利用多重PCR技术快速检测4种水稻病原细菌

对水稻中多种病原细菌的检测,使用常规方法往往耗时耗力,而多重PCR可以更加高效地进行多种细菌的检测。根据水稻细菌性谷枯病菌gyrB基因,水稻细菌性叶鞘褐腐病菌PfsI/R quorum sensing 位点以及水稻细菌性条斑病菌和水稻白叶枯病菌含铁细胞接受因子基因设计引物,建立4种水稻病菌的多重PCR检测方法,对方法进行特异性和灵敏度测试,并对采自不同地区的水稻样本进行检测。结果显示,多重PCR方法能同步地快速检测出水稻细菌性谷枯病菌、水稻细菌性叶鞘褐腐病菌、水稻细菌性条斑病菌或水稻白叶枯病菌,检测灵敏度达到103 cfu/mL的菌液浓度,利用该方法对我国不同地区的58份水稻种子进行检测,其中17个样本检测出水稻细菌性条斑病菌或水稻白叶枯病菌,未检测到水稻细菌性谷枯病菌和水稻细菌性叶鞘褐腐病菌。     

水稻白叶枯病菌群体感应系统对T3SS基因表达的调控作用分析

 为了阐明水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae, Xoo)利用DSF信号群体感应(QS)系统对毒性表达进行调控的机理,本研究对编码QS的rpf基因进行了分子鉴定, 分析了Δrpf基因突变体在水稻叶组织中的种群量变化, 利用RT-qPCR方法定量测定了编码T3SS的hrp基因转录本以及rpf基因自身表达。结果表明,rpfF、rpfC和rpfG基因与几种主要植物病原黄单胞菌同源序列高度保守;RpfF是烯脂酰辅酶A水合酶家族成员之一,RpfC含有组氨酸磷酸激酶结构域(HisKA)和磷酸受体结构域(REC),RpfG含有REC和HD-GYP结构域;Δrpf突变体在水稻叶组织中的种群量及其扩展能力均明显下降; hrp基因转录显著地受到QS调控;rpf基因表达与Xoo种群密度密切相关。因此,Xoo QS系统显著地调控了hrp基因的表达,在QS与T3SS表达之间存在一个信号通路。     

Development of a variable number of tandem repeats typing scheme for the bacterial rice pathogen Xanthomonas oryzae pv. oryzicola

Xanthomonas oryzae pv. oryzicola is an important bacterial pathogen responsible for outbreaks of bacterial leaf streak (BLS) on rice, mostly occurring in Asia and parts of Africa. To better monitor epidemics and assess population structures, efficient tools that allow the precise identification and diagnosis of pathogenic populations are needed. In this study, we explored variable numbers of tandem repeats (VNTR) as a fast, reliable, and cost-effective molecular typing tool. Screening of three X. oryzae pv. oryzicola genome sequences (Philippine strain BLS256, Chinese strain GX01, and Malian strain MAI10) predicted 28 candidate VNTR loci. Primer pairs for polymerase chain reaction (PCR) amplification of all 28 loci were designed and applied to a panel of 20 X. oryzae pv. oryzicola strains originating from Asia and Africa. Sequencing of PCR amplicons revealed 25 robust and polymorphic VNTR loci that are shared among Asian and African X. oryzae pv. oryzicola strains. A dendrogram constructed from 25 VNTR loci indicated that most Asian strains are clearly discriminated from African strains. However, in agreement with previous reports, one strain from Mali is related to Asian strains, pointing to a possible introduction of Asian strains to the African continent. The new VNTR-based tool described here is useful for studies of population structures and epidemiological monitoring of X. oryzae pv. oryzicola.     

Type-II secretion pathway structural gene xpsE, xylanase- and cellulase secretion and virulence in Xanthomonas oryzae pv. oryzae

Rice bacterial blight caused by Xanthomonas oryzae pv. oryzae is one of the most important diseases in rice-growing areas worldwide. Four virulence-deficient mutants were identified from a transposon mutagenesis library of X. oryzae pv. oryzae . Sequence-analysis revealed that the transposon of the four mutants inserted at different sites in the same ORF, which is homologous to the xpsE gene encoding a component of the type-II secretion system in many bacterial pathogens. Extracellular enzymes, such as xylanase and cellulase, were not secreted to the extracellular space in the mutants. Analysis of the protein profile of the extracellular, periplasmic and intracellular fractions indicated that at least two secreted proteins accumulated in the periplasmic space in the mutants. After genetic complementation of these mutants with a functional xpsE gene, the xpsE gene could express normally and the pathogenicity of the mutants and their secretion of extracellular enzymes were restored. Western blot analysis with an anticellulase antiserum also showed that cellulase was secreted normally in the complemented strains. The results show that the type-II secretion pathway structural gene xpsE is required for xylanase and cellulase secretion and full virulence in X. oryzae pv. oryzae .     

大豆斑疹病菌胞外纤维素酶编码基因的鉴定及其调控分析

 植物病原黄单胞菌的胞外纤维素酶基因具有差异性和多样性。大豆斑疹病菌(Xanthomonas axonopodis pv. glycines,Xag)具有胞外纤维素酶活性,但其编码基因和调控特性尚不明确。为了鉴定Xag的胞外纤维素酶编码基因,本研究从NEAU001菌株基因组内筛选出6个候选的纤维素酶编码基因。通过异源表达实验发现,engXCA和egl2基因分别编码的蛋白EngXCA和Egl2具有水解羧甲基纤维素的能力。酶活性测定实验表明,engXCA和egl2基因决定Xag的胞外纤维素酶活性。另外,对已知的10个毒性调控基因突变株进行胞外纤维素酶活性测定,揭示Xag胞外纤维素酶活性受DSF(diffusible signal factor)信号通路和全局性调控子Clp(Crp-like protein)正调控。荧光定量PCR结果进一步证实,RpfF和Clp参与正调控engXCA和egl2基因的mRNA水平。这些结果暗示,DSF信号通路可能通过Clp调控engXCA和egl2基因的表达,实现对Xag胞外纤维素酶活性的调控。     

Rep-PCR技术对中国水稻条斑病菌的遗传多样性初析

 采用Rep-PCR技术,对30个水稻细菌性条斑病菌株(Xanthomonas oryzae pv.oryzicola)进行遗传多样性分析,同时对李氏禾条斑病菌等其它10个参试菌株也进行了比较。Rep-PCR是利用一些基于细菌的短的重复序列引物(ERIC和BOX)的DNA扩增特性,2种引物组合的电泳图谱结合并分析,以水稻细菌性条斑病菌各自的指纹谱型在相似率80%时可分为6簇,初步表明我国水稻细菌性条斑病菌群体的遗传分化明显;发现自然界存在的弱或无毒性菌株与毒性菌株的Rep-PCR指纹图谱差异很大;毒性菌株的遗传分簇与其致病性具有一定的相关性。用ERIC扩增水稻条斑病菌基因组DNA的指纹比BOX更为多样,两者对菌株的分辨率不同。因此,Rep-PCR技术可有效地用于监测水稻细菌性条斑病菌的遗传变异,还可应用于菌株的鉴定和分类学研究。     

水稻细菌性条斑病菌拮抗细菌的筛选、评价与应用研究

从江苏省南京、泰州、扬州和宿迁等地区水稻田采集水稻病叶、健叶、根围土等样品90份,分离纯化得到1173个细菌分离物,对其进行水稻细菌性条斑病菌Xanthomonasoryzaepv．oryzicola皿内抑制试验,共获得12株拮抗能力较强的细菌,其中4株拮抗细菌抑菌圈直径大于27mm。盆栽试验结果表明,12株拮抗能力较强的细菌中有4株对水稻细菌性条斑病菌防效大于50％,其中菌株Lx-1l盆栽防效达62．5％。大面积示范试验结果表明菌株Lx．11田间防效达60．2％,显著高于化学药剂20％叶枯唑的防效（51．2％和45．8％）。形态特征、理化特性和分子鉴定分析确定菌株Lx．11为解淀粉芽孢杆菌Bacillusamyloliquefaciens。     

Involvement of Phosphoglucose Isomerase in Pathogenicity of Xanthomonas oryzae pv. oryzae

ABSTRACT Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight of rice, was subjected to transposon mutagenesis to generate mutants defective in pathogenicity. A novel mutant 74M913 was attenuated in virulence but retained its ability to cause the hypersensitive response in leaf blight-resistant rice and tomato. Cloning and sequence analysis revealed that the transposon in 74M913 was inserted in a gene homologous to the phosphoglucose isomerase (pgi) gene of X. axonopodis pv. citri. Growth of the mutant in a synthetic medium containing fructose or xylose as a sole carbohydrate source was much reduced, indicating the transposon disrupted pgi function. The interaction between expression of pgi and hypersensitive response and pathogenicity (hrp) genes was investigated because we had demonstrated previously that expression of hrp genes of X. oryzae pv. oryzae is induced in a synthetic medium containing xylose. However, pgi and the hrp gene (hrcU) were expressed independently. This study suggests that PGI is involved in pathogenicity of X. oryzae pv. oryzae.     

我国水稻白叶枯病和条斑病发生与防控研究进展

培育和种植抗病品种和研发新型绿色杀菌剂是防治水稻白叶枯病和条斑病的有效措施。最近几年有关稻黄单胞菌Xanthomonas oryzae致病效应蛋白调控水稻抗(感)病性研究取得了突破性进展, 为水稻抗性品种培育提供了新思路、新策略。本文对稻黄单胞菌-水稻互作系统中已知的TALE效应蛋白与水稻抗(感)病基因(R 或 S)的对应关系进行了归纳, 就tal基因与水稻R或S基因的协同进化进行了分析, 结合生物农药和高效低毒杀菌剂的应用现状, 提出了我国水稻白叶枯病和水稻条斑病绿色防控关键策略。     

水稻黄单胞菌致病相关基因插入突变体系的建立

 水稻白叶枯病菌（Xanthomonas oryzae pv. oryzae, Xoo）和条斑病菌（Xanthomonas oryzae pv. oryzicola, Xoc）是水稻上的模式病原菌,分别引起水稻白叶枯病（bacterial blight, BB）和细菌性条斑病（bacterial leaf streak, BLS）。为了精确和高效地实现目的基因的突变,本研究利用pK18mobGⅡ载体,建立了一套适于Xoo和Xoc目的基因定点插入的突变体系。通过同源片段与目的基因间的同源重组,成功获得了Xoo和Xoc的hrcV和hrpF突变体。PCR和Southern杂交证实：pK18mobGⅡ携带不同大小的同源片段,能够整合于hrcV和hrpF的特定位点;200～400 bp的同源片段能够获得最佳的突变效率;两亲接合的转化效率是电转化的5～100倍。毒性测定结果显示,hrcV基因决定着Xoo在水稻上的致病性。致病相关基因插入突变体系的建立为研究水稻黄单胞菌与水稻互作中致病相关基因的功能奠定了遗传学研究基础。     

在水稻悬浮细胞系中非亲和白叶枯病菌繁殖受到抑制的现象及其机理的研究

 研究了水稻白叶枯病菌在水稻悬浮细胞系中的繁殖动力学特征以及悬浮细胞胞外液中各类物质对白叶枯病菌繁殖的影响。结果表明,水稻-白叶枯病菌非亲和互作与亲和互作相比病原菌繁殖的数量明显减少。胞外液中粗提蛋白对白叶枯病菌繁殖不具有抑制作用。非亲和互作胞外液中二元酚含量显著增高,并且水稻细胞防卫反应基因的转录和翻译活性明显增强。表明非亲和互作中白叶枯病菌繁殖受到抑制与水稻细胞防卫反应的启动相关。     

西藏砂生槐生物碱抑菌及杀虫活性的测定

用MTT比色法、抑菌圈法对西藏高原植物砂生槐生物碱进行了室内离体抑菌活性测定。结果表明，其对水稻白叶枯病菌KS-6-6、JXOⅢ、PX099，以及水稻条斑病菌RS105、丁香假单胞菌PPB2038、马铃薯软腐病菌E的最低抑菌浓度分别达10．53、7．20、15．79、15．79、5．30、30．77mg/ml；对核果假尾孢、茄匍柄霉、褐孢霉的EC50值分别为12．90、15．79、12．55mg/ml。采用小叶碟添加法测定了对菜青虫的毒杀作用，其LD50值为326．59mg/L。     

水稻白叶枯病菌和细菌性条斑病菌的分子标记筛选及检测

 水稻白叶枯病菌（Xanthomonas oryzae pv. oryzae）和细菌性条斑病菌(Xanthomonas oryzae pv. oryzicola)是水稻种子产地检疫中最重要的两种检疫对象,且同属于水稻黄单胞杆菌。本研究基于生物信息学技术构建比较基因组学算法对两种病原的全基因组序列比对分析,得到一系列能够区分两种病原的特异性PCR引物。结合简单的PCR技术及全自动DNA分析系统,我们选取了12对引物分别对23株水稻白叶枯病菌和5株水稻细菌性条斑病菌及其它相关菌株进行验证。结果获得了2对显性标记(Xoo-Hpa1和Xoc-ORF2)以及3对共显性分子标记(M568、M897和M1575)可以达到理想的区分检测两种病原的效果。分子标记的检测灵敏度从5×10⁴到5 × 10⁷cfu·mL^-1不等,且从水稻种子浸提液中也能成功地检测水稻白叶枯病菌和细菌性条斑病菌。本研究丰富了检测标记的靶位点,并有效的结合了高通量检测的手段对多位点联合分析,增强了检测的可靠性,有望在今后的植物检疫及病原鉴定中发挥着重要的作用。     

Genotypic and Pathotypic Diversity in Xanthomonas oryzae pv. oryzae in Nepal

ABSTRACT Among the 171 strains of Xanthomonas oryzae pv. oryzae (the bacterial blight pathogen of rice) collected from eight rice-producing zones in Nepal, 31 molecular haplotypes were distinguished using two polymerase chain reaction-based assays. Six common haplotypes represented nearly 63% of the strains, and some haplotypes were geographically dispersed. Multiple correspondence analysis divided the collection into five putative genetic lineages. Lineages 1, 2, and 4 were the most frequently detected and occurred in diverse geographic populations. Twenty-six pathotypes (virulence phenotypes) of X. oryzae pv. oryzae were identified using 11 near-iso-genic rice lines, each containing a single gene for resistance. The 26 pathotypes grouped into five clusters, and cluster 1 contained wide virulence spectrum strains from all geographic populations. Although molecular variation was greatest between strains of different virulence phenotypes, some variation was observed among strains with identical virulence. There was a weak correlation (r = 0.52) between molecular haplotypes and virulence phenotypes. There are two major groups of X. oryzae pv. oryzae in Nepal. One group consists of strains with high molecular polymorphism and many pathotypes that are either virulent to the 11 major resistance genes or avirulent only to Xa21. Strains in the second group have low molecular polymorphism and are avirulent to Xa4, xa5, Xa7, and Xa21.     

hrp基因关键调控因子HrpG在水稻白叶枯病菌和条斑病菌中的交叉互补性研究

 水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae, Xoo)和条斑病菌(X. oryzae pv. oryzicola, Xoc)在非寄主烟草上产生过敏反应(HR)和在寄主水稻上具有致病性,是由hrp致病岛决定的,其中HrpG为关键调控因子。Xoo和Xoc侵染水稻途径和在水稻上产生病害症状的不同,是否与hrpG基因有关,还不清楚。本研究利用反向遗传学方法获得了Xoo和Xoc的hrpG突变体PΔhrpG和RΔhrpG,并用hrpG_Xoo基因和hrpG_Xoc基因分别互补上述2个突变体,获得了相应的互补菌株。致病性测定结果显示,PΔhrpG和RΔhrpG突变体丧失了在水稻上的致病性和在非寄主烟草上产生HR的能力,而hrpG_Xoo基因和hrpG_Xoc基因可分别互补上述突变体至野生型水平。利用GUS报告基因检测基因启动子活性发现,hrpG_Xoo和hrpG_Xoc的启动子活性没有显著差别;RT-PCR和Western杂交结果显示,hrpG基因交叉互补菌株中HrpG调控的下游基因hrpX、hpaR、hrcT、hpa2和hrpD6的表达没有差异,且III型分泌系统分泌蛋白Hpa2的分泌性没有受到影响。这些结果表明,稻黄单胞菌hrpG基因可在Xoo和Xoc中交叉互置,位于其上游和下游的调控途径可能相似,而决定Xoo和Xoc在水稻上的侵染途径以及所致病害症状差异可能与hrpG基因位点无关。     

水稻条斑病菌异柠檬酸脱氢酶在致病性中的功能分析

 异柠檬酸脱氢酶(isocitrate dehydrogenase,IcdH)催化异柠檬酸转化成α-酮戊二酸,参与碳代谢途径末端的三羧酸(tricarboxylic acid,TCA)循环。然而,编码异柠檬酸脱氢酶基因是否参与水稻条斑病菌(Xanthomonas oryzae pv. oryzicola,Xoc)的致病性,我们并不清楚。为了阐明IcdH的作用,通过同源重组技术获得了Xoc的icdH基因缺失突变体(RΔicdH),并对该突变体进行了相关功能研究。研究表明:该突变体不能利用苹果酸、丙酮酸和柠檬酸,在寄主水稻上的生长能力和致病力相对于野生型均显著降低,其游动性也显著减弱; 功能互补子恢复RΔicdH的上述表型至野生型水平; Real-time PCR结果显示,六碳单糖、蔗糖、苹果酸、丙酮酸与柠檬酸能显著诱导icdH基因的转录表达;与水稻细胞互作时icdH基因受诱导表达,并受HrpX和HrpG负调控。这些结果说明:icdH基因是Xoc获取碳源和在寄主水稻上具有致病性所需的。     

水稻白叶枯病菌室内链霉素抗性菌株的诱导及其抗性分子机制和风险评价

通过紫外光照射诱导获得了6株水稻白叶枯病菌Xanthomonas oryzae pv.oryzae抗链霉素突变体。测得链霉素对水稻白叶枯病菌敏感菌株ZJ173及其抗性突变体的最低抑制浓度(MIC)分别为0.10和600 μg/mL;对敏感菌株的有效抑制中浓度(EC50)为0.03 μg/mL,对抗性菌株的平均EC50值为11.64 μg/mL,平均抗性倍数为388。通过PCR扩增了敏感菌株ZJ173及5株抗性菌株的rpsL基因(编码S12核糖体蛋白)和rrs基因(编码16S rRNA),并检测了strA基因是否存在。序列分析表明,5株被测抗性菌株的rpsL基因均发生了突变,其中4株在氨基酸43位、1株在88位,均由赖氨酸突变为精氨酸,而rrs基因未发生突变,strA基因未被检测到。表明实验室诱导获得的水稻白叶枯病菌抗性菌株对链霉素的抗药性是由rpsL基因突变引起的。抗性风险研究表明,抗性突变体的抗药性在无药剂压力下可稳定保持,其致病性、生长速率与敏感菌株相比无明显差异,竞争性低于或略低于敏感菌株,抗性自发突变率较高,且抗性突变为单一位点突变,病害循环为多循环,因此由rpsL基因突变引起的水稻白叶枯病菌对链霉素的抗性风险较高。     

拌种灵、叶枯唑和萎锈灵对病菌的毒力机制比较

苯氨基甲酰类杀菌剂是最早开发的一类内吸性杀菌剂 [1 ] ,能有效地防治一些真菌病害 ,持效性长 ,抗药性风险低 [2 ] 。对叶枯唑产生抗药性的水稻白叶枯病菌 ( Xanthomonas oryzae pv.oryzae)对拌种灵同样具有抗药性 [3] 。近年来 ,拌种灵被有效地用于防治果园中柑桔溃疡病 ( X.citri) [4]。目前对噻唑类杀菌剂防治细菌病害的作用机制尚不清楚 ,初步认为这类药剂最初的作用靶点可能与致病性相关因子有关 [3] 。我们对 3种具有相似结构的杀菌剂拌种灵、叶枯唑和萎锈灵在离体条件下对病原菌的毒力机制进行了初步研究 ,同时也对拌种灵防治柑桔…