectives of this study were to describe the occurrence of potato ring rot in Norwegian potato production and to evaluate the effectiveness of the eradication measures employed for the control of the disease. This survey showed that the overall ring rot situation did improve considerably during the project period (1999–2008), both in relation to prior periods, and when comparing the surveying and monitoring periods. Problem areas where monitoring must be continued remain. 相似文献
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临夏地区马铃薯环腐病气候动态预测模式 总被引：2，自引：0，他引：2
温度、湿度和降水等气象要素是马铃薯环腐病发生的主要环境影响因子。为了探索临夏地区马铃薯环腐病和建立温、湿度引起发病的预测模型,利用临夏地区马铃薯种植资料和同期气象观测资料,通过相关因子诊断、数理统计和多元回归方法,分析临夏地区马铃薯环腐病不同发病阶段和发病区域气候条件以及对马铃薯产量及其构成因素的影响;把马铃薯环腐病划分为以临夏市为代表的半干旱区和以和政县为代表的阴湿气候区,从发病早期到晚期,划分为两个区域两个阶段,建立了临夏地区马铃薯环腐病发病区域不同发病阶段的预测模式。实现马铃薯环腐病气象预测;各预测模型的预测准确率达到73%以上,为临夏马铃薯推广栽培及病虫害预测提供了科学依据。 相似文献
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Effect of the ring rot pathogen and latent potato viruses on ring rot symptoms and yield of potatoes
The ring rot bacterium,Corynebacterium sepedonicum (Spieck. and Kotth.) Skapt. and Burkh., and latent potato viruses (potato virus S and potato virus X) were investigated for their effect on ring rot symptom development on potato plants in the greenhouse and on symptom development and yield of potatoes in the field. Both virus-free (VF) and virus-infected (VI) Red Pontiac stem cuttings root-inoculated with ring rot bacteria in the greenhouse developed typical (T) ring rot symptoms, and symptom severity did not differ between VF and VI plants. In a field study, both VF and VI Russet Burbank seed pieces knife-inoculated with ring rot bacteria produced plants with atypical (A) and T ring rot symptoms as well as a combination of both types. The data suggest that more A than T symptoms develop on VI plants and more T than A symptoms develop on VF plants. Combined infection with the ring rot pathogen and the latent potato viruses resulted in greater yield losses of total and marketable Russet Burbank tubers than infection with the bacterial or viral pathogens alone. 相似文献
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Netted Gem potato plants infected with either the ring rot bacterium (Corynebacterium sepedonicum) or the potato leaf roll virus alone exhibited typical symptoms of the respective diseases. Plants infected with both the bacterium and the virus exhibited severe leaf roll. Typical ring rot symptoms were masked in dual infections, thereby increasing the difficulty in detection of the bacterial disease. The combined yield-reducing effect of the two pathogens was greater than that of the potato leaf roll virus and was similar to that caused by ring rot alone. 相似文献
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S. H. De Boer J. D. Janse D. E. Stead J. Van Vaerenbergh A. R. McKenzie 《Potato Research》1992,35(2):207-216
Summary An indirect sandwich enzyme-linked immunosorbent assay (ELISA) was used to monitor potato stems of three cultivars for the
presence of the ring rot pathogen,Clavibacter michiganensis subsp.sepedonicus, during the growing season and in progeny tubers after harvest. The highest ELISA values were obtained with the highest concentration
of bacteria used to inoculate seed pieces in all cultivars tested. Low ELISA values were obtained for stems and progeny tubers
selected from plants grown from seed inoculated at lower bacterial concentrations. Estimates of bacterial densities in stems
and progeny tubers by immunofluorescence indicated that low ELISA values were most probably caused by low bacterial numbers.
It is suggested that the sensitivity of ELISA for detecting the ring rot pathogen in potato stems and progeny tubers is a
function of the concentration of bacteria in individual seed pieces. 相似文献