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1.
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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马铃薯晚疫病种薯带菌的分子检测 总被引：2，自引：1，他引：2
从马铃薯块茎上分离纯化得到晚疫病菌、银腐病菌、癌肿病菌、红腐病菌、黑点病菌和镰刀菌等并提取它们的全基因组DNA,用引物08-3和08-4对这些真菌的DNA进行PCR扩增,以检验该引物扩增晚疫病菌DNA的特异性。取马铃薯种薯的芽眼、芽及其他不同部位并用NaOH法提取晚疫病菌DNA,再用引物08-3和08-4进行特异性扩增以检验种薯是否带晚疫病菌。结果表明:(1)引物08-3和08-4有很强的特异性,只能扩增出大小为257bp的马铃薯晚疫病菌DNA片段;(2)用该引物能扩增出种薯芽眼组织中大小为257bp的晚疫病菌DNA,说明通过PCR特异扩增,能直接检测出马铃薯种薯中潜伏的晚疫病菌。(3)本次检测的本地感病品种米拉有10%的种薯带晚疫病菌。该研究为种薯传播晚疫病菌的分子检测提供了依据和方法。 相似文献
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接骨木镰刀菌是马铃薯干腐病的主要致病菌,给黑龙江省马铃薯造成了一定的经济损失。采用马铃薯干腐病引物LDJ1对马铃薯干腐病、马铃薯晚疫病、马铃薯早疫病、马铃薯黑痣病的致病菌的ITS序列进行测定,只有马铃薯干腐病菌扩增出560 bp特异性片段。再分别对接骨木镰刀菌、燕麦镰刀菌和茄病镰孢变种的ITS序列进行BLAST比对分析,针对接骨木镰刀菌设计了特异引物L2,扩增特异性产物为278 bp,该引物所建立的PCR检测体系可检测DNA含量在50 pg/μL以上的目的基因。 相似文献
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常规和巢式PCR对柑橘黄龙病菌的检测灵敏度比较 总被引：3，自引：0，他引：3
根据柑橘黄龙病(citrus Huanglongbing,HLB)病菌16SrDNA、16S/23S核糖体基因间隔区(ribosomal intergenic region,RIR)、核糖体蛋白β操纵子(β-operon)和外膜蛋白(out membrane protein,OMP)基因序列设计了8对引物,通过常规和巢式PCR方法对各引物的检测灵敏度进行了比较。结果表明,不同引物的检测灵敏度不同。对于上述任何基因,巢式PCR的灵敏度均比常规PCR至少高103倍;对于同一种基因,扩增短片段的引物比扩增长片段的引物灵敏度高或相当;在扩增产物大小相同时,用以扩增核糖体蛋白β操纵子基因的引物较其他三种稍高。对不同症状柑橘样品的检测进一步验证了该结果。因此,对于柑橘黄龙病的检测,可优先考虑使用巢式PCR方法,若使用常规PCR,则宜选用具有高灵敏度的扩增小片段引物。 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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建立检测马铃薯环腐病菌NCM-ELISA方法并组装成试剂盒,应用于检测、检疫和流行病学调查具有实践意义。本研究以马铃薯环腐病菌为抗原,制备兔抗血清,利用碱性磷酸酯酶标记的羊抗兔血清(GAR-AP)为酶标二抗,建立了马铃薯环腐病菌NCM-ELISA快速检测方法。结果表明:抗血清的最佳工作浓度为1:400,最低检出菌液浓度为1.0×106个.mL-1,特异性也比较强。因此,该方法具有敏感性高、特异性强、速度快、实用方便等特点,可以应用于马铃薯环腐病菌的快速检测和诊断。 相似文献
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马铃薯软腐病是威胁马铃薯块茎的细菌性病害之一,为了提高马铃薯的品质与产量,减少贮藏期马铃薯块茎的腐烂,对马铃薯进行软腐病检测与鉴定是十分必要的。利用16SrDNA PCR对马铃薯软腐病原菌的进行检测,证实16SrDNA PCR检测技术是可行可靠的。在对PCR引物的筛选中,要根据所检测的对象进行有效、合理的设计,才能达到快速有效的目的。以马铃薯软腐病菌为代表,利用16SrDNA PCR进行鉴定,达到了预期的目的,为病原菌的快速鉴定方法提供了依据。 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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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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G. A. Nelson 《American Journal of Potato Research》1980,57(12):595-600
The survival of the ring rot bacterium,Corynebacterium sepedonicum, on contaminated surfaces and in infected stems was investigated by root inoculating potato stem cuttings with aqueous suspensions prepared from these materials. The pathogen survived for 24 mo on contaminated surfaces of burlap, kraft paper, and polyethylene plastic held at 12% relative humidity (RH) at either 5 or 20°C. It persisted for less than 14 mo on surfaces held at 94% RH at either temperature. Infectious ring rot bacteria were also recovered from dried, infected potato stems held for 26 mo in an unheated machine shed. These results emphasize the importance of strict sanitation and disinfestation procedures in maintaining potato seed stocks free of bacterial ring rot. 相似文献
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G. A. Nelson 《American Journal of Potato Research》1985,62(1):23-28
The survival ofCorynebacterium sepedonicum, the bacterial ring rot pathogen, in infected potato stems and on burlap surfaces held at various freezing and above-freezing temperatures was investigated by root-inoculating potato stem cuttings with aqueous suspensions prepared from these materials. Infectious bacteria were recovered from dried Russet Burbank stems held for 44 mo in an unheated machine shed and from dried Warba stems held for 55 mo. Inoculum from dried stems of all cultivars held for 63 mo did not incite symptoms; however, inoculum from Russet Burbank stems incited symptomless infection in 10% of the inoculated plants. This 63-mo survival period exceeds a 26-mo survival period previously reported. Ring rot bacteria survived and remained infectious for at least 18 mo on burlap surfaces subjected to temperatures of ?40° to ?5°C, alternating temperatures of -5° to 5°C, or a constant temperature of 5°C. Inoculum from burlap stored at the lower temperatures caused the most severe symptoms. These results stress the need for keeping crop debris away from potato operations and for using proper decontamination procedures to maintain potato seed stocks free of ring rot. 相似文献
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The ring rot bacterium,Corynebacterium sepedonicum (Spieck. and Kotth.) Skapt. and Burkh. [Clavibacter michiganense subsp.sepedonicum (Spieck et Kotth.) Davis et al.], and latent potato viruses (potato virus S and potato virus X) were investigated for their effect on atypical (ATYP) ring rot symptom development on Russet Burbank potato plants at different temperatures. Plants grown at 21 C from stem cuttings root-inoculated withC. sepedonicum developed typical wilting and chlorotic symptoms of ring rot that were equally severe on virus-free (VF) and virus-infected (VI) plants. All VF and VI plants grown at 15 C from inoculated stem cuttings exhibited ATYP symptoms that included extreme stunting, resetting, and chlorotic symptoms of ring rot. More severe ATYP symptoms developed on VI than on VF plants. Up to 5 wk after inoculation withC. sepedonicum, ATYP symptom severity ratings of both VF and VI plants increased and declined thereafter. The ATYP severity ratings were highly correlated with fresh weight of plants with high severity ratings being associated with low fresh weights. These results emphasize the need to determine the role of temperature and viral pathogens on ring rot symptomology in existing and newly developed potato cultivars and thereby enable better field detection of bacterial ring rot. 相似文献
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Some of the factors that affected ring rot development in potato plants grown from stem cuttings and root-inoculated withCoryncbacterium sepedonicum were investigated. Isolates ofC. sepedonicum cultured and stored for a, year on agar media were as virulent as those that had been cultured for almost 5 months. Isolates cultured for periods over 2 years gradually lost their virulence; however, decline in virulence was not as rapid as had been previously reported. One isolate became more virulent by inoculating and recovering it from a susceptible potato plant. Ring rot symptoms developed more rapidly in the cultivar Red Pontiac than in the cultivar Netted Gem; however, disease severity eventually reached the same level in both cultivars. The more mature the plants were when inoculated, the greater was the rate of ring rot development. Duration of exposure of wounded roots to inoculum of the pathogen did not affect disease development. No ring rot bacteria were recovered from plants originating from apical cuttings of ring-rot-infected potato plants that exhibited partial wilting. By this technique, plants infected withC. sepedonicum were freed from this pathogen quickly and effectively. 相似文献