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。     

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。     

水稻病原菌Burkholderia plantarii群体感应和群体淬灭研究进展

植物伯克霍尔德菌Burkholderia plantarii是引起水稻秧苗细菌性立枯病的重要病原菌之一,其侵染性、繁殖力及适应性均很强,严重威胁中国水稻生产。文章围绕B.plantarii的发生、危害及致病机理,着重论述了细菌群体感应系统(quorum sensing,QS)的生理功能及其在B.plantarii致病力调控方面的最新研究进展,并进一步从根际微生物互作角度,综述了种间信号分子对病原菌群体淬灭(quorum quenching)的作用机制,同时结合种间信号分子的独特性,展望了其在新型微生物杀菌剂研发中的重要性和应用潜力。     

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。     

群体感应系统对甜瓜果斑病菌MH21致病力的影响

 以N-乙酰高丝氨酸内酯（N-acyl-homoserine lactone, AHL）为信号分子的群体感应（quorum-sensing, QS）系统是很多病原细菌的重要致病性调控因子。本文自甜瓜果斑病菌——西瓜食酸菌（Acidovorax citrulli）菌株MH21中克隆到AHL信号合成基因luxI_MH21,并构建了其缺失突变体MΔluxI_MH21及转化有AHL信号降解酶编码基因aiiA和aidH的工程菌株MAiiA和MAidH。信号检测结果显示MΔluxI_MH21、MAiiA和MAidH菌株均无AHL信号产生,同时细菌的游动能力及在基本培养基中的生长速率均显著下降,但对细菌生物膜形成和在非寄主植物烟草上诱导过敏性坏死反应的能力没有影响。盆栽条件下,经低浓度（10⁴ CFU/mL）MΔluxI_MH21、MAiiA和MAidH菌株处理的甜瓜种子萌发后幼苗死亡率显著低于野生型MH21和luxI_MH21基因互补菌株MΔluxI_MH21HB的处理;而高浓度细菌（10⁸ CFU/mL）处理种子后,除MAidH菌株处理引起的死苗率明显低于野生型MH21处理,其他菌株与MH21没有显著差异。子叶注射试验也得到相似的结果,以低浓度细菌（10⁴ CFU/mL）注射甜瓜子叶后发现MΔluxI_MH21、 MAiiA和MAidH菌株甜瓜子叶中的繁殖速率及对子叶的致病力与野生型MH21相比均显著下降;而高浓度细菌（10⁸ CFU/mL）处理子叶时,MΔluxI_MH21和MAiiA菌株与野生菌MH21相比致病力无显著差异,仅有MAidH菌株的致病力明显下降。说明QS系统影响菌株MH21在低细菌浓度下对甜瓜幼苗的致病力,这种作用可能与影响细菌生长有关。     

群体感应信号物质对吡咯伯克霍尔德氏菌JK-SH007在杨树内定殖的影响

为了明确杨树溃疡病生防菌吡咯伯克霍尔德氏菌JK-SH007的群体感应系统是否与其内生定殖相关，本文通过群体感应指示菌紫色杆菌CV026和液相色谱串联四级杆飞行时间质谱技术（HPLC-Q-TOF-MS）确定其群体感应信号物质种类，并利用结晶紫染色、菌体回收、GFP标记等技术，研究其群体感应信号物质对该菌株生物膜及在杨树苗内的定殖能力的影响。结果表明，菌株JK-SH007产生的群体感应信号物质为含8个碳原子酰基侧链的辛酰基-L-高丝氨酸内酯（C₈-HSL）。群体感应信号物质C₈-HSL的添加对该菌株生物膜及在杨树苗内的定殖能力有影响，并且呈现低浓度促进，较高浓度抑制的规律。C₈-HSL的添加与菌株JK-SH007生物膜的OD值和菌体量具有显著相关性，相关系数分别为0.923和0.756。当添加1.0% C₈-HSL终浓度达到5×10^{-8 g/L}时，菌株JK-SH007生物膜的形成量达到峰值。荧光显微镜镜检发现，当添加100 μL C₈-HSL时，杨树组培苗根和茎中GFP荧光标记的菌株JK-SH007的定殖数量明显较CK多。菌株JK-SH007的群体感应与其在杨树内的内生定殖能力密切相关，群体感应信号物质C₈-HSL具有增强菌株JK-SH007内生定殖的能力，同时也表明该物质有利于该菌株生防效果的发挥。     

假单胞菌WX14群体感应淬灭酶基因的克隆及其功能研究

本研究采用报告菌平板法及β-半乳糖苷酶活性测定并筛选到10株具有较强降解酰基高丝氨酸内酯（AHLs）能力的细菌,其中菌株WX14降解AHLs能力较强,且菌体和菌体外泌物均具有降解活性。利用PCR法从菌株WX14中克隆到hacA和hacB两个群体感应淬灭酶基因,基因全长分别为2286和2370 bp,氨基酸序列比对分析结果表明,HacA属于阿库来菌素A酰化酶蛋白家族,HacB属于青霉素G酰化酶蛋白家族。这两个基因编码产物均为N末端亲核（N-terminal nucleophile,Ntn）水解酶家族成员。将hacA和hacB导入到软腐果胶杆菌Pectobacterium carotovorum后,可减弱该细菌在马铃薯块和白菜上的致病性,因此这2种AHLs降解酶对依赖于群体感应的软腐病有一定防病作用。经16S rDNA序列同源性分析鉴定,3株为假节杆菌Pseudarthrobacter spp.、1株节杆菌Paenarthrobacter sp.、4株假单胞菌Pseudomonas和2株芽胞杆菌Bacillus spp.。     

拮抗性细菌生物防治植物土传病害的研究进展

经过60多年的研究,利用细菌防治植物土传病害已有很大进展,已明确18个属细菌具有这种生防潜力,而研究最多的为Agrobacterium、Bacillus和Pseudomons。作为一种土传病害的生防细菌在根部定殖受根际压力势、温度、pH、植物基因型、病菌侵染等因素影响,而细菌的表面多糖、纤毛、化学吸引素、耐渗透性、对复杂碳水化合物的利用是重要根际感应特性。生防菌通过营养基质的竞争和生态位的排斥、产生嗜铁索、抗生素等机制防治病害。最后讨论了细菌生防的存在问题及发展方向。     

郭予元院士在创建中国主要粮棉作物多病虫复合群体综合防治技术体系上的学术贡献——纪念郭予元院士诞辰90周年

本文回顾了郭予元院士在20世纪80-90年代对中国主要粮棉作物多病虫复合群体综合防治技术体系研究与实践中的重要工作, 包括组建粮棉作物多病虫复合群体综合防治技术体系的创新性思路、创建主要粮棉作物多病虫复合群体综合防治技术体系, 以及对21世纪我国粮棉作物多病虫复合群体综合防治技术体系提出的发展前景和目标等, 其中粮棉作物多病虫复合群体综合防治技术体系实施后取得了显著成效。郭予元院士在我国主要粮棉多病虫复合群体综合防治领域的相关学术思想及贡献, 对我国农作物病虫害综合防治、可持续治理、绿色防控策略制定以及植物保护学科的发展产生了深远的影响, 为我国实现农作物生物灾害可持续控制做出了重要贡献。     

<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.     

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.     

The critical role of biofilms in bacterial vascular plant pathogenesis

Bacterial life is a combination of two lifestyles, mobile and social. In the social lifestyle, cells are usually embedded in a self-produced matrix and attached to biotic or abiotic surfaces. These communities can be organized as either single or multilayered structures termed biofilms. Biofilms evolved to cope with the harsh environmental conditions that bacteria encounter within the host, mostly from the host’s defence response. In plant pathogenic bacteria, biofilms participate in the whole process of pathogenicity, from the first step of invasion to the full colonization of plant tissues. The specific role that biofilms play in the pathogenicity process of plant bacterial pathogens is poorly understood. In this review, the role of biofilms in the pathogenic process of major vascular plant pathogens is examined. In addition, quorum sensing signals and components that are essential for biofilm formation and therefore, for pathogenesis, are addressed. Although, in certain systems, further research is required, experimental evidence in the literature indicates that biofilms are, in most cases, essential for pathogenesis.