番茄溃疡病菌PCR快速检测技术

番茄溃疡病是一种严重危害番茄生产的细菌性病害，许多国家将其列为检疫性病害。利用ITS通用引物扩增了番茄溃疡病菌（Clavibacter michiganensis subsp．michiganensis）的ITS序列，并进行克隆测序。根据序列比较结果设计了引物BT1和BT2，该引物特异性好，能专一扩增出268bp电泳条带，而马铃薯环腐病菌等不同亚种、不同属的细菌及健康的番茄材料均无扩增条带。从接种但未显症番茄苗叶片及人工模拟染菌种子上提取总DNA，以此为模板均能稳定地扩增出特异性目的条带。该方法直接对种子或植株进行检测，不需进行病原菌分离培养，快速简便，适用于出入境检验检疫及种苗健康检测领域。     

稻曲病菌遗传多样性与群体结构的初步分析

 利用随机扩增多态性DNA (random amplified polymorphic DNA,RAPD)初步分析了稻曲病菌(Ustilaginoidea virens)的群体遗传结构。从1 60个随机引物中筛选32个扩增带型清晰、重复性好的引物,对不同年份采自辽宁、云南、湖北和浙江等水稻种植区的5 6个菌株进行扩增。32个引物扩增出2 2 3条带,绝大多数引物对不同年度采自不同稻区的菌株扩增的DNA谱型相同,大多数菌株间相似性系数达0.80以上。根据扩增DNA片段的多态性,从空间分布来看,来源于北方、长江流域和南方的菌株难以划分出明显的地理宗谱;不同年度的菌株DNA多态性也无明显的差异。上述结果初步表明稻曲病菌遗传稳定,寄主选择作用(寄主的基因型及其时空分布)对稻曲病菌变异的影响较小。但是尚需采用其它的分子技术测试更多的菌系,才能较系统地分析我国稻曲病菌系的遗传变异及群体结构特点。     

玉米圆斑病菌(Bipolaris zeicola)遗传多样性ISSR分析

利用简单序列重复间隔区(inter-simple sequence repeats,ISSR)标记对玉米圆斑病菌(Bipolaris zeicola)的遗传多样性进行了分析。筛选出9个扩增多态性好且稳定的通用引物,共扩增出47条DNA条带,大小分布于250~2 000bp之间,其中多态性条带为33条,为总条带数的70.21%。遗传距离为0.91处时,所有菌株被聚为6个组。ISSR标记可以揭示菌株间的亲缘关系及差异性,可用于玉米圆斑病菌遗传多样性研究。此外,通过分子标记划分的类群与利用寄主反应型之间存在一定相关性,但其关系并不密切。     

我国玉米灰斑病菌遗传多样性的ISSR分析

为明确我国发生的玉米灰斑病菌地理差异及遗传结构,利用简单序列重复区间(ISSR)对玉米灰斑病菌遗传多样性进行了分析,并利用尾孢菌特异引物对分离自四川、云南、湖北、贵州等西南地区的16个玉米灰斑病菌菌株进行了分子鉴定。结果显示,通过ISSR标记筛选出10个扩增多态性好且稳定的通用引物,共扩增出81条DNA条带,均为多态性条带,扩增片段大小在200~2 000 bp之间,菌株遗传相似系数为0.19~1.00。在遗传相似系数为0.19时,供试菌株被聚为2大类群,来自西南地区和东北地区的菌株各自聚为一组,在DNA水平上表现出明显差异,认为是2类不同的致病类群。分子鉴定结果显示引起西南各地区玉米灰斑病的主要致病菌均为玉米尾孢菌Cercospora zeina。表明我国玉米灰斑病菌存在丰富的遗传多样性,ISSR标记可揭示出玉米灰斑病菌株间的亲缘关系及遗传差异性,可用于其遗传多样性研究。     

绿僵菌菌株遗传多态性与地理来源及寄主种群分化的关联性

采用RAPD-PCR分子标记技术分析了51株不同地理来源、寄主来源的绿僵菌Metarhizium anisopliae菌株的遗传多态性。从94条RAPD引物中筛选出18条引物,对所有试验菌株进行RAPD-PCR扩增,共获得96条扩增片段,其中81条片段表现多态性,占84.1%。聚类分析表明,供试的51株菌株间的相似性系数范围为0.52~0.98,表明菌株间存在丰富的遗传多态性。供试菌株在相似性系数0.7的水平可分为4个组群。按菌株DNA多态性与地理及寄主来源的聚类分析表明,大多数菌株的DNA多态性与地理或寄主有一定的相关性,即长期的地理环境和寄主适应性可能形成了种群的分化。     

应用UP-PCR进行玉米丝黑穗病菌遗传多样性研究

本研究探讨了UP PCR技术在玉米丝黑穗病菌遗传多样性分析中的利用可行性。在13个通用引物中,筛选出9个扩增多态性好且稳定的通用引物,共扩增出113条DNA条带,大小分布于250～2 000 bp之间,其中多态性条带为95条,为总条带数的84.07%。遗传距离为0.76处时,所有丝黑穗病菌菌株被聚为6个组。利用UP-PCR技术,可以充分展现玉米丝黑穗病菌菌株间的亲缘关系及差异性,可用于玉米丝黑穗病菌遗传多样性研究,为有效地开展玉米丝黑穗病菌的遗传进化甚至探讨病菌致病性的生理分化提供了一个新的技术方法。     

甘薯茎腐病菌的遗传多样性及致病力差异分析

为了解不同地区甘薯茎腐病菌Dickeya dadantii种群遗传多样性水平及致病力差异,采用重复序列PCR基因指纹(repetitive element palindromic PCR,REP-PCR)技术和薯片接种方法,对采自广东省、广西壮族自治区和重庆市的6个市区县的59株菌株进行分析。结果表明,5对引物对59株菌株扩增出41个清晰的条带,其中36个为多态性条带,每对引物的扩增条带数在4～10之间,平均为7.2。在物种水平上,有效等位基因数、Nei’s基因多样性指数和Shannon信息指数分别为1.4768、0.2801和0.4186,其中湛江种群多样性最高,南宁种群多样性最低;当遗传相似系数为0.79时,59株菌株可被划分为5个类群,类群划分与菌株来源地间有一定的相关性。此外,不同地区病菌种群间存在明显的致病力差异,其中合浦种群与湛江种群致病力最强,万州种群致病力较弱。表明甘薯茎腐病菌种群具有丰富的遗传多样性,不同地区的病菌种群存在明显的遗传多样性与致病力差异。     

黑龙江省水稻纹枯病菌的致病力分化与AFLP分析

为了明确黑龙江省水稻纹枯病菌遗传多样性,为水稻抗病育种和水稻纹枯病的综合防治提供依据。本文对采自13个水稻种植地区的29个水稻纹枯病菌菌株进行了致病力测定和AFLP分析。结果表明9对AFLP引物对供试菌株扩增出396条带,其中多态性带187条,占总扩增带数的47.22%。黑龙江省水稻纹枯病菌的遗传距离变化在0.50~0.92之间,平均为0.71,群体遗传多样性较为丰富。UPGMA法可以将供试菌株分成4个AFLP聚类组群(Ⅰ、Ⅱ、Ⅲ和Ⅳ),相同地理来源的菌株基本上聚集在同一组群内,表明AFLP类群划分与菌株的地理来源有较强的相关性。黑龙江省水稻纹枯病菌致病性分化较为明显,并且AFLP类群划分与菌株的致病性鉴定之间存在一定相关性。     

西瓜枯萎病菌遗传多样性的相关序列扩增多态性分析

对30个西瓜枯萎病菌Fusarium oxysporum f.sp.niveum菌株基因组DNA进行相关序列扩增多态性(SRAP)分子标记分析,以探究其遗传多样性与地理来源的关系。采用尖孢镰刀菌西瓜专化型Fusarium oxysporumf.sp.niveum0、1、2号生理小种的基因组DNA为模板,对225对SRAP引物进行筛选,筛选出20对多态性、重复性较好且条带清晰的引物,对30个菌株进行PCR扩增,共扩增出386条带,其中多态性条带有371条,多态性比率为96.11%,平均每对引物扩增出19.3个位点和18.55个多态性位点。UPGMA法聚类分析结果显示,供试菌株两两之间的遗传相似系数范围为0.69~0.90,平均为0.79,说明尖孢镰刀菌西瓜专化型的遗传多样性较为丰富。基于SRAP标记聚类分析表明,30个菌株在遗传相似系数为0.70处被划分为3个类群,I类群包含24个菌株,其中18个来自湖南省,Ⅱ类群只包含1个来自黑龙江省哈尔滨市的菌株,它和另一个来自黑龙江地区的菌株被划分到不同的类群,且遗传距离相对较远;Ⅲ类群包含了5个菌株,其中3个来自海南三亚,其余两个来自湖南省。根据菌株的分布情况来看,菌株的聚集与地理来源没有明显的相关性。     

应用UP-PCR进行玉米丝黑穗病菌遗传多样陛研究

本研究探讨了UP-PCR技术在玉米丝黑穗病菌遗传多样性分析中的利用可行性。在13个通用引物中,筛选出9个扩增多态性好且稳定的通用引物,共扩增出113条DNA条带,大小分布于250~2000bp之间,其中多态性条带为95条,为总条带数的84．07％。遗传距离为0．76处时,所有丝黑穗病菌菌株被聚为6个组。利用UP-PCR技术,可以充分展现玉米丝黑穗病菌菌株间的亲缘关系及差异性,可用于玉米丝黑穗病菌遗传多样性研究,为有效地开展玉米丝黑穗病菌的遗传进化甚至探讨病菌致病性的生理分化提供了一个新的技术方法。     

番茄溃疡病菌的PCR-DHPLC快速检测

根据番茄溃疡病菌ITS序列,设计并合成了PCR-DHPLC检测引物,对番茄溃疡病菌及其他病菌共10个标准菌株进行了PCR-DHPLC检测。结果表明,番茄溃疡病菌的PCR-DHPLC检测图谱出现了特异性吸收峰,而其他病菌均未在相同洗脱时间出现吸收峰,说明这种方法具有检测番茄溃疡病菌的特异性。灵敏度实验结果表明,PCR-DHPLC体系与PCR-琼脂糖凝胶电泳体系的检测灵敏度一致。研究表明,PCR-DHPLC方法是一种特异、灵敏、快速的番茄溃疡病菌检测方法。     

进境玉米中玉米内州萎蔫病菌的PCR检测

 为了快速准确检测进境玉米样品中的玉米内州萎蔫病菌Clavibacter michiganensis subsp. nebraskensis(Cmn), 根据GenBank中Cmn的16S-23S序列设计引物CM1/CM4和引物PSM1/CM3。引物PSM1/CM3仅能从供试的4株Cmn菌株中扩增获得208 bp的预期产物, 而其他36株对照菌株均不能扩增出预期条带。灵敏度测试结果表明引物CM1/CM4和PSM1/CM3组合的巢式PCR方法的检测灵敏度高于常规PCR, 检测灵敏度可达40 fg DNA或6.8 CFU目标细菌。常规PCR和巢式PCR方法对进境美国玉米样品的阳性检出率分别为8%和24%, 试验结果表明所建立的PCR方法可用于玉米样品中Cmn的快速检测。     

不同来源番茄溃疡病菌致病力差异研究

采用打顶法接种、半选择性培养基再分离发病植株中的病原菌,以及特异性PCR验证方法,对来自3个国家9个不同地区的46株番茄溃疡病菌进行了致病性测定,以病情指数评价不同菌株的致病力。结果显示,分离自我国河北滦平县、内蒙古包头市等地的24株菌株的病情指数达到75以上,属于强致病力水平;11株菌株的病情指数为50~75,属于中等致病力;而9株菌株的病情指数为50以下,属于弱致病力;检测同时证实,有2株属于无致病力菌株。强致病力、中等致病力、弱致病力和无致病力菌株占供试菌株总数的比例分别为52.2%、23.9%、19.6%和4.3%,表明供试的46株番茄溃疡病菌存在不同程度的致病力差异。     

Survival of Clavibacter michiganensis ssp. michiganensis in infected tomato stems under natural field conditions in California, Ohio and Morocco

The survival and half-life of Clavibacter michiganensis ssp. michiganensis ( C. michiganensis ), the causal agent of bacterial canker of tomato, were determined in infected plant debris under natural field conditions in California, Ohio and Morocco using a semiselective agar medium. The organism survived significantly longer in tomato stems left on the soil surface than in stems buried in the soil at all locations studied. The pathogen was recovered in high amounts from tomato stems left on the soil surface for 314 days in Ohio and California, USA, and for 194 and 132 days in Melk Zhar and Aït Melloul, Morocco, respectively; it was recovered from stems buried in the soil for up to 314 days in Ohio, up to 240 days in California, and up to 60 days in Aït Melloul and Melk Zhar. The half-life of the pathogen in stems left on the soil surface ranged from 23·2 to 24·8 days in the USA, and from 7·8 to 12·3 days in Morocco, whereas the half-life in buried stems ranged from 14·0 to 16·7 days in the USA and from 3·7 to 9·5 days in Morocco. Based on the half-life data, the predicted survival times of C. michiganensis in stems on the soil surface in Ohio, California, Melk Zhar and Aït Melloul would be up to 822, 770, 424 and 261 days, respectively, while the predicted survival times in stems buried in the soil would be 541, 497, 305 and 128 days, respectively. These results show that the survival and half-life of C. michiganensis in plant debris are relatively long and are influenced by both tissue exposure and geographic location.     

苜蓿萎蔫病菌TaqMan探针实时荧光PCR检测方法的建立

苜蓿萎蔫病菌是我国对外检疫性二类有害生物，目前国内尚无发生6在出入境捡验检疫中主要是采用生物学和血清学方法进行检测，劳动强度大，耗费时间长。根据苜蓿萎蔫病菌与其它细菌菌株16SrDNA序列差异，设计出对苜蓿萎蔫病菌具有稳定点突交特异性探针，利用该探针对棒形杆菌属4个种及其它属细菌进行了实时荧光PCR检测实验。结果表明，只有苜蓿萎蔫病菌能检测到荧光信号，其它细菌没有荧光产生。该方法特异性强，灵敏度高，能检测到21．4fg质粒DNA，比常规PCR灵敏100倍，而且整个过程只需要2～3h。该方法可有效地应用于进出境病原菌检测之中。     

Use of the DNA sequence of the intergenic spacer region between the 16S and 23S rRNA genes for the identification of Clavibacter michiganensis subsp. insidiosus at the molecular level

A study has been performed to identify Clavibacter michiganensis subsp. insidiosus at the molecular level, using the polymerase chain reaction (PCR) technique with oligonucleotide primers based on specific sequence recognition of the intergenic spacer region between the 16S and 23S rRNA genes. The pair of primers was designed on the basis of available DNA sequence data for that region in C. m. insidiosus and other bacteria. Using this pair of primers, a large amount of an amplified DNA fragment of 218 bp in length was obtained from C. m. insidiosus. The specificity of this amplification was proved by PCR analysis, using the above-mentioned pair of primers and templates from different bacteria, some related to C. m. insidiosus. The PCR products were analysed using agarose gel electrophoresis.     

Comparative study of genetic diversity of Clavibacter michiganensis subsp. michiganensis isolates from the Canary Islands by RAPD-PCR, BOX-PCR and AFLP

Molecular characterization of seedborne pathogens is an important issue when discerning their origin and tracking the spread of a disease. In the Canary Islands (Spain), Clavibacter michiganensis subsp. michiganensis (Cmm) was first detected in 2002, causing severe losses in many tomato-growing areas. Fifty four strains of this bacterium isolated from 2002 to 2007 and 19 strains from different countries were characterized for genetic diversity. RAPD-PCR, BOX-PCR and AFLP provided differentiation among Cmm strains whereas no differences were observed with ERIC-PCR, REP-PCR and 16S-23S ITS PCR-RFLP. RAPD-PCR and BOX-PCR revealed high homogeneity among the Canary Island strains (>80 and >75% of similarity, respectively) which could not be grouped based on tomato cultivar, location or year of isolation. By contrast, strains of Cmm from other countries displayed high diversity, providing several clusters, most of which were composed of a single strain. Similarly, AFLP analysis of 29 selected strains of Cmm gave the same profile for the Canarian ones (>90% of similarity) whereas high polymorphism was obtained with strains from different countries. Moreover, two strains, one from the USA and another from Spain, were related to the Canarian strains, according to RAPD-PCR (>60% of similarity), BOX-PCR (>75%) and AFLP analysis (>90%), suggesting a common origin. The circumstances under which the Cmm outbreaks occurred in the Canary Islands and the high homogeneity observed among the Canarian strains would suggest that the bacterium was introduced into the region from only one origin.     

Molecular typing and spread of Clavibacter michiganensis subsp. michiganensis in greenhouses in Japan

Clavibacter michiganensis subsp. michiganensis (CMM) strains collected between 2005-2008 from greenhouses in different locations in Okayama Prefecture, Japan, were fingerprinted by repetitive sequence-based polymerase chain reaction (rep-PCR) with ERIC and BOX primers. One hundred and eighty strains from eight different locations in Okayama were differentiated into four haplotypes (A to D) based on rep-PCR. Regardless of the year of isolation, location or cultivar of tomato, the strains in each greenhouse and location belonged to the same haplotype, suggesting the strains originated from the previous greenhouse population. Based on Morisita's index of dispersion ( I _δ ), the distribution of diseased plants in the greenhouses, where disbudding and defoliation using either scissors or by hand were carried out in the same direction to promote the spread of CMM, occurred in an aggregated distribution in a quadrant along a row of plants, but the distribution of diseased plants indicated a random distribution in a quadrant along a furrow of plants (two adjoining rows of plants). These results showed that disbudding and defoliation contribute highly to the secondary spread of bacterial canker in commercial greenhouses.     

PCR技术快速检测玉米内州萎蔫病菌研究

玉米内州萎蔫病是北美玉米生产中的严重病害,为防止其传入我国,本研究采用Clavibacter michiganensis不同亚种的转录间隔区序列,设计了特异性的PCR引物,对C.michiganensis种下不同亚种的DNA进行了PCR扩增反应,结果表明,只有玉米内州萎蔫病菌的特异性扩增得到大小为170bp的目标片段,且仅有此一条明显的PCR扩增产物,C.michganensis种下其他亚种无扩增产物。PCR反应的灵敏度为4.36×105cfu/mL和1.56×102pg,可以满足检疫的要求。     

Colonization and movement of GFP-labeled Clavibacter michiganensis subsp. michiganensis during tomato infection

The vascular pathogen Clavibacter michiganensis subsp. michiganensis is responsible for bacterial wilt and canker of tomato. Pathogenicity of this bacterium is dependent on plasmid-borne virulence factors and serine proteases located on the chromosomal chp/tomA pathogenicity island (PAI). In this study, colonization patterns and movement of C. michiganensis subsp. michiganensis during tomato infection was examined using a green fluorescent protein (GFP)-labeled strain. A plasmid expressing GFP in C. michiganensis subsp. michiganensis was constructed and found to be stable in planta for at least 1 month. Confocal laser-scanning microscopy (CLSM) of inoculated stems showed that the pathogen extensively colonizes the lumen of xylem vessels and preferentially attaches to spiral secondary wall thickening of the protoxylem. Acropetal movement of the wild-type strain C. michiganensis subsp. michiganensis NCPPB382 (Cmm382) in tomato resulted in an extensive systemic colonization of the whole plant reaching the apical region after 15 days, whereas Cmm100 (lacking the plasmids pCM1 and pCM2) or Cmm27 (lacking the chp/tomA PAI) remained confined to the area surrounding of the inoculation site. Cmm382 formed biofilm-like structures composed of large bacterial aggregates on the interior of xylem walls as observed by CLSM and scanning electron microscopy. These findings suggest that virulence factors located on the chp/tomA PAI or the plasmids are required for effective movement of the pathogen in tomato and for the formation of cellular aggregates.