植物罹病组织中南方、爪哇、花生根结线虫的LAMP快速检测

【目的】建立一种基于环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术,从植物罹病组织中直接检测3种常见的根结线虫,为根结线虫的监测和防治提供技术支持。【方法】分别采用3种根结线虫的种类特异性引物对所选择的根结线虫的DNA片段进行PCR扩增,扩增产物纯化、回收并测序。根据3种根结线虫的测序结果,针对种类特异区段,采用PrimerExplorerV4软件,分别设计3种根结线虫的LAMP引物。设计的引物组人工合成后,以提取的纯化种群线虫DNA为模板,分别进行引物组的特异性测试,筛选出分别针对3种根结线虫的最佳引物组。【结果】研究设计的3种根结线虫的LAMP特异性引物能够直接从植物根结中检测出南方、花生、爪哇3种常见根结线虫,LAMP快速检测体系为:dNTPS浓度为1 mmol·L~(-1),Mg~(2+)的浓度为5 mmol·L~(-1),不添加甜菜碱,反应时间为45 min。【结论】本实验建立的南方、花生、爪哇根结线虫LAMP快速分子检测方法,具有特异性强、灵敏度高、简单、快速、经济等特征,能够从罹病植物组织中快速准确地检测出南方、花生和爪哇根结线虫,具有极高的实践应用价值。     

不同产区花生青枯病菌遗传多样性的分析

花生青枯病是一种重要的毁灭性病害。本研究将菌落PCR引入到花生青枯菌的鉴定及基因组研究工作中,通过菌落PCR直接对四川省内不同花生产区收集得到的花生青枯病菌进行菌株鉴定并对供试菌株的重要功能基因phcA、hrp以及hcp进行遗传多样性的比较分析。结果表明,菌落PCR能快速有效地扩增得到不同片段大小的目标片段,通过菌落PCR产物的直接测序并进行序列比对,发现来源于不同生态环境、不同花生品种的青枯病菌株其重要的功能基因在序列上不存在差异,表明这些重要的功能基因具有高度保守的特性。菌落PCR省去了菌株的再培养以及DNA的提取等繁琐过程,不仅可实现对花生青枯菌高通量的分子鉴定,还可快速地开展基因克隆及基因组学方面的研究工作,在花生青枯菌的筛选、鉴定以及植物检疫方面具有较强的应用性,在花生青枯菌的基因组研究中具有重要的应用价值。     

噬菌体鸡尾酒联合生物有机肥防控番茄青枯病的效果研究

【背景】前期研究表明,可专性侵染青枯菌的噬菌体鸡尾酒(组合)可有效减少番茄青枯病的发生。生物有机肥虽然可降低青枯病发病率,但受田间环境影响,防控效果常不稳定。【目的】为了提高生物有机肥的防控效果,靶向抑制番茄青枯病,探究噬菌体鸡尾酒联合含有解淀粉芽孢杆菌的生物有机肥防控番茄青枯病的田间效果,以及该防控方法对番茄根际细菌群落结构的影响。【方法】将经解淀粉芽孢杆菌T-5二次发酵获得的生物有机肥(Bio-Organic Fertilizer,BOF)在春季作为基肥施入番茄大棚,开花期在番茄根部浇灌噬菌体鸡尾酒悬液,统计青枯病的发生情况和番茄根际青枯菌的数量,根据高通量测序结果分析番茄根际细菌群落的结构变化。【结果】与常规施肥相比,生物有机肥配合噬菌体鸡尾酒(BOF+P)可显著降低番茄青枯病的发病率,显著改变根际细菌群落的β多样性,提高拟杆菌门(Bacteroidetes)的相对丰度,并降低芽单胞菌门(Gemmatimonadetes)的相对丰度。【结论】噬菌体鸡尾酒可显著提升生物有机肥防控番茄青枯病的效果,具有良好的应用潜力。     

一株番茄青枯病菌拮抗细菌的筛选、发酵条件优化及田间小区防效

[背景]番茄青枯病是由青枯劳尔氏菌(Ralstonia solanacearum)引起的一种土传细菌性病害,该病原菌严重影响番茄的生产。[目的]筛选番茄青枯病的生防细菌,并将其用于病害防治。[方法]采用抑菌圈法、琼脂扩散法从湖南衡阳青枯病发病田的健康番茄根际土壤筛选对青枯劳尔氏菌具有较强拮抗能力的菌株,通过形态学观察、生理生化试验、16S rRNA基因和gyrA基因测序分析确定其分类地位;以单因素试验和正交试验对发酵条件进行优化;通过田间小区试验初探其防效。[结果]筛选的菌株TR-1被初步鉴定为贝莱斯芽孢杆菌(Bacillus velezensislezensis);菌株TR-1最佳培养基配方(g/L):可溶性淀粉20.0,大豆蛋白胨10.0,磷酸氢二钾5.0;最佳发酵条件:pH6.0-7.0,温度30-33℃,摇床转速160 r/min,发酵时长48 h,优化后TR-1无菌发酵上清液对青枯菌抑菌圈直径达2.95 cm,约为优化前的2倍;其田间小区防效为60.30%。[结论]通过对菌株TR-1发酵条件进行优化可大大提升其发酵液抑菌效果,而且菌株TR-1在田间小区试验中对番茄青枯病防效优...     

云南省烟植地青枯菌RS-22的分离及其拮抗菌的筛选和鉴定

【背景】由青枯劳尔氏菌(Ralstonia solanacearum)引起的烟草青枯病是一种重要土传病害,在我国南方烟区普遍发生。生物防控是针对烟草青枯病的一种有效防治措施,但是相关的研究报道还较少。【目的】分离云南省烟植地的青枯病原菌,筛选其拮抗菌并对其抑菌效果进行鉴定。【方法】采用平板稀释法从云南感病烟草中分离获得青枯菌,采用平板对峙法筛选青枯菌拮抗菌,筛选得到的拮抗菌通过16SrRNA基因测序比对确定菌种类型,并在实验室和大田鉴定其对青枯病的防治效果。【结果】从感病烟草茎中分离出一株强致病性青枯菌小种RS-22,该菌能侵染烟草和番茄并最终使植物死亡;筛选出12株RS-22拮抗菌,其中拮抗作用最强的是Y4;Y4被鉴定为一株解淀粉芽孢杆菌(Bacillus amyloliquefaciens),其菌体和分泌物都能抑制RS-22生长;Y4根部灌根处理能显著提高烟草和番茄对青枯菌RS-22的抗性,Y4处理能使感病烟草部分恢复正常,在云南文山州烟草种植大田施加Y4菌剂和菌剂有机肥混合物也能显著降低烟草青枯病的感病率。【结论】青枯菌RS-22具有广谱的致病性,筛选的拮抗菌Y4能显著抑制青枯菌生长,而且对青枯菌侵染植物有很好的防治效果。研究结果为进一步研究烟草青枯病的生物防控提供了新的理论依据。     

青枯假单胞菌致病性的分子生物学

青枯假单胞菌(Pseudomonas solanacearum)或称青枯菌引起许多重要经济作物如烟草、花生、番茄等植物的萎焉病。主要通过土壤传染病害,它的寄主范围很广泛,有33科100多个种,危害茄科植物为最多。     

红树内生海洋细菌CIII-1发酵条件初步研究

摇瓶法测定结果表明,用7号培养基,初始pH 7.5,装液量1/10摇瓶体积,转速150 r/min,温度25℃下培养96 h,菌株分泌产生抗番茄青枯病细菌的物质最多,抑菌圈直径最大,为红树植物内生海洋细菌CIII-1菌株产抗植物青枯病菌物质的最佳培养条件。并发现该菌株产生的抗菌物质对西瓜枯萎病菌、香蕉枯萎病菌等植物病原真菌也有较强的拮抗作用。     

红树内生海洋细菌CIII-1发酵条件初步研究

摇瓶法测定结果表明,用7号培养基,初始pH 7.5,装液量1/10摇瓶体积,转速150 r/min,温度25℃下培养96 h,菌株分泌产生抗番茄青枯病细菌的物质最多,抑菌圈直径最大,为红树植物内生海洋细菌CⅢ-1菌株产抗植物青枯病菌物质的最佳培养条件.并发现该菌株产生的抗菌物质对西瓜枯萎病菌、香蕉枯萎病菌等植物病原真菌也有较强的拮抗作用.     

基于EB1基因的环介导等温扩增（LAMP）法检测感染家蚕微粒子病的蚕卵

【目的】家蚕 Bombyx mori 微粒子病一直影响着蚕种业的健康发展,快速而准确地检测出寄生于蚕卵中的家蚕微孢子虫 Nosema bombycis 对于有效控制家蚕微粒子病危害意义重大。【方法】环介导等温扩增（loop-mediated isothermal amplification, LAMP）法是一种快速、灵敏、特异的DNA体外恒温扩增方法,本文基于LAMP检测法的原理,依据家蚕微孢子虫孢子繁殖复制相关的 EB1 基因（GenBank登录号：KF421134.1）设计LAMP引物,对LAMP反应体系的最佳反应温度、内外引物浓度比、检测反应的特异性、灵敏度等进行研究,建立了一种检测蚕卵感染家蚕微粒子病的LAMP检测方法。【结果】结果表明,基于EB1 基因建立的LAMP检测方法在63℃恒温下在1.5 h内就可完成对样品的有效检测,本LAMP法对家蚕微孢子虫孢子DNA的检测灵敏度为5.0×10^-3 ng/μL,对EB1-pMD^TM19-T质粒标准品的检测灵敏度为1.0×10² copies/μL,同时对人工感染家蚕微粒子病单个母蛾产下蚕卵的1/8卵圈量或1粒蚕卵均能检出阳性结果。上述结果都分别应用凝胶电泳法、恒温荧光检测仪以及SYBR GreenⅠ显色肉眼观察法得到同步判定。【结论】本研究建立的基于EB1基因LAMP法可用于蚕卵微粒子病的检测,LAMP法为蚕种质量检验及成品卵微粒子病现场检疫提供了新技术。     

油橄榄青枯病菌的接种试验

油橄榄青枯病是油橄榄的主要病害,在我国的大部分林地都有发生和危害,严重影响油橄榄的生长发育和生存。1978年从我所油橄榄(Olea europaea L.)林地发病植株中分离出来的致病菌株、病原菌经鉴定为Pseudomonas sola nacearum Smith,简称油P_(?),根据青枯病菌寄主范围,我们采用蕃茄,茄子,辣椒,芝麻,花生,油橄榄,芭蕉、蓖麻等寄主植物进行了接种试验,并以花Ps为对比菌株。结果表明,油P和花Ps均能导致蕃茄、茄子、油橄榄、花生、辣椒,烟草致病,不侵染芭蕉、芝麻和蓖麻,但两菌株表现发病速度有快慢之别,致病性有所差异,发病症状也有不同。因此,我们认为油Ps可否是枯病菌假单胞菌油橄榄的一个小种,有待研究。根据接种试验,油橄榄林地不宜套种花生,烟草,蕃茄,茄子等作物,可套种草本的芝麻油料作物。     

Development of ELISA for the detection of Ralstonia solanacearum in tomato: its application in seed health testing

Bacterial wilt caused by Ralstonia (formerly Pseudomonas) solanacearum is worldwide in distribution. It is one of the most destructive bacterial diseases of economically important crops. The serological assays so far developed for the detection of R. solanacearum were able to provide information as to the presence or absence of the pathogen in soil and plant materials. However, they could not discriminate between virulent and avirulent strains of the pathogen and were not specific to strains and races. In the present investigation, virulent bacterial cells (encapsulated with mucin) from tomato seeds were used as antigen and polyclonal antisera were developed in rabbit using a classical immunization protocol. Antisera thus developed were examined for the antibody titre, sensitivity, specificity, rapidity and the efficacy of the antibody in identifying the virulent and avirulent strains of the pathogen and the potential for application of this assay to the screening of infected plant materials and seeds. Our results indicate that the anti-tomato R. solanacearum: (i) has a good antibody titre of 1:10,000; (ii) can detect as few as 100 bacterial cells/ml; (iii) is tomato-specific (it reacted with tomato R. solanacearum, and not with isolates from chilli or eggplant); (iv) is reactive to all isolates of R. solanacearum from tomato; (v) is not cross-reactive with non-pseudomonads; (vi) is virulent strain-specific as it recognizes the virulent exopolysaccharide component as an antigenic determinant; (vii) reactivity could be correlated well with the degree of infection in tomato seeds and plant materials. The enzyme linked immunosorbent assay developed is sensitive, specific and rapid, therefore suitable for the detection of R. solanacearum isolates from tomato seeds during routine assays.     

PCR-based specific detection of Ralstonia solanacearum by amplification of cytochrome c1 signal peptide sequences

A polymerase chain reaction (PCR)-based method was developed to detect the DNA of Ralstonia solanacearum, the causal agent of bacterial wilt in various crop plants. One pair of primers (RALSF and RALSR), designed using cytochrome c1 signal peptide sequences specific to R. solanacearum, produced a PCR product of 932 bp from 13 isolates of R. solanacearum from several countries. The primer specificity was then tested using DNA from 21 isolates of Ralstonia, Pseudomonas, Burkholderia, Xanthomonas, and Fusarium oxysporum f. sp. dianthi. The specificity of the cytochrome c1 signal peptide sequences in R. solanacearum was further confirmed by a DNA-dot blot analysis. Moreover, the primer pair was able to detect the pathogen in artificially inoculated soil and tomato plants. Therefore, the present results indicate that the primer pair can be effectively used for the detection of R. solanacearum in soil and host plants.     

Amplification of RNA by NASBA allows direct detection of viable cells of Ralstonia solanacearum in potato

AIMS: The objective of this study was to develop a Nucleic Acid Sequence Based Amplification (NASBA) assay, targeting 16S rRNA sequences, for direct detection of viable cells of Ralstonia solanacearum, the causal organism of bacterial wilt. The presence of intact 16S rRNA is considered to be a useful indicator for viability, as a rapid degradation of this target molecule is found upon cell death. METHODS AND RESULTS: It was demonstrated by RNase treatment of extracted nucleic acids from R. solanacearum cell suspensions that NASBA exclusively detected RNA and not DNA. The ability of NASBA to assess viability was demonstrated in two sets of experiments. In the first experiment, viable and chlorine-killed cells of R. solanacearum were added to a potato tuber extract and tested in NASBA and PCR. In NASBA, only extracts spiked with viable cells resulted in a specific signal after Northern blot analysis, whereas in PCR, targeting 16S rDNA sequences, both extracts with viable and killed cells resulted in specific signals. In the second experiment, the survival of R. solanacearum on metal strips was studied using NASBA, PCR-amplification and dilution plating on the semiselective medium SMSA. A positive correlation was found between NASBA and dilution plating detecting culturable cells, whereas PCR-amplification resulted in positive reactions also long after cells were dead. The detection level of NASBA for R. solanacearum added to potato tuber extracts was determined at 104 cfu per ml of extract, equivalent to 100 cfu per reaction. With purified RNA a detection level of 104 rRNA molecules was found. This corresponds with less than one bacterial cell, assuming that a metabolically active cell contains ca 105 copies of rRNA. Preliminary experiments demonstrated the potential of NASBA to detect R. solanacearum in naturally infected potato tuber extracts. CONCLUSIONS: NASBA specifically amplifies RNA from viable cells of R. solanacearum even present in complex substrates at a level of 100 cfu per reaction. SIGNIFICANCE AND IMPACT OF THE STUDY: The novel NASBA assay will be particularly valuable for detection of R. solanacearum in ecological studies in which specifically viable cells should be determined.     

Enrichment double-antibody sandwich indirect enzyme-linked immunosorbent assay that uses a specific monoclonal antibody for sensitive detection of Ralstonia solanacearum in asymptomatic potato tubers

Sensitive and specific routine detection of Ralstonia solanacearum in symptomless potato tubers was achieved by efficient enrichment followed by a reliable double-antibody sandwich indirect enzyme-linked immunosorbent assay based on the specific monoclonal antibody 8B-IVIA. This monoclonal antibody reacted with 168 typical R. solanacearum strains and did not recognize 174 other pathogenic or unidentified bacteria isolated from potato. The optimized protocol included an initial enrichment step consisting of shaking the samples in modified Wilbrink broth for 72 h at 29 degrees C. This step enabled specific detection by the enzyme-linked immunosorbent assay of 1 to 10 CFU of R. solanacearum per ml of initial potato extract. Analysis of 233 commercial potato lots by this method provided results that coincided with the results of conventional methods.     

A polyphasic approach for studying the interaction between Ralstonia solanacearum and potential control agents in the tomato phytosphere.

Ralstonia solanacearum biovar 2, the causative agent of brown rot in potato, has been responsible for large crop losses in Northwest Europe during the last decade. Knowledge on the ecological behaviour of R. solanacearum and its antagonists is required to develop sound procedures for its control and eradication in infested fields.A polyphasic approach was used to study the invasion of plants by a selected R. solanacearum biovar 2 strain, denoted 1609, either or not in combination with the antagonistic strains Pseudomonas corrugata IDV1 and P. fluorescens UA5-40. Thus, this study combined plating (spread and drop plate methods), reporter gene technology (gfp mutants) and serological (imunofluorescence colony staining [IFC]) and molecular techniques (fluorescent in situ hybridization [FISH], PCR with R. solanacearum specific primers and PCR-DGGE on plant DNA extracts). The behaviour of R. solanacearum 1609 and the two control strains was studied in bulk and (tomato) rhizosphere soil and the rhizoplane and stems of tomato plants.The results showed that an interaction between the pathogen and the control strains at the root surface was likely. In particular, R. solanacearum 1609 CFU numbers were significantly reduced on tomato roots treated with P. corrugata IDV1(chr:gfp1) cells as compared to those on untreated roots. Concomitant with the presence of P. corrugata IDV1(chr:gfp1), plant invasion by the pathogen was hampered, but not abolished.PCR-DGGE analyses of the tomato rhizoplane supported the evidence for antagonistic activity against the pathogen; as only weak R. solanacearum 1609 specific bands were detected in profiles derived from mixed systems versus strong bands in profiles from systems containing only the pathogen. Using FISH, a difference in root colonization was demonstrated between the pathogen and one of the two antagonists, i.e. P. corrugata IDV1(chr:gfp1); R. solanacearum strain 1609 was clearly detected in the vascular cylinder of tomato plants, whereas strain IDV1 was absent.R. solanacearum 1609 cells were also detected in stems of plants that had developed in soils treated with this strain, even in cases in which disease symptoms were absent, indicating the occurrence of symptomless infection. In contrast, strain 1609 cells were not found in stems of several plants treated with either one of the two antagonists.The polyphasic analysis is valuable for testing antagonistic strains for approval as biocontrol agents in agricultural practice.     

Specific and sensitive detection of Ralstonia solanacearum in soil on the basis of PCR amplification of fliC fragments

Ralstonia solanacearum is the causative agent of bacterial wilt in many important crops. A specific and sensitive PCR detection method that uses primers targeting the gene coding for the flagella subunit, fliC, was established. Based on the first fliC gene sequence of R. solanacearum strain K60 available at GenBank, the Ral_fliC PCR primer system was designed; this system yielded a single 724-bp product with the DNAs of all of the R. solanacearum strains tested. However, R. pickettii and four environmental Ralstonia isolates also yielded amplicons. The Ral_fliC PCR products obtained with 12 strains (R. solanacearum, R. pickettii, and environmental isolates) were sequenced. By sequence alignment, Rsol_fliC primers specific for R. solanacearum were designed. With this primer system, a specific 400-bp PCR product was obtained from all 82 strains of R. solanacearum tested. Six strains of R. pickettii and several closely related environmental isolates yielded no PCR product; however, a product was obtained with one Pseudomonas syzygii strain. A GC-clamped 400-bp fliC product could be separated in denaturing gradient gels and allowed us to distinguish P. syzygii from R. solanacearum. The Rsol_fliC PCR system was applied to detect R. solanacearum in soil. PCR amplification, followed by Southern blot hybridization, allowed us to detect about one target DNA molecule per PCR, which is equivalent to 10(3) CFU g of bulk soil(-1). The system was applied to survey soils from different geographic origins for the presence of R. solanacearum.     

青枯菌诱导的花生基因表达谱SSH分析

以抗青枯病花生种质‘J4’和‘中花6号’、感青枯病花生品种‘中花12号’为材料,用强产青枯菌毒菌株(Ralstonia solanacearum)对其根系分别接种,采用抑制差减杂交(SSH)技术检测花生根系应答侵染的基因表达谱变化,并对文库中差异基因进行Real-time PCR分析。结果表明:经菌液PCR检测对挑选出的1 036阳性克隆片段进行测序及片段整合分析,获得162条花生基因,有功能注释的基因58条,其中44条基因参与了细胞结构(6%)、信号转导(12%)、抗病防御(5%)、转录调控(12%)等生理过程。用Real-time PCR技术对7个基因在‘中花6号’和‘中花12号’中的表达模式分析结果表明,6个基因在青枯菌侵染早期在抗病材料‘中花6号’中呈上调表达,可能与青枯病抗性直接相关。     

产肠毒素大肠杆菌快速检测方法的建立和评价

目的利用环介导等温扩增(LAMP)技术,建立产肠毒素大肠杆菌(ETEC)的快速、便捷、敏感、特异的检测方法,并对该方法的特异性和敏感性进行评价,为实验动物检测和细菌性腹泻的诊断提供技术支持。方法根据GenBank公布的产肠毒素大肠杆菌的LT毒素基因序列(S60731.1)设计外引物和内引物进行LAMP扩增,对LAMP特异性和敏感性与PCR方法做比较。结果建立的LAMP方法检测最低浓度为100 pg/μL,灵敏度是PCR的10倍以上并具有较高的特异性,利用该方法对27份猴腹泻样品进行LAMP和PCR方法检测,发现PCR检出率为33.3%,LAMP(60 min内)结果与PCR相同,而LAMP(90 min内)检出率为92.6%,约是PCR检出率的3倍。结论建立了一种用于检测肠毒性大肠杆菌(ETEC)的LAMP检测方法,该方法特异性强,灵敏度高,方便快捷,适合于ETEC临床快速检测。     

Static suppression of tomato bacterial wilt by bacterial coagulation using a new functional polymer that coagulates bacterial cells and is highly biodegradable

Tomato bacterial wilt by Ralstonia solanacearum was suppressed by coagulation of bacterial cells without disinfection using a copolymer of methyl methacrylate with N-benzyl-4-vinylpyridinium chloride in a molar ratio of 3:1 (PMMA-co-BVP) as a polymeric coagulant for bacterial cells. When 10 mg/kg of PMMA-co-BVP was added to soil before transplanting of tomato seedlings, and 2 mg/kg was supplemented once a week after transplanting, a 51% reduction of appearance and a 54% reduction of index of symptoms were observed. PMMA-co-BVP did not exhibit bactericidal activity against R. solanacearum, and coagulation of the bacterial cells appeared to reduce the opportunity for infectious contact of roots of tomato with cells of R. solanacearum, and resulted in disease suppression. PMMA-co-BVP was shown to be highly biodegradable, and the half-life was 5.1 d when treated with activated sludge in soil.