生防菌对青枯雷尔氏菌的致弱特性

青枯雷尔氏菌(Ralstonia solanacearum)的致病性与弱化指数存在着特定的相关性,弱化指数＜0.60时,菌株回接番茄组织培养苗的发病率为100%,定义为强致病力菌株;弱化指数＞0.80,回接发病率为0,定义为无致病力菌株,弱化指数介于0.60和0.80之间,回接发病率5.3%～100%,菌株的致病性为不确定性,定义为过度性菌株.010703-01菌株进行培养致弱、物理致弱、化学致弱和生物致弱的实验结果表明培养时间延长至120 h未发现致弱作用继代培养11代以前未出现明显致弱;物理因子超声波处理无致弱作用;化学因子农用链霉素处理有抑制作用,但无致弱作用;生物因子15种细菌的致弱作用可用聚类分析将之分为三类,第一类具致弱特性,包括青枯病生防菌ANTI-8098A(Bacillus cereus)和球形芽孢杆菌(B.sphaercus),第二类具抑制特性,而无致弱作用,包括Bt-9408(B.thuringiensis)等3种芽孢杆菌;第三类抑制作用低和无致弱作用,包括阴沟肠杆菌(Enterobactercloacae)和未知学名的10株芽孢杆菌(Bacillus spp.)等.研究结果表明,生防菌对青枯雷尔氏菌的致弱作用与因其它因素产生的致弱作用有着本质区别.     

基于BOX-PCR和REP-PCR技术青枯雷尔氏菌遗传多样性分析

青枯雷尔氏菌(Ralstonias olanace arum)能够对许多重要作物引起致命性萎蔫病害,广泛分布在热带、亚热带以及温带地区.研究青枯雷尔氏菌的遗传多样性对于了解青枯病的发生和流行具有十分重要的意义.本研究利用青枯雷尔氏菌特异性引物鉴定84株来自福建地区不同寄主的青枯雷尔氏菌,结果表明,这些菌株均在504 bp位置出现特异性条带.同时采用BOX插入因子PCR(BOX-PCR)和重复基因外回文序列PCR(REP-PCR)对这些菌株进行基因多样性研究,基于它们所扩增出的基因指纹图谱表明,BOX-PCR扩增出19条特异性条带,REP-PCR扩增出20条特异性条带.系统聚类结果表明,青枯雷尔氏菌的遗传分化与寄主作物和地理来源都存在相关性,其中,寄主植物是在遗传差异中起主导作用.进一步分析可知,地域性的差异主要由BOX-PCR提供,寄主间的差异主要由REP-PCR提供.不同地理来源的青枯雷尔氏菌在BOX-PCR中可扩增出各自特异性条带,在REP-PCR中同样具有与各个寄主相对应的特异性条带,利用这些特异性条带可以很容易区分不同寄主以及来源的青枯雷尔氏菌.BOX-PCR和REP-PCR多态性分析技术可为我国青枯雷尔氏菌基因多样性的研究提供另一条途径.     

福建省青枯雷尔氏菌无毒基因组成及与致病力关系分析

青枯雷尔氏菌(Ralstonia solanacearum)致病力分化严重.为了解福建省青枯雷尔雷氏菌的致病力类型及与无毒基因组成关系,本研究以弱化指数为指标,对福建省内不同地区和寄主的63个青枯雷尔氏菌进行致病力测定,结果表明,供试的青枯雷尔氏菌可分为3种致病力类型:强致病力、过渡型和无致病力;进一步对供试菌株进行无毒基因avrA、popP1和popP2的检测,3个无毒基因分布频率不同,avrA基因分布频率最高,达79.37％,popP1基因的分布频率最低为46.03％;不同寄主的菌株无毒基因组成不同,番茄(Lycopersicum esculentum)和辣椒(Capsicum annuum)寄主以分布3种和2种无毒基因为主,茄子(Solanum melongena)寄主主要分布2种无毒基因,花生(Arachis hypogaea)寄主的菌株无毒基因分布较少,只分布1种avrA基因或没有检测到无毒基因;不同地域的菌株无毒基因组成不同,以番茄寄主为例,建瓯玉山镇的菌株分布3种和2种无毒基因为主,分别占45.45％和31.82％,宁德屏南菌株中3个无毒基因均等分布,福州北峰菌株中只分布avrA和popP2基因,二者均等分布,福州营口菌株中popP2基因分布频率最高,达60.00％;不同致病力的菌株无毒基因组成不同,无致病力菌株分布3种和2种无毒基因为主,过渡型菌株分布2种无毒基因为主,而强致病力菌株分布1种无毒基因为主;青枯雷尔氏菌的无毒基因组成与致病力相关性分析表明,3个无毒基因分布均与致病力呈正相关,其中popP1基因与菌株致病力相关性最高,达显著水平,其皮尔逊相关系数(Pearson correlation coefficient,PCC)值为0.31.本研究为植物青枯病预警和防控提供重要信息.     

基于ITS序列和RAMS标记分析青枯雷尔氏菌的遗传多样性*

应用ITS序列和RAMS技术对福建省不同地域,不同寄主作物的青枯雷尔氏菌进行遗传多样性研究。结果表明,供试的21株青枯雷尔氏菌的ITS区序列有3种类型,这3种类型菌株的ITS序列只有1-2个碱基的差异,与参比菌株GMI1000的ITS区序列或者相同或者有1-2个碱基的差异。在此基础上,利用RAMS技术进一步分析表明,供试的青枯雷尔氏菌及参比菌株GMI1000的基因组DNA间存在丰富的多态性。聚类结果显示,供试菌株可聚为3个类群,同一寄主作物的菌株聚在同一类群中,同一地理来源的菌株聚在同一亚类群中,说明青枯雷尔氏菌的遗传分化与寄主作物和地理来源都存在相关性,与寄主作物的关系更密切。     

生物有机肥对烟草根际微生物群落及青枯雷尔氏菌丰度的影响

【目的】探究生物有机肥施用对烟草根际土壤细菌、真菌群落结构和青枯雷尔氏菌丰度的影响机理。【方法】选用长沙市某公司生产的生物有机肥和常规烟草专用肥,在湘西花垣县长期定位试验点连续5年开展大田试验,研究施肥对土壤理化性质和微生物群落的影响。试验设置两种施肥处理：常规烟草专用肥（CF）和生物有机肥（BOF）。【结果】与CF相比,施用生物有机肥处理的土壤烟草青枯病发病率降低了89.8%,同时青枯雷尔氏菌相对丰度也显著降低,降幅达40.1%;土壤pH、碱解氮和有效磷显著增加,分别增加了1.2%、12.1%和60.2%;施用生物有机肥后根际土壤微生物如Roseiflexaceae,Gemmatimonadaceae,Nitrospira,Ramophialophora,Preussia等显著富集,且这些潜在有益菌与青枯雷尔氏菌相对丰度呈显著负相关关系。通过ABT预测模型分析发现潜在有益菌是影响青枯雷尔氏菌相对丰度的最主要生物因子。【结论】连续5年的试验结果表明,施用生物有机肥不仅改善了作物生长的土壤环境,显著提高了土壤pH和土壤速效养分含量,还促使潜在有益菌在根际土壤中富集,抑制了青枯雷尔氏菌的生...     

青枯雷尔氏菌Tn5转座子无致病力突变株构建及其生物学特性

青枯雷尔氏菌(Ralstonia solanacearum)是植物细菌性青枯病的病原菌,为研制植物疫苗菌剂提供遗传稳定且突变位点明确的青枯雷尔氏菌无致病力菌株,本研究利用EZ-Tn5转座子随机插入青枯雷尔氏菌(Rs91)构建青枯雷尔氏菌无致病力突变体库,通过电击转化,筛选获得13株具有无致病力菌株形态的突变株。研究了其中5株突变株的插入位点和生物学特性,结果表明,其插入位点分别位于phcA和pchS基因,其生长速率和相对胞外多糖含量显著低于Rs91,而最适pH值和温度未改变。其发酵液经分光光度计扫描,结果显示,突变株在同一波长下的光吸收值均大于Rs91,经聚类分析,显示它们与Rs91可聚为不同的3类,即Rs91为第Ⅰ类,phcA基因突变株为第Ⅱ类,phcS基因突变株为第Ⅲ类。经番茄(Lycopersicon esculentum)盆栽苗致病力检测,15d后均未发病,确定为无致病力青枯雷尔氏菌。本研究为研制防治青枯病的植物疫苗菌剂提供了基础资料。     

Suppression of Ralstonia solanacearum in soil following colonization by other strains of R. solanacearum

The effect of prior colonization of a sterile loam soil and a sterile clay loam soil by individual soil bacteria on the subsequent growth of a bacterial wilt pathogen Ralstonia solanacearum YU1Rif43 (tRNA type III: Seal et al. 1992: Appl. Environ. Microbiol., 58, 3759–3761) was investigated. Various strains, belonging to the same type, the same species, the same genus, Gram-negative, Grampositive, or fungi, were used. The degree of suppression of the growth of R. solanacearum YU1Rif43 was markedly different depending on the species that had previously colonized the soil, hereafter referred to as priorcolonists. All the strains belonging to R. solanacearum type III suppressed the growth of R. solanacearum YU1Rif43 markedly, while strains of R. solanacearum type I and type II showed a moderate suppressive effect on R. solanacearum YU1Rif43. The suppressive effect of the strains belonging to species other than R. solanacearum, including fungal strains, was relatively limited, or some strains did not show any suppressive effect. The production of bacteriocin did not appear to be related to the strong suppressive effect of the R. solanacearum type III strains. Possible mechanisms for the suppressive effect of priorcolonists on R. solanacearum YU1Rif43 are discussed in relation to nutrients and physical sites in soil available for growth.     

Effect of calcium concentration in nutrient solution on development of bacterial wilt and population of its pathogen Ralstonia solanacearum in grafted tomato seedlings

We examined the effect of the calcium (Ca) concentration in the nutrient solution on the development of bacterial wilt and the population of its pathogen Ralstonia solanacearum in tomato (Lycopersicon esculentum Mill. cv. Momotarou) seedlings grafted onto the rootstock of a highly resistant cultivar (cv. Hawaii 7998). The grafted seedlings were cultured in a nutrient solution containing Ca at concentrations of 0.4, 4.4, and 20.4 mm, and inoculated with the pathogen by stem puncture at the base of the stem of the rootstock, and the disease incidence was recorded for a period of 21 d. In another experiment, xylem exudates were collected from decapitated scions of the Ca-treated seedlings 5 d after inoculation, and the population of the pathogen in the exudates was counted by plating on a selective medium. The grafted tomato seedlings were highly resistant to bacterial wilt, when cultured in the nutrient solution containing Ca at 20.4 mm. The population of the pathogen in the xylem exudates decreased with increasing concentration of Ca in the solution. However, even in the presence of Ca at a high concentration, infection with the virulent pathogen was observed in the xylem of the scion.     

Effect of calcium concentration in nutrient solution before and after inoculation with Ralstonia solanacearum on resistance of tomato seedlings to bacterial wilt

We previously reported that calcium (Ca) nutrition in tomato (Lycopersicon esculentum Mill.) significantly affected the resistance to bacterial wilt caused by Ralstonia solanacearum Smith. To elucidate the mechanisms underlying the Ca-dependent resistance, the effect of the Ca concentration in the nutrient solution applied before and after inoculation with the pathogen on the resistance of tomato seedlings to bacterial wilt was studied. One week before inoculation, seedlings were transferred to nutrient solutions containing Ca at concentrations of 0.4, 4.4, or 20.4 mM. Soon after inoculation, the seedlings that were treated with each concentration of Ca before inoculation were transferred to solutions containing the same three concentrations of Ca. Although the disease development was not affected by the concentration of Ca in the solution before inoculation, a higher concentration of Ca after inoculation reduced the disease severity. This result suggests that the concentration of Ca in the host, especially in the cell walls, before infection may not be directly involved in the Ca-dependent resistance of tomato seedlings to bacterial wilt.     

青枯假单胞菌的一段12.8kb DNA带有特异性控制病菌在花生上致病的基因

在本工作中,通过平板筛选分离得到两个来自马铃薯的青枯假单胞菌菌株Ｔ２００２和Ｔ２０５３（对花生不致病）的利福平抗性自发突变株Ｔ２０１６和Ｔ２０１７,通过三亲本接合,ｐＧＸ１２５２分别以９．０７×１０－７、２．５４×１０－６频率被导入到Ｔ２０１６和Ｔ２０１７中,所得接合子Ｔ２１２３（Ｔ２０１６／ｐＧＸ１２５２）和Ｔ２１２４（Ｔ２０１７／ｐＧＸ１２５２）均分别能使花生汕油５２３发生青枯病,但它们侵染的植株出现病症的时间比Ｔ２００５晚３～５ｄ,说明ｐＧＸ１２５２能扩大Ｔ２０１６和Ｔ２０１７的寄主范围使之包括花生。从侵染Ｔ２１２３、Ｔ２１２４的病株中重新分离病菌,四环素抗性检测和质粒检测表明所有细菌均含有ｐＧＸ１２５２,说明在植物体内无抗生素选择压力情况下ｐＧＸ１２５２能在Ｔ２０１６、Ｔ２０１７中稳定存在。本工作进一步证实ｐＧＸ１２５２带有特异性控制青枯假单胞菌在花生上致病的与寄主专一性有关的基因。     

我国有机污染场地现状分析及展望

以有机污染场地为研究对象,调查分析了全国277个有机污染场地的行业类别、污染类型以及有机污染物种类分布情况,发现主要贡献行业为化学原料及化学品制造行业,贡献率为37.9％;污染类型中以各类有机污染物复合污染为主;除了不同有机污染物之间的相互复合,有机污染物与重金属复合污染特征明显,占总场地58.5％.有机污染物种类分布...     

Ralstonia eutropha菌株镉抗性调节基因CzcR的克隆与序列测定

G细菌镉抗性决定子是一个编码4个蛋白的CzcCBAD操纵子,参照GenBank已登录Czc基因序列进行引物设计,利用PCR技术,从可在含350mg/LCdCl2的培养基上生长的抗镉菌株Ralstoniaeutropha质粒中,扩增出长度约为1200bp的革兰氏阴性细菌镉抗性系统中的抗性调节基因CzcR,然后将其亚克隆到pGEM-T-easy载体上,并转化至受体菌中,构建重组质粒,采用碱性裂解法提取质粒DNA后,经EcoRI酶切分析和核苷酸序列分析,其与GenBank中登录的CzcR基因序列相似性高达98%,显示其具有正确的CzcR基因核苷酸序列。镉抗性基因的获得将大大推动对微生物抗重金属机理的研究,并为进一步构建耐镉基因工程菌,高效净化重金属污染环境打下坚实的基础。     

Experimental Study of Heat Transfer in Soil to Inhibit Microbial Growth Using the Underground Release of Hot Water and a Hydraulic Head Method

This research aimed to study the effectiveness of heat transfer in soil using the underground release of hot water with a hydraulic head. Pressure heads of 1.0, 1.5 and 2.0 m were utilized for underground release with a nozzle to investigate heat transfer in soil necessary to inhibit microbial growth through underground hot water release using a pressure head of liquid. The nozzle was made of stainless steel pipe (SUS304). The proposed system was designed to inhibit microbes of Ralstonia solanacearum with a hot water volume flow of 300, 400 and 500 ml min^?1, while temperature was kept constant at 70 ºC. The hot water released underground provided higher soil temperature with shorter working time. The research found that the number of layers and diameter of drilled holes had a significant effect on soil temperature distributed at various depth levels (y-plan), resulting in enhanced performance (100%) for the inhibition of microbes in an x-plan radius of 15 cm for a shorter time of 20–30 min with one nozzle.