PCR-based sensitive detection of Ralstonia solanacearum from soil,eggplant, seeds and weeds

Ralstonia solanacearum is an economically important, bacterial plant pathogen which affects a wide range of crop plants. R. solanacearum survives in the soil for many years and weeds serve as symptomless carrier. One of the important aspects in controlling R. solanacearum is its early detection. In this study, detection threshold of R. solanacearum in the soil was standardised using polymerase chain reaction (PCR) method. The minimum threshold limit ranged between 6.8 × 10 and 3.6 × 10² CFU g^?1 of soil. Using this standardised protocol R. solanacearum was detected from the rhizosphere soil of eggplants showing varying degrees of wilt. PCR method was quite sensitive to detect R. solanacearum from the xylem fluid of eggplant. Presence of R. solanacearum in the soil infected with capsicum wilt was also demonstrated successfully and the minimum detection limit was 4 × 10² CFU g^?1 of soil. The bacterium was not detected from the eggplant seeds collected during 2006 and 2007 seasons. However, the bacterium was detected from the weed (Alternanthera sessilis) grown in the eggplant field indicating the possibility of weeds serving as symptomless carrier. Using our method, it is possible to detect R. solanacearum from soil, plant and weeds grown in the field at an early stage so that proper management strategies could be taken to prevent the infection and further spread of the pathogen.     

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。     

植物病原细菌Ralstonia solanacearum GMI1000中分泌蛋白信号肽分析

利用SignalP 3.0、TMHMM 2.0、TargetP 1.01、LipoP 1.0和PSORTb蛋白分析软件并结合L值计算,对植物病原细菌Ralstonia solanacearum GMI1000菌株基因组中的全部3 440个ORFs进行了分析预测,确定其中186个ORFs所编码蛋白质的N-端有信号肽序列,且它们的氨基酸残基相对保守.其中134条具有分泌型信号肽,22条具有RR-motif型信号肽,30条具有信号肽酶Ⅱ型信号肽.对各类信号肽及其结构域的长度作了系统的分析.未发现Prepilin-like信号肽和细菌素和信息素信号肽.     

青枯菌hrp基因的研究进展

青枯菌的hrp基因可诱发植物的超敏反应.对其基因组全序列测定表明:hrp基因簇位于基因组的大质粒上,共有20多个基因组成.从青枯菌中分离得到的可直接诱发植物超敏反应的效应蛋白主要为pop基因编码,它由hrp基因编码的类型Ⅲ蛋白分泌通道释放.目前的研究表明:(1)在hrp基因簇中,hrpY、hrpX及hrpV与分泌通道的一种纤毛的组装有关;(2)hrpB是整个类型Ⅲ蛋白分泌通道基因的转录激活子并作用于基因组中的其它效应基因;(3)hrpG是植物信号对hrp,基因的表达进行级联调控的组分之一.     

青枯菌致病性与基因组之间的关系

青枯菌是引起植物毁灭性青枯病的病原菌。青枯菌基因组约5.8Mb,具有高(G C)含量和约5120个可能的编码基因。该菌基因组由3.7Mb的染色体和2.1Mb的大质粒所组成,其独特的基因组构成与Ⅲ型分泌系统等主要的致病因子密切相关。综述了青枯菌的致病性与其基因组之间关系的新近研究进展。     

青枯菌特异性噬菌体的研究进展与应用

烟草等茄科植物青枯病的防治是一个世界性难题,传统的化学防治、合理轮作、抗病品种等措施无法有效控制该病的发生。噬菌体用于细菌性病害的防治已有很长历史,近年来利用噬菌体防治青枯菌引发的青枯病方面的研究越来越受重视。我们简要综述了青枯菌噬菌体的研究进展,并对青枯菌噬菌体生物防治的应用前景进行了展望。     

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.     

Rapid and effective detection of anthrax spores in soil by PCR

AIMS: To detect Bacillus anthracis DNA from soil using rapid and simple procedures. METHODS AND RESULTS: Various amounts of B. anthracis Pasteur II spores were added artificially to 1 g of soil, which was then washed with ethanol and sterile water. Enrichment of the samples in trypticase soy broth was performed twice. A DNA template was prepared from the second enrichment culture using a FastPrep instrument. The template was then used for nested and real-time polymerase chain reaction (PCR) with B. anthracis-specific primers, to confirm the presence of B. anthracis chromosomal DNA and the pXO1/pXO2 plasmids. CONCLUSIONS: One cell of B. anthracis in 1 g of soil could be detected by nested and real-time PCR. The usefulness of the PCR method using field samples was also confirmed. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that this could be a useful method for detecting anthrax-spore contaminated soil with high sensitivity. Its application could have great impact on the progress of epidemiological surveillance.     

Possible Mechanisms Limiting Movement of Ralstonia solanacearum in Resistant Tomato Tissues

The distribution and appearance of Ralstonia solanacearum in the upper hypocotyl tissues of root‐inoculated tomato seedlings of resistant rootstock cultivar LS‐89 (a selection from Hawaii 7998) and susceptible cultivar Ponderosa were compared to clarify the mechanism that limits the movement of the bacterial pathogen in resistant tomato tissues. In stems of wilted Ponderosa plants, bacteria colonized both the primary and the secondary xylem tissues. Bacteria were abundant in vessels, of which the pit membranes were often degenerated. All parenchyma cells adjacent to vessels with bacteria were necrotic and some of them were colonized with bacteria. In stems of LS‐89 plants showing no discernible wilting symptoms, bacteria were observed in the primary xylem tissues but not in the secondary xylem tissues. Necrosis of parenchyma cells adjacent to vessels with bacteria was observed occasionally. The pit membranes were often thicker with high electron density. The inner electron‐dense layer of cell wall of parenchyma cells and vessels was thicker and more conspicuous in xylem tissues of infected LS‐89 than in xylem of infected Ponderosa or mock‐inoculated plants. Electron‐dense materials accumulated in or around pit cavities in parenchyma cells next to vessels with bacteria, and in vessels with bacteria. Many bacterial cells appeared normal in vessels, except for those in contact with the pit membranes. These results indicate that R. solanacearum moves from vessel to vessel in infected tissues through degenerated pit membranes and that restricted movement in xylem tissues was the characteristic feature in LS‐89. The limitation in bacterial movement may be related to the thickening of the pit membranes and/or the accumulations of electron‐dense materials in vessels and parenchyma cells.     

培养条件对青枯雷尔氏菌脂肪酸组成的影响

应用气相色谱技术测定不同温度、培养时间、pH值等培养条件下青枯雷尔氏菌(Ralstonia solanacearum)脂肪酸的结果表明: 青枯雷尔氏菌强致病力菌株Rs-J.1.4-010704-01v的脂肪酸种类有14~34种, 主要特征脂肪酸为C16:1ω7c/C15:0 ISO 2OH(10.644 min), C16:0(10.950 min), C18:1ω7c(14.177 min), 所占总百分比含量为总脂肪酸的55.66%~75.69%; 该菌脂肪酸的种类与含量随着培养条件的改变而发生变化,     

茄科雷尔氏菌的蛋白分泌系统及其特征

茄科雷尔氏菌利用自身的分泌系统能向胞外分泌上百种蛋白, 其中Ⅱ型和Ⅲ型分泌系统通过不同机制将分泌蛋白靶定到胞外或宿主细胞, 是决定茄科雷尔氏菌对宿主产生致病性的主要因素。其中Ⅲ型分泌系统不依赖Sec信号转导系统但必须依赖于宿主细胞的识别激活, 并在病原菌对宿主细胞的特异性识别和细菌在宿主细胞的生长增殖中发挥功能。到目前为止, 已经从茄科雷尔氏菌的GMI1000株系中鉴定出两类在宿主细胞中存在靶标, 并由Ⅲ型分泌系统分泌的效应蛋白Pop2和Gala蛋白家族。主要就茄科雷尔氏菌Ⅲ型分泌系统的基本特征以及效应蛋白及其宿主靶标的相互作用进行综述。     

过量表达辣椒CaNPR1提高烟草对青枯菌的抗性

对水稻和拟南芥等模式植物的研究表明,NPR1(nonexpressor of pathogenesis-related genes 1)是依赖于SA通路的防御反应调节基因,但在辣椒和烟草等茄科作物中该蛋白的功能还鲜有报道.研究从辣椒cDNA文库中分离获得一个NPR1的类似物全长cDNA(CaNPR1),并获得了其超表达的转基因烟草T1代株系.研究结果表明,这些株系与其野生型植株没有明显表型差异,但却表现出较野生型植株更高的抗青枯菌侵染活性.同时,研究还发现CaNPR1的超表达还显著提高了防御相关基因的表达,表明NPR1在不同植物间具有较强的功能保守性.     

粤、闽、贵烟草青枯病菌分离株及其致病力

【目的】从粤、闽、贵3地烟草青枯病株中分离青枯病菌,测定其致病力并探讨判断致病力的方法。【方法】采用氯化三苯基四氮唑(简称TTC)培养基和蛋白质电泳相结合对青枯病菌致病力进行测定,并分析菌株生化型。【结果】已分离出烟草青枯病菌59株,依据致病力进行划分,无致病力菌株5株,有致病力菌株54株。不同致病力菌株蛋白电泳特定条带存在差异。粤、闽、贵3地烟草青枯病菌有致病力菌株均占主导地位,广东有致病力菌株比例略大,福建次之,贵州最小。按生化型划分,59株青枯病菌中1株属于生化型Ⅳ外,其他58株属于生化型Ⅲ或其亚型生化型Ⅲ-1。【结论】烟草青枯病菌致病力存在差异,SDS-PAGE蛋白指纹中的特定条带可作为判定青枯病菌有无致病力的依据。     

Measurement of Horizontal and Vertical Movement of Ralstonia solanacearum in Soil

Two model systems were constructed to measure horizontal and vertical movement of bacteria in soil. These systems were applied to measuring movement of Ralstonia solanacearum (race 1, biovar 3), a causal agent of bacterial wilt of tomato, in andosol and sand at 28°C. The first system was used to measure horizontal movement of the bacteria in soil packed in a narrow horizontal frame. Suspension of the pathogen was applied to soil at one end of the frame, and bacterial number per gram of soil was measured over distance from the inoculation point after 4 days. Horizontal movement of R. solanacearum in supersaturated soil, but without flow, was possibly due to diffusion and the front advanced at 2.2 cm/day in andosol, and at 8.1 cm/day in sand. Using the same experimental system, but applying water inflow to one end of the frame only, the bacterium was detected at the front of water in andosol and sand. The front of the distribution advanced at 20.4 cm/h in andosol and 66.3 cm/h in sand. In the second experimental system, a cylinder of soil packed in a short tube was soaked with water, and soil at the top of the tube was inoculated with bacterial suspension. Immediately, soil cylinders were turned upward, and the bacterial number per gram of soil was measured along vertical distance from the inoculation point after 7 days. Using the system with andosol, the capillary water front rose to 32.5 cm over 7 days after inoculation, and R. solanacearum reached to 18.8 cm height. In sand, capillary water rose to 20.0 cm and the bacteria reached to 16.3 cm height.     

Random Genome Sequencing of Ralstonia solanacearum Strain IVIA 1602 and Comparative Analysis with Strain GMI1000

Ralstonia solanacearum is a β‐proteobacterium which affects several hundred plant species and provokes important agronomic losses. Five biovars of this bacterium have been described and they show behavioural differences. In this study a random sequencing of the genome of R. solanacearum strain IVIA 1602 (race 3, biovar 2), isolated from potato, was performed. The resulting 730 Genomic Survey Sequences (GSSs), representing 6.38% of the complete genome, were compared against the completely sequenced genome of strain GMI1000 (race 1, biovar 3), isolated from tomato, which is the only strain of this species sequenced until now. This comparative analysis showed, as expected, a high degree of similarity, but it also revealed strain‐specific regions of the genome as well as a number of insertion/deletion events and chromosomal rearrangements. All together, this comparative analysis gives an overview of the genomic divergence between these two biovars of the R. solanacearum species complex.     

Studies on the biosynthesis of ralfuranones in Ralstonia solanacearum

Ralfuranones, aryl-furanone secondary metabolites, are involved in the virulence of Ralstonia solanacearum in solanaceous plants. Ralfuranone I (6) has been suggested as a biosynthetic precursor for other ralfuranones; however, this conversion has not been confirmed. We herein investigate the biosynthesis of ralfuranones using feeding experiments with ralfuranone I (6) and its putative metabolite, ralfuranone B (2). The results obtained demonstrated that the biosynthesis of ralfuranones proceeded in enzymatic and non-enzymatic manners.     

茄科雷尔氏菌脂酰-CoA合成酶的功能鉴定

【目的】茄科雷尔氏菌是一种常见的农作物致病菌,引起植物青枯病。研究其脂肪酸代谢途径将有助于寻找新的抗菌药物靶点,为防治青枯病害提供新的思路。【方法】利用大肠杆菌FadD序列,进行同源比对发现茄科雷尔氏菌GMI1000中RSc2857(RsFadD)具有较高的相似性,推测其具有脂酰-CoA合成酶活性。采用PCR扩增方法获得RsfadD基因,连入表达载体pBAD24M后互补大肠杆菌fadD突变株,并检测转化子的生长情况。RsfadD与pET-28b连接后,在大肠杆菌BL(DE3)中表达,并利用Ni-NTA纯化获得带有组氨酸标签的RsFadD,体外测定RsFadD的活性。利用同源重组方法,获得RsfadD敲除突变株,分析突变株的生长性状。【结果】RsfadD异体互补大肠杆菌fadD突变株,恢复突变株在以脂肪酸为碳源的基础培养基上生长。体外活性测定RsFadD具有脂酰-CoA合成酶活性,对不同链长的脂肪酸都具有活性,但活性低于大肠杆菌FadD。RsfadD突变株在添加不同链长脂肪酸的基础培养上仅能微弱生长,而在丰富培养基上生长无差异。【结论】茄科雷尔氏菌中RsfadD编码脂酰-CoA合成酶,在脂肪酸利用过程中发挥重要作用。但RsfadD突变株在基础培养基上微弱生长,说明茄科雷尔氏菌基因组中还有其他的脂酰-CoA合成酶基因。以上研究结果为进一步研究茄科雷尔氏菌中脂酰-CoA合成酶以及脂肪酸利用机制奠定了基础。     

不同青枯病抗性的番茄品种内生细菌生理群数量研究

本论文对青枯病抗性不同的番茄品种其内生细菌生理群数量变化进行了研究,结果表明,番茄内生细菌生理群数量的变化随品种抗性、生育期和季节的不同而变化.在7大类生理群细菌中,氨化细菌的数量最多,且在幼苗期以后,高抗青枯病番茄品种中数量明显高于高感品种,初步认为,氨化细菌可能是影响青枯病发生的关键性微生物.番茄抗病品种在不同生育期,其内生细菌的总体数量要比感病品种多,呈交替波动变化.氨化细菌、硝化细菌、固氮细菌和反硫化细菌平均数量均表现为在夏季高于冬季,硫化细菌的数量则冬季高于夏季,厌气性细菌数量最少.     

抗青枯病基因RRS1的研究进展

RRS1是被发现的第一个青枯病抗性基因,能介导多个Ralstonia solanacearum小种的广谱抗性反应,也是至今第一例依赖NDR1蛋白的TIR-NBS-LRR类R基因。该文综述了目前分子机制研究最为详尽的拟南芥抗青枯病基因RRS1的克隆、功能和表达作用模式的研究进展,对其他作物青枯病抗性的分子机理研究提供科学的依据和启示。