蔬菜土传病原菌三重PCR检测体系的建立与应用     

刘芮池  程有普  柴阿丽  石延霞  谢学文  帕提古丽  李宝聚 《中国农业科学》2019,52(12):2069-2078

【目的】 针对生产中危害严重的3种蔬菜土传病原菌瓜果腐霉（Pythium aphanidermatum）、尖镰孢（Fusarium oxysporum）、大丽轮枝菌（Verticillium dahliae）,建立三重PCR（triplex PCR）检测体系,为蔬菜土传真菌病害的早期诊断和鉴定提供技术与方法。【方法】 筛选3种病原菌特异性引物组合,通过设定不同的PCR退火温度、引物浓度、循环次数以及延伸时间,探索影响三重PCR扩增的因素,建立蔬菜土传病原菌三重PCR检测体系,并对体系灵敏度进行检测。为了验证建立体系的稳定性,选择2×Taq Master PCR Mix（北京博迈德生物技术有限公司）和TaKaRa Taq酶（大连宝生物工程有限公司）2个不同公司的试剂,分别利用C1000 Touch ^TM型（赛默飞世尔科技有限公司）与Aeris ^TM型（新加坡艺思高科技有限公司）热循环仪进行三重PCR反应,比较检测结果的可重复性。对田间采集的35份病害样本和149份土壤样本,分别进行三重PCR和病原菌分离检测,以确定所建立得三重PCR检测体系的适用性。 【结果】 三重PCR反应体系中引物对AsAPH2B/AsPyF、FOF1/FOR1、VActF/VActR可分别扩增出瓜果腐霉、尖镰孢、大丽轮枝菌长度为163、328和530 bp的特异性目的片段。反应体系（25 μL）：0.12 μmol·L ^-1 AsAPH2B/AsPyF,0.16 μmol·L ^-1 FOF1/FOR1,0.24 μmol·L ^-1 VActF/VActR,2×Taq Master PCR Mix 12.5 μL,退火温度为60.8℃,35个循环。该体系对病原菌纯培养物的检测灵敏度为10 ^-1ng·μL ^-1,对土壤中大丽轮枝菌、尖镰孢和瓜果腐霉的检测灵敏度分别为10 ⁵、10 ⁶个孢子/g土壤和10 ^-2mg菌丝/g土壤。分别采用2×Taq Master PCR Mix和TaKaRa Taq酶,利用C1000 Touch ^TM型和Aeris ^TM型热循环仪进行三重PCR,扩增结果一致,说明三重PCR体系检测稳定性好。对田间采集的病害和土壤样本进行检测,25份病害样本和71份土壤样本中检测出带菌,三重PCR检测与病原菌分离培养结果一致。 【结论】 本研究建立的瓜果腐霉、尖镰孢、大丽轮枝菌三重PCR检测体系具有灵敏度高、稳定性和重复性好的特点,能够快速、准确地检测田间病株及其根围土壤中的瓜果腐霉、尖镰孢、大丽轮枝菌,为蔬菜土传病害的早期预防和流行监测提供了有效的技术手段。  相似文献   

韩国大丽轮枝菌形态学及分子生物学特性的初步研究(英文)     

尚飞  金洪琪 《延边大学农学学报》2012,34(1):60-66

通过显微镜观察对大丽轮枝黄萎病菌株进行初步分类,并对该菌种轮枝状分生孢子梗的形态进行详细描述,确认了微菌核的存在。筛选出快速鉴定该菌种的特异性引物Vd3/Vd4。同时,利用10对RAPD引物对实验菌株进行分析,结果表明,轮枝黄萎病菌种间存在着高度的遗传多样性。  相似文献   

棉花品种抗黄萎病性与体内生化成分相关分析   总被引：12，自引：0，他引：12  

夏正俊  顾本康  吴蔼民  李经仪  陈轶林  陈小波 《植物保护学报》1994,21(4):305-310

对棉花不同抗感黄萎病性品种生化成分分析结果表明:棉花品种在无菌土中体内可溶性还原糖含量相应地高于接菌土中的含量。蕾期抗耐品种平均含量为2.276％,高于感性品种的1.417％,而铃期趋势相反。叶片单宁含量高于根部。在无菌土中,抗耐品种叶片单宁含量平均为3.58％,高于感病品种的3.28％;而在接菌条件下趋势相反。根部棉酚含量显著高于叶部,但两者极显著相关。在无菌土和接菌土中,抗性品种棉酚含量均高于耐性品种,而耐性品种又高于感性品种。棉株体内棉酚含量与棉花品种抗黄萎病性显著正相关。同工酶谱测定显示,过氧化物酶在抗感品种的无病症叶片中,酶带特征基本一致,叶片显症后表现主酶带区加宽,主酶带数增多,变化趋势与症状严重度正相关。在显症条件下,抗性品种酯酶同工酶活性略强于感性品种;与无症叶相比,显症叶的SOD酶带数由原来的4—6条减少至3—4条,抗性品种的酶带数少于感性品种1—2条。  相似文献   

蜡蚧轮枝菌防治植物病虫害研究进展   总被引：4，自引：0，他引：4  

杨晓昱  于蓉蓉 《中国植保导刊》2005,25(5):9-11

蜡蚧轮枝菌在蔬菜病虫害防治中有很大开发潜力。就蜡蚧轮枝菌的致病性、环境条件对其生长的影响、储藏期存活率及在生产中应用的概况的研究进展进行了综述。研究进展表明,该种真菌既可以防治粉虱、蚜虫、蓟马等蔬菜害虫,又可以防治蔬菜的白粉病、锈病等。  相似文献   

陆地棉对黄萎病抗性的分子标记研究   总被引：14，自引：0，他引：14  

王红梅  张献龙  贺道华  林忠旭  聂以春  李运海  陈伟 《植物病理学报》2005,35(4):333-339

 利用陆地棉标准系TM-1和常抗棉2个陆地棉品种杂交并自交,获得109个F₂单株及F_2:3家系为作图群体,以SSR、RAPD和SRAP 3种分子标记进行抗黄萎病性状的分子标记筛选。结果从1611对(条)引物中仅筛选到70对(条)多态性引物,获得75个多态性位点并进行标记间的连锁性分析。75个标记构建了一个包括15个连锁群,全长535 cM的陆地棉品种间分子标记遗传连锁图,标记间平均距离为11.15 cM,有27个标记不能进入任何连锁群。连锁群的标记数最少2个,最多6个;长度从1.0 cM到92.7 cM不等。对其F_2:3家系的成株期抗黄萎病性状即平均病情指数的分布进行分析,显示其呈正态分布,进一步说明陆地棉对黄萎病的抗性为数量遗传;单标记分析及复合区间作图,检测出与抗黄萎病性相关的3个QTL,分别位于第3、5、6连锁群上,贡献率分别为14.15%、3.45%和18.78%。另外,对该群体生长过程中黄萎病不同发病高峰期的病情也进行了分析。  相似文献   

棉花落叶型黄萎病菌的检测技术     

傅正擎  吴蔼民  顾本康  何孔旺  顾春燕 《植物保护学报》2001,28(1):28-32

采用Nit硝酸还原酶缺陷型突变体技术，共得到棉花黄萎病菌Nit突变体301个，其中A型155个，B型97个，C型29个。共检测到落叶型棉花黄萎病菌11株，除以前报道的6株外，发现了5个新菌株。针刺接种后，在棉株上均引起典型的落叶型症状。通过聚丙烯酰受凝胶电泳比较落叶型和非落叶型的8个代表菌株的蛋白质电泳图谱的差异，发现在浓度为7.5%的凝胶中，落叶型菌株有两条特异性的蛋白，其迁移率分别为Rf5=0.135,Rf9=0.405。用非落叶型菌株DF4、落叶型菌株SY12菌体的可溶性蛋白制备获得两个抗血清。用间接ELISA法检测，即能区分棉花黄萎病菌的立枯、枯萎、炭疽病菌，落叶型和非落叶型黄萎病菌均呈阳性反应，但落叶型黄萎病菌的OD值高于非落叶型黄萎病菌。  相似文献   

Plant Host Range of Verticillium longisporum and Microsclerotia Density in Swedish Soils     

Anna Johansson  Jan-Kees C. Goud  Christina Dixelius 《European journal of plant pathology / European Foundation for Plant Pathology》2006,114(2):139-149

Verticillium longisporum is a soil-borne fungal pathogen causing vascular wilt of Brassica crops. This study was conducted to enhance our knowledge on the host range of V. longisporum. Seven crop species (barley, oat, oilseed rape, pea, red clover, sugar beet and wheat) and five weed species (barren brome, black-grass, charlock, cleavers and scentless mayweed) all common in southern Sweden were evaluated for infection by response to V. longisporum. Oat, spring wheat, oilseed rape, scentless mayweed and charlock inoculated with V. longisporum in a greenhouse showed stunting to various degrees close to the fully ripe stage. Based on the extent of microsclerotia formation, explants were separated into four groups: for pea and wheat, <5% of the samples had formed microsclerotia; for scentless mayweed, 5–10%; for oat, 10–20%; and for charlock and oilseed rape >80%. The results suggest that plant species outside the Brassicaceae can act as reservoirs of V. longisporum inoculum. Soil inoculum densities in nine fields were monitored over a period of 12 months, which ranged from 1 to 48 cfu g⁻¹ soil. Density of microsclerotia was lowest just after harvest, reaching its maximum six months later. No significant correlation between inoculum density in soil and disease incidence on oilseed rape plants was found. However, the data suggest that a threshold of 1 cfu g⁻¹ soil is needed to cause disease on oilseed rape. Species identification based on microsclerotia morphology and PCR analysis showed that V. longisporum dominated in soil of seven, and V. dahliae in two of the nine fields studied.  相似文献   

Genetic variability and virulence among Verticillium albo-atrum isolates from hop     

Sebastjan Radišek  Jernej Jakše  Branka Javornik 《European journal of plant pathology / European Foundation for Plant Pathology》2006,116(4):301-314

Verticillium wilt, caused by Verticillium albo-atrum or V. dahliae, is an important disease of many worldwide crop species. In Europe, V. albo-atrum isolates infecting hop express different levels of virulence, inducing mild or lethal disease syndromes, and it is therefore an attractive model for studying the virulence of this pathogen. In this work, eleven amplified fragment length polymorphism (AFLP) primer combinations were used to analyze genetic variability among 55 V. albo-atrum hop isolates from four European hop growing regions, as well as isolates from other hosts and V. dahliae isolates. Cluster analysis divided V. albo-atrum and V. dahliae isolates into two well-separated groups. Within the V. dahliae cluster, isolates were separated without host specific grouping, although no host adapted isolates were included. In V. albo-atrum, the alfalfa isolates were distinct from isolates of other hosts, where a high association with virulence was observed in hop and tomato isolates. All lethal hop isolates were genetically different from mild hop isolates. The lethal hop isolates from England and Slovenia expressed the same virulence phenotype, although they showed a different AFLP pattern. The mild hop isolates formed two subgroups, to which isolates clustered irrespective of geographical location. These data suggest multiple origins of V. albo-atrum hop isolates, and the possible appearance of new virulent isolates in the future in other hop growing regions.  相似文献   

苜蓿黄萎病菌在我国的适生性分析研究   总被引：2，自引：1，他引：2  

邵刚  李志红  张祥林  耿建  孙楠  张国珍 《植物保护》2006,32(5):48-51

本文以适生性评判指标分析为基础,采用生物学建模与GIS分析相结合的方法,依据我国677个气象站点的逐月气温数据以及全国土壤pH等值线图,对苜蓿黄萎病菌在我国的适生性作了分析研究。结果表明,苜蓿黄萎病菌在我国的适生性强,适生范围较广,每年4~9月份,该病菌在我国西北、东北以及华北地区的适生程度处于较高水平。鉴于此,我国应加强苜蓿黄萎病菌的植物检疫措施力度,严防该病的传入或扩散。  相似文献   

Cloning of DNA fragments specific to the pathotype and race of <Emphasis Type="Italic">Verticillium dahliae</Emphasis>     

Toshiyuki Usami  Shu Ishigaki  Hiroko Takashina  Yuko Matsubara  Yoshimiki Amemiya 《Journal of General Plant Pathology》2007,73(2):89-95

Japanese isolates of Verticillium dahliae, a causal agent of wilt disease in many plants, are classifiable into pathotypes based on their pathogenicity. Because these pathotypes are morphologically indistinguishable, establishing a rapid identification method is very important for the control of this pathogen in Japan. For cloning DNA fragments that are useful for identification and specific detection of V. dahliae pathotypes, we performed random amplified polymorphic DNA (RAPD) analyses using various isolates. One polymerase chain reaction (PCR) product, E10-U48, was specific to isolates pathogenic to sweet pepper. The other product, B68-TV, was specific to race 1 of isolates pathogenic to tomato. The specificity of these sequences was confirmed by genomic Southern hybridization. Further analyses revealed that the region peripheral to B68-TV obtained from the genomic DNA library includes the sequence specific to all isolates pathogenic to tomato (races 1 and 2). Moreover, sequence tagged site (STS) primers designed from B68-TV and its peripheral region showed race-specific and pathotype-specific amplification in a PCR assay. The probes and primers obtained in this study are likely to be useful tools for the identification and specific detection of pathotypes and races of V. dahliae. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession number AB095266.  相似文献