群体感应淬灭——防治植物细菌病害的新策略

群体感应（quorum sensing,QS）是细菌的一种调控机制,指细菌通过感应特定信号分子的浓度来感知周围环境中自身或其它细菌的数量,并调整相关基因的表达以适应环境的变化。多种植物病原细菌利用QS系统调控致病因子的表达,因此,QS系统可以作为细菌病害防治的新靶点。对细菌QS调控机制的干扰和破坏称为群体感应淬灭（quorum quenching）。本文介绍了QS与植物病原细菌致病性的关系,以及近年来群体感应淬灭研究的新进展。     

细菌群体感应抑制剂研究进展

多重耐药菌的出现已成为农业防治植物病害的一大难题,目前急需发展新的防治策略。群体感应 (quorum sensing, QS) 是一种微生物之间普遍存在的依赖于菌体密度的沟通协调机制,控制着细菌的生长、增殖、致病性、生物被膜形成及相关群体活动行为。群体感应抑制剂在抑制细菌毒性基因表达时不会对细菌生长产生压力,从而避免了细菌耐药性的产生。这一新颖的抑菌机制使其在开发新型农药方面有很大潜力。本文着重介绍了细菌群体感应机制、天然的及合成的细菌群体感应抑制剂种类及其应用。     

申氏杆菌37-1中群体感应淬灭内酯酶AiiS的功能分析

许多植物病原细菌通过群体感应（quorum sensing,QS）系统调控相关毒性因子的表达,而群体感应淬灭（quorum quenching,QQ）是通过干扰QS系统,达到防治植物细菌性病害的重要策略之一。本研究利用原位培养法分离得到2000多株不同菌株形态的植物根围细菌,结合QS系统信号分子检测平板筛选到7株具有QQ活性的候选细菌,其中菌株37-1可完全降解信号分子。16S rDNA序列分析表明,菌株37-1属于Shinella sp.。全基因组序列分析发现菌株37-1中存在一个可能的QQ降解酶编码基因aiiS（autoinducer inactivation gene from Shinella sp.）。系统发育分析表明AiiS属于α/β水解酶家族蛋白。液相色谱-串联质谱分析进一步表明AiiS可水解N-乙酰高丝氨酸内酯（N-acylhomoserine lactone,AHL）类QS信号分子中的内酯健,生成酰基高丝氨酸,因此AiiS属于AHL内酯酶。将aiiS基因导入胡萝卜软腐果胶杆菌Pectobacterium carotovorum subsp.carotovorum Z3-3中,可显著降低该菌AHL信号分子和果胶酸盐裂解酶的产生及其在白菜、马铃薯和胡萝卜上的致病性。以上结果表明菌株37-1中AiiS蛋白是一种AHL内酯酶;病原细菌中异源表达aiiS基因可有效干扰相关病原细菌中QS系统的调控功能,表明AiiS蛋白具备开发为潜在新型生防制剂的价值。     

Heterodera filipjevi许昌群体SCAR标记的建立

菲利普孢囊线虫Heterodera filipjevi是禾谷类作物上重要的病原线虫之一,严重影响禾谷类作物的产量和品质。本课题组前期研究发现菲利普孢囊线虫许昌群体是一个新的致病型,其在小麦上的致病力强于其他多个群体,危害更为严重。本研究旨在建立菲利普孢囊线虫许昌群体的快速、准确的分子检测体系,为Heterodera filipjevi许昌群体的监测和防控及抗病品种的选育利用奠定基础。本研究采用随机扩增多态性DNA(RAPD)和序列特征扩增区域(SCAR)的方法,对黄淮麦区5个重要小麦孢囊线虫致病型共9个线虫群体进行RAPD分析和SCAR标记转化,并通过增加除黄淮麦区外的线虫群体验证所获得的致病型相关分子标记的特异性和有效性。本研究共筛选了331条RAPD引物,筛选出2个菲利普孢囊线虫许昌群体相关的RAPD标记,引物S86可以扩增出1条约550 bp的多态性片段,引物S178可以扩增出1条约1 200 bp的多态性片段,并将这2个RAPD标记成功转化为SCAR标记。SCAR标记的特异性检测结果表明这两个SCAR标记只在许昌群体上有特异性扩增,可以用于许昌群体的分子检测。     

哈密瓜细菌性果斑病菌群体感应系统的检测

本文利用群体感应信号报告菌Agrobacterium tumefaciens NTL4(pZLR4),在LB报告平板上对燕麦食酸菌西瓜亚种的19个菌株进行了初步检测,发现18个菌株有群体感应信号产生.用琼脂条法对菌株Pslb-94进一步检测,证实菌株Pslb-94存在群体感应系统.提取了该菌株信号物质,反相薄层层析(TLC)检测证明该菌株能产生群体感应信号分子.     

酰基高丝氨酸内酯酶SS10的酶学特性及其抗软腐病功能的初探

 N-酰基高丝氨酸内酯(AHLs)作为细菌群体感应中的信号分子参与调节植物病原细菌致病因子的表达。N-酰基高丝氨酸内酯酶(简称AHL内酯酶)通过水解AHL生成酰基高丝氨酸, 使AHL失去活性, 阻断病原菌的群体感应机制, 使病原菌失去致病力。利用谷胱甘肽-琼脂糖亲和层析柱和凝血酶处理获得纯化的重组AHL内酯酶SS10, 分子量约为28 kD, 其反应的最适pH值为8.0, 在30℃以下稳定性很高。动力学和底物特异性分析表明:AHL内酯酶SS10对所检测的8种AHL具有很强的催化活性, 表明该酶的底物谱可能较宽, 并且具有催化裂解酰基高丝氨酸内酯键的特异性。纯化的AHL内酯酶SS10可以显著降低胡萝卜软腐欧文氏菌胞外果胶酶、多聚半乳糖醛酸酶的产生量。致病性测定表明, 该重组蛋白对胡萝卜软腐病菌具有较强的抗病活性。     

蚕豆赤斑病病原菌鉴定

蚕豆赤斑病是蚕豆的重要病害之一,严重影响蚕豆生产。本研究通过形态学和分子特征,对采自甘肃、青海、江苏、四川、河北、重庆等6个省市的蚕豆赤斑病病原菌进行鉴定。结果表明,在6个省市均鉴定到蚕豆葡萄孢(Botrytis fabae)、灰葡萄孢(B.cinerea)和拟蚕豆葡萄孢(B.fabiopsis)3种病原菌。致病力测定表明:不同地理来源的3种病原菌分离物都存在致病力差异,但均以强致病力分离物为主。本研究是继湖北省之后在其他省市首次发现拟蚕豆葡萄孢。     

四川籼稻区稻瘟病菌群体遗传结构

应用rep-PCR分子指纹技术对2000～2002年采自四川6个籼稻自然生态区的137个稻瘟病菌菌株进行了DNA分子指纹扩增和聚类分析,共获得73个不同的DNA指纹图谱(单元型)和62条分子量不等的DNA带型.结果显示,无论以何种遗传相似水平划分,四川稻瘟病菌的群体结构都表现很突出的优势宗谱,又存在着具有较多遗传多样性的次要小宗谱和特异性宗谱,蕴含着极其丰富的遗传信息;在0.19遗传相似水平,所有供试菌株可以划分成37个遗传宗谱,层次较为丰富.四川稻瘟病菌群体结构具有明显的时空特点,不同年度间稻瘟病菌群体存在一定的亲缘关系,又各自拥有当年的特异性宗谱;在空间上,不同稻作区表现出从复杂到简单的病菌群体变化特点.稻瘟病菌的遗传宗谱与生理小种致病型不存在一一对应的关系,作者认为将二者横向比较没有可比性.     

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。     

水稻转录因子OsBTF3对不同病原菌和信号分子的基因表达反应

 转录因子基因OsBTF3在水稻品种日本晴悬浮细胞中受白叶枯病菌(Xanthomonas oryzae pv.oryzae,Xoo)诱导表达。为了阐明OsBTF3在水稻叶组织中的表达特征,本研究利用RT-Q-PCR技术,对经3种亲和性病原菌[水稻白叶枯病菌(Xoo)、水稻条斑病菌(Xooc)和稻瘟病菌(Mg)]接种和4种信号分子[脱落酸(ABA)、水杨酸(SA)、茉莉酸甲酯(MeJA)、乙烯(ETH)]诱导处理的水稻叶片中OsBTF3的转录本进行了定量分析。结果表明,OsBTF3对Xoo、Xooc和Mg侵染的基因表达反应均显著地受到诱导,但反应速度和强度略有差异。而4种信号分子对OsBTF3表达的诱导作用差异较大,ABA诱导活性最强,MeJA和ETH次之,SA诱导作用不显著。因此,OsBTF3基因表达不仅具有病原菌Xoo、Xooc和Mg的诱导性,而且也具有信号分子MeJA、ETH和ABA的应答性。     

Bacteria from the citrus phylloplane can disrupt cell–cell signalling in Xanthomonas citri and reduce citrus canker disease severity

Xanthomonas citri subsp. citri (Xcc) is the causal agent of citrus canker, a disease that affects almost all types of citrus crops. Production of particular Xcc pathogenicity factors is controlled by a gene cluster rpf, which encodes elements of a cell–cell communication system called quorum sensing (QS), mediated by molecules of the diffusible signal factor (DSF) family. Interference with cell–cell signalling, also termed quorum quenching, either by signal degradation or over‐production, has been suggested as a strategy to control bacterial disease. In this study, three bacterial strains were isolated from citrus leaves that displayed the ability to disrupt QS signalling in Xcc. Pathogenicity assays in sweet orange (Citrus sinensis) showed that bacteria of the genera Pseudomonas and Bacillus also have a strong ability to reduce the severity of citrus canker disease. These effects were associated with alteration in bacterial attachment and biofilm formation, factors that are known to contribute to Xcc virulence. These quorum‐quenching bacteria may represent a highly valuable tool in the process of biological control and offer an alternative to the traditional copper treatment currently used to treat citrus canker disease.     

<Emphasis Type="Italic">N</Emphasis>-acyl-homoserine lactone-mediated quorum sensing blockage,a novel strategy for attenuating pathogenicity of Gram-negative bacterial plant pathogens

Quorum sensing is a bacterial communication mechanism by which bacteria sense their own population size and couple specific gene expression to cell density. In Gram-negative bacteria, the most commonly used quorum sensing signals are N-acyl homoserine lactones (AHLs). It is now apparent that many pathogenic bacteria employ quorum sensing to control premature expression of virulence factors. This control is thought to decrease the likelihood that the plant host would detect the pathogens presence and activate its defense system. Novel strategies that target bacterial quorum sensing systems in order to control plant bacterial diseases are discussed.     

Identification of <Emphasis Type="Italic">N</Emphasis> −3-hydroxyoctanoyl-homoserine lactone production in <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> 5064, pathogenic to broccoli,and controlling biosurfactant production by quorum sensing

Quorum sensing controls a number of key processes in growth, reproduction and virulence of many gram-negative bacteria via signalling molecules or autoinducers. It can control, for instance, the production of pectic enzymes which are virulence factors in disease. Pseudomonas fluorescens 5064 produces biosurfactants which are important for bacterial establishment on the plant surface prior to causing disease in broccoli. The aim of this paper was to determine if biosurfactant production in this bacterium is controlled via quorum sensing. To do this, 35 surfactant-minus Tn5 mutants of P. fluorescens 5064 were screened for their abilities to produce a quorum sensing signal. Six of these biosurfactant-deficient mutants showed a large reduction in quorum sensing signal production and varied in their abilities to cause disease. In one mutant, 6423, which contains a single Tn5 insertion, the production of the signal was almost eliminated. Synthetic quorum sensing signal and quorum sensing signal extracted from wild type P. fluorescens 5064 restored biosurfactant production by addition to the culture in mutant 6423. The quorum sensing signal in wild type P. fluorescens 5064 was identified by high pressure liquid chromatography and mass spectrometry as N -3-hydroxyoctanoyl-homoserine lactone.     

Silencing of Erwinia amylovora sy69 AHL‐quorum sensing by a Bacillus simplex AHL‐inducible aiiA gene encoding a zinc‐dependent N‐acyl‐homoserine lactonase

Quorum sensing in Gram‐negative bacteria is regulated by diffusible signal molecules called N‐acyl‐l ‐homoserine lactones (AHLs). These molecules are degraded by lactonases. In this study, six Bacillus simplex isolates were characterized and identified as a new quorum‐quenching species of Bacillus. An aiiA gene encoding an AHL‐lactonase was identified based on evidence that: (i) it showed high homology with other aiiA genes of Bacillus sp.; (ii) the deduced amino acid sequence contained two conserved regions, ¹⁰⁴SHLHFDH¹¹¹ and ¹⁶⁵TPGHTPGH¹⁷³, characteristic of the metallo‐β‐lactamase superfamily; and (iii) the protein had zinc‐dependent AHL‐degrading activity. Additionally, the expression of the aiiA gene was significantly up‐regulated by 3‐oxo‐AHL. The AHL‐lactonase inhibited multiplication of the 3‐oxo‐C6‐AHL‐producing plant pathogen Erwinia amylovora sy69 both in vitro and in planta. The results provide support for the use of the quorum‐quenching functionality of B. simplex in the integrated control of the devastating fire blight pathogen.     

Management of bakanae and bacterial seedling blight diseases in nurseries by irradiating rice seeds with atmospheric plasma

The control of seedborne rice seedling diseases in the seed beds is important to avoid epidemics in rice nurseries and paddies, which may result in severe yield loss. Recently, irradiation with plasma containing electrons, creating positive or negative ions and neutral species, has been shown to have an antimicrobial effect, probably via generation of reactive oxygen species. This study examines whether two seedborne rice seedling diseases, bakanae disease caused by the fungal pathogen Fusarium fujikuroi, and bacterial seedling blight caused by Burkholderia plantarii, are suppressed by irradiation of infected rice seeds with atmospheric plasma. Seed germination and seedling growth were not inhibited in plasma‐treated healthy seeds. When F. fujikuroi‐infected rice seeds were irradiated with plasma after being immersed in sterile distilled water, bakanae disease severity index and the percentage of plants with symptoms were reduced to 18.1% and 7.8% of non‐irradiated control, respectively, depending on the duration of plasma irradiation. The bacterial seedling blight disease index was also reduced by plasma irradiation in vacuum‐inoculated seeds to 38.6% of the non‐irradiated control, and in infected seeds harvested from spray‐inoculated heads of rice plants to 40.1% of the control. Therefore, plasma irradiation seems to be effective in controlling two independent seedborne rice seedling diseases.     

Burkholderia gladioli associated with symptoms of bacterial grain rot and leaf-sheath browning of rice plants

Rice plants with bacterial leaf-sheath browning and grain rot were observed in Fukuoka Prefecture in Japan during the autumn seasons of 1995 and 1996. Burkholderia spp. were consistently isolated from the infected leaf sheaths and grains. These isolates were pathogenic and induced symptoms of seedling rot, grain rot, and leaf-sheath browning in rice plants, as well as in some orchidaceous plants (cymbidium, dendrobium, and oncidium leaves), gladiolus leaves, and onion bulbs. On the basis of morphological, physiological and pathological tests, and species-specific polymerase chain reaction, the isolates were identified as belonging to either Burkholderia glumae or Burkholderia gladioli. B. gladioli, as well as B. glumae, attacked rice plants after artificial inoculation and reproduced the symptoms similar to those after natural infections. We confirmed that rice is an additional natural host of B. gladioli. It is clarified that bacterial grain rot of rice is caused not only by B. glumae but also by B. gladioli.