黄瓜细菌性白枯病病菌检测试纸条的研制

应用胶体金免疫层析技术研制了黄瓜细菌性白枯病病菌[Pseudomonas viridiflava (Burkholder 1930) Dowson1939]检测试纸条.采用柠檬酸三钠法还原氯金酸制备胶体金,标记黄瓜细菌性白枯病病菌多克隆抗体,将金标抗体喷涂在结合垫上,将黄瓜细菌性白枯病病菌抗体和羊抗兔二抗包被在硝酸纤维素膜上作检测线和质控线,组装制成黄瓜细菌性白枯病病菌检测试纸条.用试纸条检测黄瓜细菌性白枯病病菌的结果表明,制备的试纸条特异性好,与其他常见植物病原细菌等无交叉反应,对黄瓜叶片中黄瓜细菌性白枯病病菌的最低检测限为106 cfu/mL,能在5～15 min内快速检测出黄瓜细菌性白枯病病菌,适合田间现场快速检测黄瓜细菌性白枯病病菌.     

基于胶体金免疫层析法快速检测蓝莓中的百菌清残留

为建立蓝莓样品中百菌清残留快速筛查方法，以农药百菌清为目标分析物，系统研究了胶体金标记参数及样品前处理方法对胶体金免疫层析方法 (colloidal gold immuno-chromatographic assay, GICA)的影响。结果表明：以25 nm的胶体金颗粒标记百菌清单克隆抗体作为检测探针，分别将包被原百菌清-BSA (1 mg/mL)和羊抗鼠IgG抗体(0.1 mg/mL)包被于硝酸纤维膜(NC膜)，形成检测线(T线)和质控线(C线)，组装成百菌清胶体金免疫层析检测试纸条。蓝莓样品经酸化乙腈提取，双蒸水(dd H2O)稀释后，应用该纸条对蓝莓中百菌清残留肉眼观察检出限(LOD)为0.1 mg/kg (T线完全消线)，可实现15 min内蓝莓中百菌清的定性与半定量分析，同时，试纸条对样品中4-羟基百菌清、五氯硝基苯、多菌灵和腐霉利的检测不存在交叉反应。蓝莓中百菌清添加回收试验的胶体金免疫层析检测试纸条测试结果与超高效液相色谱-三重四级杆串联质谱仪(UPLC-MS/MS)方法的检测结果一致。这两种方法都可以成功地应用于蓝莓中百菌清的检测，胶体金免疫层析检测试纸条有助于现场检...     

黄瓜细菌性角斑病菌交叉引物恒温扩增检测技术

黄瓜细菌性角斑病是我国黄瓜生产上的重要病害之一,其病原菌为Pseudomonas syringae pv.lachrymans。根据该病原菌甘油醛-3-磷酸脱氢基因保守序列设计引物和探针,建立了交叉引物恒温扩增和核酸试纸条检测技术。菌体DNA检测灵敏度可达0.55 ng,纯菌直接检测灵敏度基本可达到单个细菌。所测试的5株黄瓜细菌性角斑病菌和染病黄瓜叶片均为阳性,其他13株对照菌株均为阴性。该方法灵敏度高,且操作简单,对设备要求低等,适合基层实验室应用。     

建兰花叶病毒和齿兰环斑病毒联合免疫胶体金试纸条的研制及应用

为了实现兰花种植苗圃和口岸一线直接检测样品中建兰花叶病毒Cymbidium mosaic virus(CymMV)和齿兰环斑病毒Odontoglossum ringspot virus(ORSV)的目的,我们研制了上述两种病毒联合免疫胶体金检测试纸条。采用柠檬酸三钠还原法制备胶体金颗粒,标记单抗C10并固定于胶金垫上;将抗体5B7和4F3分别固定于硝酸纤维素膜上作为检测线,将羊抗鼠抗体包被固定于硝酸纤维素膜上作为质控线,经优化后组装胶体金试纸条。结果表明研制的联合试纸条特异性强,能够在10min之内同时检出两种病毒,操作简便。对两个种植苗圃的93份兰花样品进行实际检测所得结果与ELISA的检测结果符合性好(Kappa值分别为84.6%和86.7%),能满足兰花样品中建兰花叶病毒和齿兰环斑病毒的初筛要求。     

利用GICA-PCR快速检测瓜类果斑病菌

瓜类果斑病菌(Acidovorax avenae subsp.citrulli,Aac)是瓜类作物上重要的病原细菌,为我国进境植物检疫性有害生物。胶体金免疫层析试纸条方便快捷,应用广泛。该方法使用不当会出现假阳性问题,仅适用于病原菌的初筛检测。本研究将Aac胶体金免疫层析方法(GICA)与PCR技术相结合,建立了GICA-PCR检测方法。检测结果表明,该方法在蛋白与核酸2个层面上从发病西瓜叶片上检测到瓜类果斑病菌,有效解决了试纸条检测的假阳性问题,提高了瓜类果斑病菌检测的准确性,值得推广应用。     

番茄环斑病毒和烟草环斑病毒复合型胶体金免疫层析试纸条的研制

采用柠檬酸三钠还原法制备胶体金颗粒,标记番茄环斑病毒和烟草环斑病毒的兔多克隆抗体,用微定量喷头在硝酸纤维素膜上喷好2条病毒检测线（T线）和1条羊抗兔抗体质控线（C线）,制成复合型免疫层析检测试纸条。一张试纸条在10min内,可同时检测出这两种病毒。试纸条检测番茄环斑病毒和烟草环斑病毒粗提纯液,检测灵敏度在1μg/mL,试纸条检测番茄环斑病毒和烟草环斑病毒的混合病汁液可稀释1000倍（重量/体积）。用健康叶片和缓冲液对照测试,试纸条结果都为阴性。     

4种葫芦科植物病毒复合胶体金免疫层析试纸条的研制

使用柠檬酸三钠还原法制备胶体金颗粒,在pH值7.6,蛋白用量10～15μg/mL条件下,制备形成4种葫芦科植物病毒多克隆抗体的稳定胶体金蛋白复合物;设计了双向复合试纸条,使用微定量喷头在试纸条两侧的硝酸纤维素膜上分别喷2条病毒检测线和1条质控线,组装制成免疫层析检测试纸条。结果表明经过条件优化后制备的双向复合试纸条特异性好,可在10min内同时检测4种葫芦科植物病毒,且对不同病毒阳性材料的检测灵敏度可达到稀释103倍以上。     

番茄褐色皱果病毒胶体金免疫试纸条的研制

 番茄褐色皱果病毒(tomato brown rugose fruit virus, ToBRFV)是一种新发病毒,严重威胁番茄的安全生产。为了快速、简便地检测该病毒,我们制备了ToBRFV胶体金免疫试纸条。本研究以ToBRFV粒子为免疫原,通过杂交瘤技术制备了17个抗ToBRFV的单克隆抗体。将不同单抗两两组合分别作为胶体金标记抗体和硝酸纤维素膜检测线上的捕获抗体,共获得272个配对组合的胶体金试纸条。通过特异性测定筛选到一组配对抗体制备的试纸条能够在5 min内特异识别ToBRFV,而与番茄斑驳花叶病毒、番茄花叶病毒、烟草花叶病毒、黄瓜花叶病毒、辣椒轻斑驳病毒、马铃薯X病毒、马铃薯Y病毒、番茄褪绿病毒、番茄斑萎病毒、番茄黄化曲叶病毒等无交叉反应。灵敏度分析表明,该试纸条可从稀释12 800倍的番茄叶片病汁液中检测到ToBRFV,也可检测到50 ng ToBRFV粒子。本研究制备的胶体金试纸条使用方便,灵敏度高,特异性强,适合田间大批量样品检测,可用于ToBRFV的精准监测及早期预警。     

检测南方水稻黑条矮缩病毒胶体金免疫试纸条的建立

由白背飞虱Sogatella furcifera(Horváth)传播的南方水稻黑条矮缩病毒(Southern rice blackstreaked dwarf virus,SRBSDV)是目前我国南方水稻上危害最严重的病毒,为开发简便、快速、准确的SRBSDV病毒检测技术和检测试剂,以感染SRBSDV的植物粗提液为免疫原,利用杂交瘤技术制备了2株抗SRBSDV的单抗(14A8和15G6),并利用制备的单抗建立了可快速、特异、灵敏地检测SRBSDV的胶体金免疫试纸条。结果表明,2株制备单抗的抗体类型及亚类均为Ig G1、kappa链,单抗腹水的间接ELISA效价均达到10~(-7);Western blot分析表明,2株单抗均与SRBSDV的外壳蛋白亚基有特异反应,而不与水稻黑条矮缩病毒(Rice black-streaked dwarf virus,RBSDV)反应。以制备14A8和15G6单抗分别为捕获抗体和胶体金标记抗体,开发成能在5 min内准确、特异地检测水稻植物和白背飞虱传毒介体体内SRBSDV的胶体金免疫试纸条;灵敏度分析表明,该检测试纸条的检测水稻病叶的灵敏度达到1∶6 400倍(g/m L),检测单头携毒白背飞虱的灵敏度达到1∶51 200倍(单头/μL)。田间样品检测结果表明,该试纸条的检测结果与RT-PCR的符合率达到100%。建立的SRBSDV胶体金免疫试纸条可对南方水稻黑条矮缩病毒进行快速、特异、灵敏的诊断和检测。     

应用PCR方法快速检测黄瓜细菌性角斑病菌

黄瓜细菌性角斑病是黄瓜上的一种重要细菌病害,其病原为丁香假单胞菌黄瓜致病变种(Pseudomonas syringae pv.lachrymans),目前未见到该病害特异性PCR检测方法的报道。通过分析丁香假单胞菌(P.syringae)不同致病变种glyceraldehyde-3-phosphate dehydrogenase 1(gap1)基因序列设计得到一对Psl特异性PCR引物。利用该引物对丁香假单胞菌不同致病变种、假单胞菌属其他种及其他属的共46株菌株进行了PCR扩增,结果表明,所有不同来源的12株黄瓜细菌性角斑病菌均得到179bp的目标片段,而所有其他参试菌株均无扩增条带,PCR检测的灵敏度为7.5×103cfu/mL。利用该方法可从接种后发病的黄瓜叶片总DNA中检测到特异条带,而健康叶片无条带。该引物的PCR检测方法可直接用于植株总DNA的检测,无需进行病原菌的分离培养,快速简便,适用于进出境检验检疫及种苗健康检测等。     

Genetic diversity of Pseudomonas syringae pv. lachrymans strains isolated from cucumber leaves collected in Poland

Several strains of Pseudomonas syringae pathovar (pv.) lachrymans and related bacterial pathogens were isolated from cucumber ( Cucumis sativus ) leaves collected in central and southern Poland in 2001 and 2002. Twenty five original strains, together with five reference strains of P. syringae pv. lachrymans , pv. syringae and pv. tomato , were genetically characterized by PCR-RFLP (polymerase chain reaction − restriction fragment length polymorphism), ADSRRS (amplification of DNA fragments surrounding rare restriction sites), and PCR-MP (PCR − melting profiles) fingerprinting techniques. Genetic similarity analyses of the PCR-RFLP and ADSRRS fingerprints showed that strains of P. syringae pv. lachrymans form distinct clusters. The results also indicated that the ADSRRS and the PCR-MP fingerprinting techniques may serve as more efficient tools for evaluating genetic similarity among pathovars and strains of P. syringae than PCR-RFLP. The 25 strains showed diverse pathogenicity to cucumber seedlings and biochemical tests were varied. The syrB gene was identified in four cucumber strains, characterized as P. syringae pv. syringae .     

采用16S rDNA鉴定甜瓜细菌性叶斑病菌

从甘肃河西、新疆阿勒泰甜瓜上分离获得的2株致病细菌,通过16S rDNA序列测定以及序列同源性比较,结合病原菌落培养性状、菌体形态观察和革兰氏染色反应等,初步确定当地甜瓜细菌性叶斑病菌为丁香假单胞杆菌[Pseudomonas syringae pv.lachrymans (Smith et Bryan) Younget al.]     

Multilocus sequence typing of Pseudomonas syringae sensu lato confirms previously described genomospecies and permits rapid identification of P. syringae pv. coriandricola and P. syringae pv. apii causing bacterial leaf spot on parsley

Since 2002, severe leaf spotting on parsley (Petroselinum crispum) has occurred in Monterey County, CA. Either of two different pathovars of Pseudomonas syringae sensu lato were isolated from diseased leaves from eight distinct outbreaks and once from the same outbreak. Fragment analysis of DNA amplified between repetitive sequence polymerase chain reaction; 16S rDNA sequence analysis; and biochemical, physiological, and host range tests identified the pathogens as Pseudomonas syringae pv. apii and P. syringae pv. coriandricola. Koch's postulates were completed for the isolates from parsley, and host range tests with parsley isolates and pathotype strains demonstrated that P. syringae pv. apii and P. syringae pv. coriandricola cause leaf spot diseases on parsley, celery, and coriander or cilantro. In a multilocus sequence typing (MLST) approach, four housekeeping gene fragments were sequenced from 10 strains isolated from parsley and 56 pathotype strains of P. syringae. Allele sequences were uploaded to the Plant-Associated Microbes Database and a phylogenetic tree was built based on concatenated sequences. Tree topology directly corresponded to P. syringae genomospecies and P. syringae pv. apii was allocated appropriately to genomospecies 3. This is the first demonstration that MLST can accurately allocate new pathogens directly to P. syringae sensu lato genomospecies. According to MLST, P. syringae pv. coriandricola is a member of genomospecies 9, P. cannabina. In a blind test, both P. syringae pv. coriandricola and P. syringae pv. apii isolates from parsley were correctly identified to pathovar. In both cases, MLST described diversity within each pathovar that was previously unknown.     

广东南瓜细菌性叶枯病及其病原鉴定

 在广东省雷州市发生一种南瓜(Cucurbita moschata)叶枯病,病株叶片边缘开始出现水渍状病斑,逐步发展成大病斑,后期病斑焦枯;在叶片上也可形成近圆形水渍状病斑,伴有黄色晕圈,后期病斑联合形成不规则大枯斑;叶柄和匍匐茎被侵染后呈水渍状腐烂。从病斑上分离到一种细菌,在KB培养基上,菌落为椭圆形,乳白色,半透明,边缘参差不齐,紫外灯照射下产生荧光反应。致病性测定结果表明,该病原细菌可侵染6个南瓜品种引起与田间症状相同的叶枯病。生理生化试验结果表明,该病原细菌与丁香假单胞丁香致病变种(Pseudomonas syringae pv. syringae)的特性一致。应用假单胞菌属特异引物Ps-for/Ps-rev和丁香假单胞丁香致病变种组群特异性引物Group III-F/Group III-R,可从该病原细菌中扩增出预期大小分别为1 018 bp和750 bp的目的片段。应用丁香致病变种syrB基因特异性引物B1/B2,可从该病原菌中扩增出预期大小为750 bp的丁香霉素基因片段。基于16S rDNA与gyrB基因序列系统进化分析均表明,南瓜叶枯病菌株与已报道的P. syringae pv. syringae菌株HS191(CP006256)亲缘关系最近,二者聚类在一起形成一个小分支。人工接种条件下,该病原细菌还可侵染西葫芦、丝瓜、茄子、番茄、菜豆、扁豆等植物。这些结果表明,引起广东省南瓜叶枯病的病原为丁香假单胞丁香致病变种(Pseudomonas syringae pv. syringae)。这是首次在中国发现丁香假单胞丁香致病变种引起南瓜叶枯病。     

Factors that Affect Spread of Pseudomonas syringae in the Phyllosphere

ABSTRACT Successful spread of an organism to a new habitat requires both immigration to and growth on that habitat. Field experiments were conducted to determine the relative roles of dispersal (i.e., immigration) and bacterial multiplication in spread of Pseudomonas syringae pv. syringae in the phyllosphere. To study spread, individual plots consisted of three nested concentric squares with the inner 6 m(2) planted to snap beans serving as the sink. Each sink, in turn, was surrounded by a barrier zone, usually 6 m wide, which was surrounded by a 6-m-wide source area. The source areas were planted with snap bean seeds inoculated with doubly marked strains derived from wild-type P. syringae pv. syringae B728a. The treatments were designed to test the effects of the nature and width of the barrier zone and suitability of the habitat in the sinks on spread of P. syringae pv. syringae. The marked strains introduced into the source areas at the time of planting were consistently detected in sink areas within a day or two after emergence of bean seedlings in the sources as assessed by leaf imprinting and dilution plating. The amounts of spread (population sizes of the marked strain in sinks) across barrier zones planted to snap bean (a suitable habitat for growth of P. syringae pv. syringae), soybean (not a favorable habitat for P. syringae pv. syringae), and bare ground were not significantly different. Thus, the nature of the barrier had no measurable effect on spread. Similarly, spread across bare-ground barriers 20 m wide was not significantly different from that across barriers 6 m wide, indicating that distance on this scale was not a major factor in determining the amount of spread. The suitability of the sink for colonization by P. syringae pv. syringae had a measurable effect on spread. Spread to sinks planted to clean seed was greater than that to sinks planted with bean seeds inoculated with a slurry of pulverized brown spot diseased bean leaves, sinks planted 3 weeks before sources, and sinks planted to a snap bean cultivar that does not support large numbers of P. syringae pv. syringae. Based of these results, we conclude that the small amount of dispersal that occurred on the scale studied was sufficient to support extensive spread, and suitability of the habitat for multiplication of P. syringae pv. syringae strongly influenced the amount of spread.     

Genetic characterization by fluorescent AFLP of Pseudomonas savastanoi pv. savastanoi strains isolated from different host species

The genetic diversity of 71 Pseudomonas savastanoi pv. savastanoi strains isolated from different host species and from diverse geographical regions was determined by fluorescent amplified fragment length polymorphism (f-AFLP) analysis. The study was carried out using three different selective primer combinations. Strains of P. syringae pv. syringae , P. syringae pv. phaseolicola , P. syringae pv. glycinea , P. syringae pv. tagetis and P. amygdali were also included as outgroups. Based on cluster analysis of f-AFLP data, all P. savastanoi pv. savastanoi strains showed a high degree of similarity, grouping in a cluster and forming a taxon clearly separate from outgroup strains. AFLP analyses failed to support placing strains of P. savastanoi pv. savastanoi , P. syringae pv. phaseolicola and P. syringae pv. glycinea in the same species. Strains of P. savastanoi pv. savastanoi formed subclusters that correlated with the host species. Strains identified within these subclusters were related to the geographical region where the strains were isolated. Strains of P. savastanoi pv. savastanoi from olive were divided into two subclusters. Strains from oleander were differentiated from those from ash and were divided into two additional subclusters, distinct from olive strains. Three strains isolated from jasmine showed a high level of similarity among them but, at a lower Dice similarity coefficient, were linked to a subcluster including olive strains. Finally, two strains isolated from privet were similar to strains from olive and were included in the same subcluster.     

Detection of New Ethylene-Producing Bacteria, Pseudomonas syringae pvs. cannabina and sesami, by PCR Amplification of Genes for the Ethylene-Forming Enzyme

ABSTRACT Strains of Pseudomonas syringae (78 strains and 43 pathovars) and other strains (79) of plant and insect origin were examined for the presence of the ethylene-forming enzyme gene (efe) by polymerase chain reaction (PCR) assay. The sequence of the efe gene of P. syringae pv. phaseolicola PK2 was used to design two primer sets for amplification of the gene. In addition to P.syringae pv. phaseolicola (the "kudzu strain") and P.syringae pv. glycinea, which were efficient ethylene producers, several strains of P.syringae pvs. sesami and cannabina generated PCR products of the predicted size. A DNA probe of the efe gene, isolated from strain PK2, hybridized to these PCR products, indicating homology to the P.syringae pv. phaseolicola efe gene. PCR restriction fragment length polymorphism analyses suggested that these four pathovars harbor a similar efe gene. Furthermore, the probe hybridized to an indigenous plasmid of P.syringae pv. cannabina, suggesting that the efe gene could be located on a plasmid in this pathovar, but did not hybridize to plas-mids of P.syringae pv. sesami strains. P.syringae pvs. sesami and cannabina strains produced ethylene in King's medium B at levels similar to those of P.syringae pvs. phaseolicola and glycinea. Thus, two new ethylene-producing bacteria were detected by the PCR assay.