水稻基腐病细菌毒素的遗传特性和产毒相关的分子标记

[目的]水稻基腐病(Erwinia chrysanthemi pv.zeae)是水稻上重要的细菌病害之一,本论文对该病菌的毒素遗传特性和产毒相关的分子标记进行了研究.[方法]通过化学诱变方法,筛选基腐细菌去质粒的突变体Ech7-mu1;应用RAPD技术,筛选产毒素相关的分子标记.[结果]毒素活性测定结果表明,野生菌Ech7和去质粒菌株Ech7-mu1都能产生毒素.从260条随机引物中,筛选出引物K10,该引物能从不产生毒素的突变株Ech7-4中扩增出大小为2139bp的DNA特异片段,但不能扩增野生菌Ech7,将该片段克隆,测序分析,设计特异引物,在突变体Ech7-4中获得了与毒素产生相关的SCAR分子标记(标记符合率为100%).该基因片段有5个ORFs,其中2个ORFs分别编码NADH-黄素还原酶和N-乙酰转移酶,另外2个不完整的ORFs编码的蛋白分别与Pseudomonas aerginosa(ZP00136947)和Yersinia Pestis(ZP01177873)的抗菌素代谢转运蛋白通透酶(DMT)具有66%和46%的同源率.[结论]水稻基腐细菌毒素的生物合成是由染色体基因编码,与质粒无关.不产生毒素的突变菌株基因突变的位点位于SCAR标记DNA的3'末端.     

生防细菌对水稻的促生性及诱导抗性研究

研究了4株生防菌对水稻白叶枯病的抑制作用和菌悬液浸种、浸芽、浇苗和包衣4种处理对水稻生长的促进作用,及对水稻体内过氧化物酶POD、多酚氧化酶PPO、苯丙氨酸解氨酶PAL 3种保护酶的诱导表达作用。结果表明,4个菌株均对水稻幼苗有促生及诱导抗病性的作用。其中,WY2菌株诱导水稻抗病性和对水稻的促生性都要优于其他3株菌株。水稻幼苗接种生防细菌24 h后再接种病原菌,生防细菌能促进植物体内保护酶PAL、POD、PPO活性的提高,进而诱导植物抗病性的提高。     

伯氏致病杆菌IDP16 蛋白抑制大蜡螟的免疫反应

[目的]从伯氏致病杆菌(Xenorhabdus bovienii)胞外组分中分离纯化出能够抑制大蜡螟(Galleria mellonella)免疫反应的一种蛋白,研究其在昆虫病原线虫及其共生菌致病过程中的作用.[方法]采用硫酸铵沉淀和柱层析的方法对活性蛋白进行分离和纯化,通过体内注射并观察血淋巴黑化进行活性蛋白的筛选;采用荧光微球和琼脂糖小球评价活性蛋白对血细胞吞噬、包被作用的影响;采用双向电泳结合质谱分析对活性蛋白进行鉴定,设计引物用PCR的方法克隆其编码基因,利用pET 30a载体进行原核表达,以亲和层析纯化重组蛋白.[结果]纯化得到一个昆虫免疫抑制蛋白,命名为IDP16,该蛋白可显著抑制大蜡螟血淋巴中的多酚氧化酶活性,降低血细胞的吞噬和包被作用.克隆得到其编码基因并进行了原核表达,重组蛋白仍具有免疫抑制活性.[结论]伯氏致病杆菌产生的IDP16蛋白能够抑制昆虫的免疫反应,在共生菌和宿主昆虫互作过程中起着重要的作用.     

火龙果致腐菌的分离鉴定及生物拮抗防腐措施

[背景]红心火龙果在常温下贮藏3 d鳞片就会出现黄化、萎蔫、果柄发霉,贮藏8d果实腐烂率高于55％,贮藏16 d果实腐烂率达95.5％以上.[目的]对红心火龙果主要致腐微生物进行分离鉴定,并筛选出防腐效果最佳的拮抗菌株.[方法]采用传统纯培养法对红心火龙果的致腐微生物进行分离纯化;通过致腐菌回接火龙果试验,找出主要致腐...     

氨基茚磷酸(AIP)和氨基氧乙酸(AOA)对茉莉酸甲酯诱导的烟草一些酶活性的影响

用茉莉酸甲酯(MJ,1mg ml^-1)处理培养在含0．1mmol／L AIP(水杨酸合成抑制剂)和／或1mmol／L AOA(乙烯合成抑制剂)的MS培养基上的烟草愈伤组织,测定某些酶的活性。结果表明：MJ明显提高过氧化物酶(POD)、β1,3-葡聚糖苷酶和几丁质外切酶的活性,略微促进苯丙氨酸解氨酶(PAL)、多酚氧化酶(PPO)的活性,抑制几丁内切酶的活性,而AOA和AIP则明显抑制MJ对POD和β1,3-葡聚糖苷酶活性的诱导作用,但对MJ诱导的PAL和PPO的活性影响很小,AOA和AIP可能作为逆境因子促进PAL的活性。AOA能部分解除MJ对几丁内切酶的抑制作用,但对MJ诱导的几丁外切酶的影响较小,而AIP抑制几丁内切酶的活性,也抑制MJ对此酶的诱导作用。因此我们认为：MJ对POD、PPO和几丁内切酶的影响可能是通过乙烯途径,对β1,3-葡聚糖苷酶和几丁外切酶的影响可能是通过水杨酸(SA)途径,而对PAL的影响可能是通过其它途径。     

抗烟草黄瓜花叶病毒多糖筛选及其对烟草防御酶活性的影响

[目的]筛选出对烟草黄瓜花叶病毒病有良好抑制作用的多糖并探索其对烟叶防御酶活性的影响.[方法]采用半叶法,测定了安络小皮伞多糖等21种真菌多糖在枯斑三生烟上对CMV的钝化、预防及治疗效果,并测定了抗病毒多糖处理后普通烟NC-89体内防御酶的变化.[结果]安络小皮伞多糖对CMV具有较好的钝化及预防效果,其200倍液与等量供试病毒液混合30 min后接种,钝化效果为83.41%;喷施安络小皮伞多糖200倍液24 h后接毒处理,预防效果可高达93.15%.安络小皮伞多糖对CMV防治机理的研究表明,多糖处理后烟草相关防御酶POD、PAL和PPO活性增强,其中喷施安络小皮伞多糖24 h后接毒处理的酶活增加最为显著,该处理烟苗的POD、PAL和PPO的酶活峰值分别可增加至对照的2.74、3.45和2.82倍.[结论]安络小皮伞多糖通过增强烟草体内防御酶活性而提高烟草对烟草黄瓜花叶病毒病的抗性.     

不同分离方法对子实体形成和粘细菌分离的影响

[目的]基于模拟原位环境策略、可培养粘细菌的营养策略及细菌互作网络,改良分离培养基,以提高分离粘细菌的多样性.[方法]通过添加土壤浸提液、使用不同种类的诱导菌和改变诱导菌的接种方式设置分离方法,同时以传统的分离方法作对照.[结果]改良的分离方法比对照组诱导出了更多粘细菌子实体种类,采自4个地区的9份样品共分离纯化出40...     

群体感应抑制剂对海洋生态功能菌生物膜形成的影响

[目的]研究天然群体感应抑制剂(Quorum sensing inhibitors,QSI)分子对海洋生态功能菌生物膜形成的影响.[方法]以对污损生物幼虫附着具有诱导作用的海洋细菌为目标菌,通过在其生物膜的形成过程中添加天然群体感应抑制剂,研究其对目标菌成膜细菌数和浮游细菌数、生物膜形态以及生物膜表面胞外多糖含量的影响.[结果]呋喃酮和吡啶在50 mg/L时,对8株目标菌的成膜有显著的抑制作用,抑制率在80％左右,吲哚、青霉烷酸和香豆素在较高浓度800 mg/L才有比较好的抑制活性.生长抑制实验结果显示,同等浓度下,QSI分子对目标菌成膜的抑制活性明显高于其对浮游细菌生长的抑制活性.结果表明,QSI分子主要通过干扰目标菌群体感应系统以抑制生物膜的形成.[结论]研究证实QSI分子在海洋菌生物膜形成过程中具有一定的调控作用.通过添加QSI可能能够间接抑制由生物膜诱导的污损生物附着,从而以新的角度研制新型抗污损物质.     

细菌素Paracin 1.7的纯化与性质

[目的]分离纯化(Lactobacillus paracasei)HD1.7所产生的细菌素并分析其特性.[方法]细菌素Paracin1.7的纯化采用色谱技术,其分子量检测采用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE),利用琼脂扩散法测定细菌素活力.[结果]Paracin 1.7分离于我国传统发酵食品酸菜发酵液中,其产生菌为副干酪乳杆菌.Paracin 1.7可以抑制其它微生物的生长,为细菌素.该菌在稳定期可产生大量Paracin 1.7.经过阳离子交换层析、凝胶过滤层析以及高效液相色谱(HPLC),对该细菌素进行了初步纯化,并经Tricine-SDS-PAGE检测其分子量大约为11 kDa.Paracin 1.7抑菌谱较广,其抑菌范围包括Proteus,Bacillus,Enterobacter,Staphylococcus,Escherichia,Lactobacillus,Microccus,Pseudomonas,Salmonella,Saccharomyces,其中有些为食品源致病菌.该细菌素在酸性及高温下稳定,对几种蛋白质酶敏感.该细菌素对敏感菌株的作用方式为抑菌.在4℃保存4个月后,Paraein 1.7的抑菌活性保持稳定.[结论]基于细菌素Paracin 1.7的性质,该细菌素可用作食品防腐剂.     

紫色红曲霉Mp-21胞内次级代谢产物的分离纯化与活性研究

[目的]紫色红曲霉(Monascus purpureus)Mp-21胞内次级代谢产物的分离纯化与活性研究.[方法]通过综合运用多种色谱分离方法对次级代谢产物进行分离纯化,最后通过多种图谱数据对化合物进行结构的解析与鉴定.对分离纯化得到的单体化合物进行抗氧化与降血糖相关酶抑制活性的测定.[结果]从紫色红曲霉Mp-21胞内...     

Physiological and pathological characteristics of Erwinia chrysanthemi isolates from potato tubers

Nine isolates of Erwinia chrysanthemi from rotting potato tubers were compared with six type or reference strains of this species. Phenotypic properties of the potato isolates closely agreed with those of Erw. chrysanthemi pv. zeae and with the characteristics proposed for Dickey's infrasubspecific subdivision IV (1979) and Samson & Nassan-Agha's biovar 3 (1978), where Zea mays was among the most common host species. Pathogenicity tests on 20 ornamental and agricultural species showed only Cyclamen sp. and Z. mays to be susceptible. In Ouchterlony double diffusion tests, antisera to whole live cells of one potato strain reacted with four of the six pathovars of Erw. chrysanthemi. Tuber isolates did not produce blackleg symptoms in inoculated stems. The rationale of intensive pathogenicity testing is discussed.     

几种野生植物提取物抑菌作用研究

本文以乙醇为溶剂,分别提取了瓦松、泽漆及一组中草药配方的有效成分;室内测定了三种提取液对5种植物病原菌(棉花枯萎病菌Fusarium oxysporum f.sp.vasinfectum;小麦赤霉病菌Fusarium graminearum Schw;西瓜枯萎病菌Fusarium oxysporum f.sp.niveum;仙人掌软腐病菌Erwinia chrysanthemi pv.chrysanthemi;魔芋软腐病菌Erwiniacarotovora pv.carotovora)的抑菌作用.结果表明:三种提取物对镰刀菌均有不同程度的抑制作用,且三种提取物的抑菌效果差异显著,其中瓦松的抑菌效果最强.当在50 mL培养基中加入5mL不同提取液时,瓦松对不同镰刀菌的相对抑制率为91.39%～100%;中草药配方提取液对不同镰刀菌的相对抑制率分别为41.11%～85.37%;而泽漆提取液对镰刀菌的相对抑制率仅为12.86%～21.23%;当在50 mL培养基中加入提取液体积降低为2 mL、1 mL、0.5mL时,三种提取液对三种镰刀菌的抑制效果骤然下降,但下降的梯度不一.对大白菜软腐病菌与魔芋软腐病菌而言也是瓦松的抑菌效果最强,中草药配方次之,泽漆的抑制效果最差.     

引起芭蕉芋细菌性软腐病的玉米迪基氏菌(Dickeya zeae)CE1的全基因组序列及比较基因组学分析

【背景】玉米迪基氏菌(Dickeya zeae)可引起香蕉、水稻等重要作物的细菌性软腐病,并造成巨大的损失。芭蕉芋抗性较好且与病虫害相关的报道很少,本研究团队首次报道了由D.zeae CE1引起的芭蕉芋细菌性软腐病。【目的】揭示CE1菌株的全基因组序列,并与同样来源于广东香蕉和水稻的D. zeae菌株作比较基因组学分析,初步探讨D. zeae种内不同病原细菌在与寄主互作过程中可能存在的遗传分化机制。【方法】采用三代测序结合二代测序对CE1菌株进行完整基因组测序,利用比较基因组学方法分析该菌株与香蕉和水稻菌株的进化关系和基因组特征差异。【结果】细菌基因组测序表明,CE1菌株的完整基因组大小为4 714 731 bp,注释预测到4 052个编码基因。与芭蕉芋和香蕉两个寄主亲缘关系类似,基因组比较分析发现来自芭蕉芋和香蕉的病菌菌株亲缘关系较近,它们在遗传进化上明显不同于水稻菌株。基因家族分析表明,编码重要致病因子如细菌分泌系统、鞭毛蛋白、胞外多糖、规律间隔成簇短回文重复序列(clustered regularly interspaced short palindromic repeats,C...     

hrpD6基因决定白叶枯病菌在烟草上的过敏反应和在水稻上的致病性

【目的】白叶枯病菌hrp基因簇由包括hrpD6在内的26个hpa-hrp-hrc基因组成,与植物互作后形成Ⅲ型分泌系统(T3S),将T3S效应分子注入寄主细胞中从而决定在非寄主上的过敏反应(HR)和在水稻上的致病性。但hrpD6基因是否参与了白叶枯病菌在非寄主上的过敏反应(HR)和在水稻上的致病性(pathogenicity)还不清楚。【方法】借助同源重组方法,本研究对白叶枯病菌hrpD6基因进行了突变。【结果】PCR和Southern杂交结果显示,hrpD6基因被成功敲除。烟草上测定结果显示,hrpD6突变体ΔPhrpD6丧失了HR激发能力。致病性测定发现,ΔPhrpD6在水稻苗期不能形成水渍症状,在成株期水稻上不具有致病性,并且细菌生长能力显著下降。功能互补结果显示,hrpD6基因可恢复ΔPhrpD6在烟草上激发HR和在水稻上的致病性以及在水稻组织中的生长能力。RT-PCR结果显示,hrpD6基因的转录表达不仅受水稻诱导,而且受hrpG和hrpX基因调控。不仅如此,hrpD6基因突变还影响T3S效应分子hpa1基因的转录表达和Hpa1蛋白的分泌,暗示hrpD6基因对hpa1基因转录表达具有调控作用。【结论】hrpD6基因的缺失导致白叶枯病菌不能激发烟草产生HR和和丧失在水稻上的致病性,主要是HrpD6对hpa1基因转录表达具有调控作用,并影响T3S效应分子Hpa1的分泌。这些结果为进一步分析hrpD6是否参与T3S分泌装置的形成和调控其它hrp基因的转录表达从而决定病菌在非寄主上的HR和在水稻上的致病性,提供了科学线索。     

Biological activities of purified harpin(Xoo) and harpin(Xoo) detection in transgenic plants using its polyclonal antibody

Many harpins have been found in plant pathogen bacteria that can elicit disease and insect resistance in plants, and promote plant growth. In this work, we overexpressed and purified Xanthomonas oryzae pv. oryzae harpin, harpinxoo, in Escherichia coli BL21/pGEX-hpa1. Harpinxoo was fused to the Cterminus of glutathione S-transferase （GST） and purified using the Bulk GST purification module and thrombin cleavage capture kit. Purified harpinxoo protein was sensitive to protease K and stable to heat treatment, and could not induce a hypersensitive response after treatment with various plant metabolic inhibitors; these characteristics were similar to harpinEa of Erwinia amylovora. The purified harpinxoo showed a similar ability to induce tobacco mosaic virus resistance in tobacco as harpinEa. Its antibody worked well in detecting the purified harpinxoo, harpinxoo in the total protein of E. coli BL21/pGEX-hpa1 and an hpal transgenic rice.     

The acyl-homoserine lactone-type quorum-sensing system modulates cell motility and virulence of Erwinia chrysanthemi pv. zeae

Erwinia chrysanthemi pv. zeae is one of the Erwinia chrysanthemi pathovars that infects on both dicotyledons and monocotyledons. However, little is known about the molecular basis and regulatory mechanisms of its virulence. By using a transposon mutagenesis approach, we cloned the genes coding for an E. chrysanthemi pv. zeae synthase of acyl-homoserine lactone (AHL) quorum-sensing signals (expI_Ecz) and a cognate response regulator (expR_Ecz). Chromatography analysis showed that expI_Ecz encoded production of the AHL signal N-(3-oxo-hexanoyl)-homoserine lactone (OHHL). Null mutation of expI_Ecz in the E. chrysanthemi pv. zeae strain EC1 abolished AHL production, increased bacterial swimming and swarming motility, disabled formation of multicell aggregates, and attenuated virulence of the pathogen on potato tubers. The mutation also marginally reduced the inhibitory activity of E. chrysanthemi pv. zeae on rice seed germination. The mutant phenotypes were rescued by either exogenous addition of AHL signal or in trans expression of expI_Ecz. These data demonstrate that the AHL-type QS signal plays an essential role in modulation of E. chrysanthemi pv. zeae cell motility and the ability to form multicell aggregates and is involved in regulation of bacterial virulence.     

Extracellular polysaccharide of Erwinia chrysanthemi A350 and ribotyping of Erwinia chrysanthemi spp

Erwinia chrysanthemi spp. are gram-negative bacterial phytopathogens causing soft rots in a number of plants. The structure of the extracellular polysaccharide (EPS) produced by the E. chrysanthemi strain A350, which is a lacZ- mutant of the wild type strain 3937, pathogenic to Saintpaulia, has been determined using a combination of chemical and physical techniques including methylation analysis, low-pressure gel-filtration and anion-exchange chromatography, high-pH anion-exchange chromatography, partial acid hydrolysis, mass spectrometry and 1- and 2D NMR spectroscopy. In contrast to the structures of the EPS reported for other strains of E. chrysanthemi, the EPS from strain A350 contains D-GalA, together with L-Rhap and D-Galp in a 1:4:1 ratio. Evidence is presented for the following hexasaccharide repeat unit: [structure: see text] All the Erwinia chrysanthemi spp. studied to date have been analyzed by ribotyping and collated into families, which are consistent with the related structures of their EPS.     

Evidence against a direct antimicrobial role of H2O2 in the infection of plants by Erwinia chrysanthemi

We have investigated the role of bacterial resistance to oxidative stress in pathogenesis. The oxyR gene from the pathogenic bacterium Erwinia chrysanthemi has been characterized. It is closely related to that found in Escherichia coli (88% overall amino acid identity). An E. chrysanthemi oxyR mutant strain was constructed by marker exchange. After induction with a sublethal dose of H2O2, this mutant was more sensitive to H2O2 and showed reduced levels of catalase and glutathione reductase activities, compared with the wild type. The oxyR mutant was unable to form individual colonies on agar plates unless catalase was added exogenously. However, it retained full virulence in potato tubers and tobacco leaves. These results suggest that the host-produced H2O2 has no direct antimicrobial effect on the interaction of E. chrysanthemi with the two plant species.     

An evaluated improvement of the extinction dilution method for isolation of ammonia-oxidizing bacteria

A simple and rapid method for extracting DNA from plants based on the use of an extraction buffer and precipitation with isopropanol was assayed to see its usefulness in detecting pathogenic bacteria in plant material. The method was compared with a phenol-chloroform standard procedure obtaining higher sensitivity levels of detection. The protocol developed was efficient for detecting a Gram-positive bacterium, Clavibacter michiganensis subsp. sepedonicus and several Gram-negative pathogenic bacteria (Ralstonia solanacearum, Erwinia amylovora, Xanthomonas axonopodis pv. citri) with a sensitivity of 10(2)-10(3) cfu/ml in spiked samples. It was also efficient to specifically identify such bacteria in naturally infected plant material. This procedure is proposed as a routine tool for detection of plant pathogenic bacteria, as well as in environmental microbiology and biotechnology studies.