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。     

利用双重PCR-DHPLC技术检测水稻细菌性谷枯病菌的研究

本研究建立了一种应用双重PCR结合变性高效液相色谱技术(polymerase chain reaction-denatured high performanceliquid chromatography,PCR-DHPLC)检测水稻细菌性谷枯病菌的方法。根据水稻细菌性谷枯病菌ITS序列(internal tran-scribed spacer)和gyrB基因序列,设计两对特异性PCR检测引物,对水稻细菌性谷枯病菌株和非水稻细菌性谷枯病菌株分别进行PCR-DHPLC及双重PCR-DHPLC检测,同时进行检测灵敏度及阳性菌株的同源性分析。结果显示,PCR-DHPLC检测的特异性强,灵敏度为菌浓度4×102cfu/mL,7株水稻细菌性谷枯病菌PCR产物同源性一致。该方法能简便、灵敏、高特异性地对水稻细菌性谷枯病菌进行高通量的自动化检测。     

利用PCR技术专化性检测水稻细菌性条斑病菌

 设计水稻细菌性条斑病菌的专化性引物,并建立相应的PCR检测体系,分别对31株水稻细菌性条斑病菌和15株水稻白叶枯病菌及其它相关菌株进行了测试。结果表明,建立的PCR检测体系可专化性检测水稻细菌性条斑病菌,而水稻白叶枯病菌和其它菌株均没有扩增信号。检测灵敏度可以达到20个细菌菌体,从自然发病和人工接种发病的水稻种子成功地检测出条斑病菌。实现了对水稻细菌性条斑病菌的快速和专化性检测。     

利用双重PCR技术快速检测水稻细菌性谷枯病菌

根据水稻细菌性谷枯病ITS和gyrB基因,设计两对特异性PCR检测引物,建立了水稻细菌性谷枯病菌的双重PCR检测方法。用该方法对水稻细菌性谷枯病菌和其它植物源性细菌进行双重PCR扩增及灵敏度测试,并对采自不同地区的水稻样本进行水稻细菌性谷枯病菌的检测。结果显示,双重PCR方法能特异性地检测出8株水稻细菌性谷枯病菌,可从含水稻细菌性谷枯病菌浓度为102cfu/mL的菌液中检测出该病菌;采用该方法对我国不同地区的水稻材料进行检测,并未发现水稻细菌性谷枯病菌。     

雪松疫霉(Phytophthora lateralis)的快速分子检测

由雪松疫霉(Phytophthora lateralis)引起的疫病是一类植物检疫性病害。为建立该病原菌的快速检测技术,本文比较分析了雪松疫霉和其他疫霉的tRNA序列,在此基础上设计了一对检测雪松疫霉的特异性引物T1/T2,该对引物从雪松疫霉中扩增得到1条192 bp的条带,而其他15种疫霉和其他真菌菌株均无扩增条带,表明该对引物对雪松疫霉具有特异性。在25μL PCR反应体系中,引物T1/T2检测灵敏度为10 pg基因组DNA;而以引物T3/T4和T1/T2进行巢式PCR扩增,能够检测到1 fg基因组DNA,使检测灵敏度提高了10 000倍。该检测体系对灭菌水中游动孢子的检测灵敏度可达0.5个游动孢子,对人工接种发病的植物组织能够特异性地检测到该病原菌。此外,进一步建立了该病原菌的实时荧光定量PCR检测体系。     

16S nested-PCR技术检测玉米细菌性枯萎病菌

 玉米细菌性枯萎病是玉米上的重要种传病害,病原菌为Pantoea stewartii subsp.stewartii。本研究设计了16S通用引物,扩增该病菌及其近似种的16S rDNA,通过序列测定和分析,针对该病菌设计了特异性引物,采用nested-PCR技术,能够准确地区别该病菌及其近似种,检测的灵敏度在DNA水平上达到10^-3 pg级,检测活菌则达到2 cfu。检测人工污染的玉米种子时,不受种子提取液中其它物质的干扰,灵敏度依然达到2 cfu。     

三重PCR检测黄瓜炭疽病菌、菌核病菌和细菌性萎蔫病菌

 本试验建立一种可同时检测黄瓜炭疽病(Colletotrichum orbiculare)、黄瓜菌核病(Sclerotinia sclerotiorum(Lib.)de Bary)和黄瓜细菌性萎蔫病(Erwinia tracheiphila)等黄瓜主要病害病原菌的三重PCR检测体系。采用正交试验设计方法, 对三重PCR的影响因素分析研究, 进行退火温度优化, 并以3个引物组、Taq DNA聚合酶、dNTP和Mg²⁺ 共6因素3水平进行多重PCR体系优化, 成功建立了适合黄瓜主要病害的三重PCR最佳检测体系, 即25 μL的反应体系中含有0.24 μmol·L^-1 CY1/CY2;0.72 μmol·L^-1 SSFWD/SSREV;0.336 μmol·L^-1 ET-P1/ ET-P2;1 U Taq聚合酶;0.15 mmol·L^-1 dNTP;1 mmol·L^-1 MgCl₂, 最适退火温度为63℃。该方法能够快速从田间黄瓜发病植株和根围土壤中将黄瓜炭疽病菌、黄瓜菌核病菌和黄瓜细菌性萎蔫病菌检测出来, 灵敏度可以达到10 pg·μL^-1。     

玉米细菌性枯萎病菌PCR检测

 根据GenBank中玉米细菌性枯萎病菌及其近似种的16S序列差异,设计了一对玉米细菌性枯萎病菌特异性引物Ps2r/Ps3r,该引物能从供试的7株玉米细菌性枯萎病菌中特异性扩增出一条268 bp的预期条带,供试的32株近似种菌株都没有扩增产物;与国内外文献报道的其它5对特异性引物相比,除引物PSA/PSB外,引物DEP1/DEP2、ES16/ESIG2c、HRP1d/HRP3c和CPSL1/CPSR2c在不同程度上对部分近似种菌株出现了扩增。试验结果表明,引物Ps2r/Ps3r和PSA/PSB能特异性扩增玉米细菌性枯萎病菌,得到预期的扩增产物。对不同系列稀释度的DNA和玉米样品中病菌的检测结果表明,由引物Ps1/Ps4和Ps2r/Ps3r组合的巢式PCR方法的检测灵敏度高于引物ITSA/ITSB和PSA/PSB组合的巢式PCR方法,也高于Bio-PCR检测方法;前者可以检测到玉米种子中300 cfu/sample的目的细菌,该检测方法在进境玉米种子样品玉米细菌性枯萎病菌的检疫中具有比较理想的应有潜力和推广价值。     

PCR-DHPLC技术快速检测玉米细菌性枯萎病菌

本研究成功建立了玉米细菌性枯萎病菌的PCR检测方法.该方法根据细菌ITS序列的特异性,设计了对玉米细菌性枯萎病菌具有稳定性点突变的特异性引物及探针,并对5株玉米细菌性枯萎病菌及18种植物原性细菌的DNA进行了PCR、实时荧光PCR及PCR结合变性高效液相色谱技术(PCR-DHPLC)检测.结果表明,几种方法特异性强,检测灵敏度均为菌液浓度102 cfu/mL,PCR-DHPLC技术具有检测成本较低、高通量、自动化程度高、污染风险小及鉴定结果准确等特点,能够满足快速、准确诊断玉米细菌性枯萎病菌的要求.     

番茄细菌性溃疡病菌的实时荧光PCR检测

 由Clavibacter michiganensis subsp.michiganensis(Cmm)引起的番茄细菌性溃疡病是一种严重危害番茄生产的种传细菌性病害。根据ITS序列多态性设计引物及TaqMan探针进行实时荧光PCR检测的结果表明,这组引物一探针能检测出所有供试的Cmm菌,对照菌均未检测到荧光信号。用接种但未显示症状的番茄苗叶片及人工处理的带菌种子提取的核酸作为模板,均能检测到病菌,其检测灵敏度比常规PCR高约100倍。实验中不需病原菌的分离培养及PCR的后续处理。该方法快速、简便、安全、准确,适用于出入境检验检疫及种子、种苗健康检测领域。     

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 .     

黄瓜细菌性角斑病免疫胶体金检测试纸条的研制

 应用免疫胶体金技术进行黄瓜细菌性角斑病(Pseudomonas syringae pv.lachrymans)的快速检测研究。为了研制黄瓜细菌性角斑病的免疫胶体金检测试纸条,通过柠檬酸三钠还原法制备胶体金,选择25 nm胶体金标记黄瓜细菌性角斑病菌多克隆抗体(CMb)。采用双抗夹心法,将CMb-胶体金复合物包被在胶体金结合垫上,将羊抗兔二抗和CMb包被在硝酸纤维素膜上,分别作为质控线(C)和检测线(T),组装成试纸条。该试纸条检测灵敏度为106 cfu/mL,与唐菖蒲疮痂病菌(Pseudomonas fluorescens biovarⅡ)等26个菌株无交叉反应,特异性强,检测时间为15 min。稳定性试验表明试纸条在37℃条件下放置15 d可保持检测结果的可靠性。用试纸条对田间采集的病叶进行检测,C线和T线清晰可见,缓冲液对照呈阴性反应。本试纸条可应用于生产上黄瓜细菌性角斑病早期快速检测,进而指导病害防治。     

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.]     

Rapid Diagnosis of Pseudomonas syringae pv. papulans, the Causal Agent of Blister Spot of Apple, by Polymerase Chain Reaction Using Specifically Designed hrpL Gene Primers

ABSTRACT The identification and detection of Pseudomonas syringae pv. papulans, the causal agent of blister spot of apple, on apple leaves and fruit was achieved by polymerase chain reaction amplification of a specific DNA fragment of the hrpL sequence. The consensus primers hrpL(1) and hrpL(2) were designed based on the alignment of pseudomonad hrpL gene sequences available in nucleic acid data banks. This primer set produced a 631-bp amplicon from 37 of the 57 pseudomonads strains tested. These strains belonged to genomospecies 1 and 2, as described by Gardan et al. (8). The amplicon obtained from 30 of these strains was digested with eight restriction enzymes. Three different restriction patterns were produced from strains belonging to genomospecies 1, resulting in A1 and A2 patterns, while strains belonging to genomospecies 2 were characterized by a B pattern. Patterns A1 and A2 differed at only two sites, a Bsp 143I site located at nucleotide 360 and a MseI site located at nucleotides 22-24. Group A2 consisted solely of P. syringae pv. papulans strains. The hrpL gene in P. syringae pv. papulans strain CFBP3323 was sequenced. Two primer sets, Pap1/Pap2 and Pap1/Pap3, were designed and tested for specificity to P. syringae pv. papulans. These primers amplified expected fragments of 242 and 303 bp, respectively. Pap1/Pap2 amplified a fragment only with P. syringae pv. papulans DNA, while Pap1/Pap3 amplified all tested strains belonging to genomospecies 1. A diagnostic procedure using the Pap1/Pap2 primer set was successful for the detection of P. syringae pv. papulans in diseased fruit and artificially inoculated leaves.     

Mixtures of plant growth-promoting rhizobacteria enhance biological control of multiple cucumber pathogens

ABSTRACT Plant growth-promoting rhizobacteria (PGPR) strains INR7 (Bacillus pumilus), GB03 (Bacillus subtilis), and ME1 (Curtobacterium flaccumfaciens) were tested singly and in combinations for biological control against multiple cucumber pathogens. Investigations under greenhouse conditions were conducted with three cucumber pathogens-Colletotrichum orbiculare (causing anthracnose), Pseudomonas syringae pv. lachrymans (causing angular leaf spot), and Erwinia tracheiphila(causing cucurbit wilt disease)-inoculated singly and in all possible combinations. There was a general trend across all experiments toward greater suppression and enhanced consistency against multiple cucumber pathogens using strain mixtures. The same three PGPR strains were evaluated as seed treatments in two field trials over two seasons, and two strains, IN26 (Burkholderia gladioli) and INR7 also were tested as foliar sprays in one of the trials. In the field trials, the efficacy of induced systemic resistance activity was determined against introduced cucumber pathogens naturally spread within plots through placement of infected plants into the field to provide the pathogen inoculum. PGPR-mediated disease suppression was observed against angular leaf spot in 1996 and against a mixed infection of angular leaf spot and anthracnose in 1997. The three-way mixture of PGPR strains (INR7 plus ME1 plus GB03) as a seed treatment showed intensive plant growth promotion and disease reduction to a level statistically equivalent to the synthetic elicitor Actigard applied as a spray.     

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.     

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.     

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.