Induction of systemic resistance byPseudomonas fluorescens Pf1 againstXanthomonas oryzae pv.Oryzae in rice leaves

When lower leaves of rice plants were inoculated with powder formulation of a saprophytic strain ofPseudomonas fluorescens, Pfl, upper leaves, in addition to the inoculated lower leaves, showed resistance to the rice bacterial blight pathogenXanthomonas oryzae pv.oryzae. When the leaves were challenge-inoculated withX. oryzae pv.oryzae 4 days afterP. fluorescens application on lower leaves, the disease intensity in upper leaves decreased from 6.7 to 1.1. When rice seeds were treated with the formulation ofP. fluorescens Pfl and sown, 30-day-old seedlings showed resistance toX. oryzae pv.oryzae and the disease intensity decreased from 6.8 to 1.2. The induced resistance was transient; leaves sprayed withP. fluorescens Pfl at 30 days after treatment and leaves of 60-day-old seedlings fromP. fluorescens-treated seeds did not show resistance to the pathogen. In field trials, seed treatment followed by foliar application of the powder formulation ofP. fluorescens Pfl effectively controlled rice bacterial blight and increased the yield. In the induced resistant leaves a sharp increase in lignification and activities of peroxidase, phenylalanine ammonia-lyase and 4-coumarate: CoA ligase was observed when the leaves were challenge-inoculated withX. oryzae pv.oryzae. An approximately threefold increase in lignin content, peroxidase activity and phenylalanine ammonia-lyase activity and a fivefold increase in 4-coumarate: CoA ligase activity were observed 5 days after challenge inoculation withX. oryzae pv.oryzae in rice leaves pretreated withP. fluorescens for 5 days. A similar increase in defense-related activities was not observed in susceptible interactions or inP. fluorescens-treated plants at later stages of interactions when no resistance to the pathogen was observed.     

云南水稻白叶枯病菌生理小种初析

从云南省 5个地州14个县市的水稻白叶枯病标本中分离水稻白叶枯病菌菌株 ,选择具有区域代表性的菌株 5 1株 ,利用已知抗病基因的近等基因系 ,在孕穗期应用剪叶法接种明确其生理小种。鉴定结果表明 ,云南省水稻白叶枯病菌小种复杂多样 ,共有 14种类型 ,暂定为YN1～YN14 ,优势生理小种为YN3、YN8、YN11,其中YN3分布在红河、德宏等 4个县市 ;YN8小种分布在大理等地区 ,小种的分布可能受地理区域的影响     

Virulence factors analysis of native isolates of Xanthomonas albilineans and Xanthomonas sacchari from Tucumán,Argentina, reveals differences in pathogenic strategies

Xanthomonas albilineans (Xa) and X. sacchari (Xs) are both sugarcane pathogens. Xa is the causal agent of leaf scald disease, but there is limited information about the pathogenicity of Xs. The aim of this work was to study virulence factors of native strains of Xa (Xa32, Xa33, and XaM6) and Xs (Xs14 and Xs15) previously isolated from sugarcane with leaf scald symptoms, to gain insight into the biology of each microorganism. We analysed epiphytic survival, sensitivity to oxidative stress, extracellular degradative enzymes, cell motilities, exopolysaccharide (EPS) characteristics, cell adhesion, biofilm development, and control of stomatal regulation of the five strains. We observed that each species presented similar phenotypes for every factor analysed. Xa strains appeared to be more sensitive to oxidative stress and presented lower epiphytic survival than Xs. All strains presented endoglucanase activity; however, we could only detect protease and amylase activities in Xs strains. Swimming and sliding were higher in Xs, but twitching was variable among species. We also observed that only Xs strains produced a xanthan-like EPS, presented a strong cell adhesion, and structured biofilm. We detected some intraspecific variations showing that higher amounts of EPS produced by Xs14 correlated with its higher sliding motility and its homogenous and more adhesive biofilm. In addition, EPSs of Xs14 and Xs15 presented differences in strand height and acetyl percentage. Finally, we found that strains of both species were able to interfere with stomatal aperture mechanism. All these differences could influence the colonization strategies and/or disease progression in each species.     

不同柑橘品种对柑橘溃疡病的抗性调查

通过柑橘Citrus sp.4类10个品种对柑橘溃疡病Xanthomonas citri(Hasse)Dowson抗病性的调查发现，不同的柑橘品种对柑橘溃疡病的抗病程度有明显差异，甜橙类，脐橙类普遍容易感病，属高度感病品种：宽皮橘类的抗病性最强；而柚类感病轻重不一，以台湾白柚抗病性最强；而沙田柚抗病性较弱。     

一个用于体外鉴定野油菜黄单胞菌Ⅲ型效应物的报告质粒的构建

许多革兰氏阴性植物病原细菌通过Ⅲ型分泌系统(T3SS),将效应物蛋白分泌并转运进入植物体内,从而引起植物发病或过敏反应.效应物是病原细菌和植物分子互作的关键物质.全基因组筛选鉴定Ⅲ型效应物蛋白并研究其功能,对于解明病原菌的致病机理、寄主范围和进化具有重要意义.利用avrBs1作为报告基因,将其过敏反应诱导结构域(AvrBs159-444)克隆到不含启动子的穿梭质粒pLAFR6上,构建一个用于Ⅲ型效应物转运体外鉴定的报告质粒pLJB,并以已经鉴定为效应物基因的XC-1553的启动子验证该报告质粒,证明pLJB工作正常.该报告质粒为进一步进行全基因组筛选鉴定野油菜黄单胞菌Ⅲ型效应物提供材料.     

Genetic transformation of Citrus sinensis cv. Hamlin with hrpN gene from Erwinia amylovora and evaluation of the transgenic lines for resistance to citrus canker

Transgenic Citrus sinensis (L.) Osb. cv. Hamlin plants expressing the hrpN gene were obtained by Agrobacterium tumefaciens (Smith and Towns) Conn-mediated transformation. hrpN encodes a harpin protein, which elicits the hypersensitive response and systemic acquired resistance in plants. The gene construct consisted of gst1, a pathogen-inducible promoter, a signal peptide for protein secretion to the apoplast, the selection genes nptII or aacC1 and the Nos terminator. The function of gst1 in citrus was evaluated in transgenic C. sinensis cv. Valencia harboring the reporter gene uidA (gus) driven by this promoter. Histochemical analysis for gus revealed that gst1 is activated in citrus leaves by both wounding and inoculation with Xanthomonas axonopodis Starr and Garces pv. citri (Hasse) Vauterin et al. Genetic transformation was confirmed by Southern blot hybridization in eight cv. Hamlin acclimatized plants. RT-PCR confirmed hrpN gene expression in seven cv. Hamlin transgenic lines before pathogen inoculation. Some hrpN transgenic lines showed severe leaf curling and abnormal growth. Six hrpN transgenic lines were propagated and evaluated for susceptibility to X. axonopodis pv. citri. RT-PCR confirmed gene expression in all six hrpN transgenic lines after pathogen inoculation. Several of the hrpN transgenic lines showed reduction in susceptibility to citrus canker as compared with non-transgenic plants. One hrpN transgenic line exhibited normal vegetative development and displayed very high resistance to the pathogen, estimated as up to 79% reduction in disease severity. This is the first report of genetic transformation of citrus using a pathogen-inducible promoter and the hrpN gene. Further evaluations of the transgenic plants under field conditions are planned. Nevertheless, the evidence to date suggests that the hrpN gene reduces the susceptibility of citrus plants to the canker disease.     

白条黄单胞菌侵染对蔗叶和蔗茎组织超微结构的影响

本研究利用普通光学显微镜、透射电镜和扫描电镜观察白条黄单胞菌[Xanthomonas albilineans(Ashby) Dowson]侵染后菌体定殖及对甘蔗叶片和蔗茎组织超微结构的影响。结果表明,白条黄单胞菌侵染后叶片的薄壁组织细胞膜裂解,木质部生长受到抑制。叶绿体形状多近圆形,基质片层解体,被膜破坏呈松弛状态,出现大量的原生质膜。扫描电镜可见白条黄单胞菌存在于各个叶肉细胞中,同时在维管束中也发现菌体定殖,特别是木质部导管。蔗茎的维管束被白条黄单胞菌堵塞,使得蔗茎木质化、木栓化严重。本研究有助于理解白条黄单胞菌对甘蔗叶片和蔗茎细胞内细胞器损伤的形态破坏,为白条病防治提供超微形态学依据。     

蔗糖水解酶注释基因XC0805与野油菜黄单胞菌8004菌株的蔗糖利用及致病相关

 野油菜黄单胞菌(Xanthomonas campestris pv.campestris,Xcc)是造成十字花科植物减产的重要病原菌,其通过水孔或伤口进入植物体内并在维管束中快速繁殖。光合产物(蔗糖)的运输主要依靠植物维管组织,同时蔗糖也是维管组织重要的能量来源。本研究利用生物信息学和遗传学等方法对Xcc中参与蔗糖利用的基因进行了分析,并研究了这些基因与其致病力的关系。Xcc 8004菌株中注释的参与蔗糖分解蛋白的编码基因有XC1002、XC1642、 XC1645和XC0805。 XC1002和XC1642的编码产物属于糖苷水解酶GH97蛋白超家族,而XC1645和XC0805的编码产物则属于GH13家族。突变分析发现除XC0805外,其他3个都不影响Xcc的蔗糖利用能力。接种实验表明,XC0805参与Xcc的侵染或在植物体内的生长,其他3个不参与致病过程。另外,Xcc 8004中预测参与蔗糖转运的基因(XC0806和XC0807)突变并不影响细菌的蔗糖利用和致病力,表明其可能存在其他的蔗糖转运通路。上述结果说明XC0805可能是Xcc 8004主要的蔗糖水解酶基因,且在致病中起重要作用。     

在小檗碱作用下水稻细菌性条斑病菌生理及转录反应分析

为探究小檗碱对水稻细菌性条斑病菌Xanthomonas oryzae pv.oryzicola的抑菌机制,测定小檗碱对水稻细菌性条斑病菌GX13菌株的毒力、致病因子、细胞结构和能量代谢的影响,并利用Illumina-HiSeq高通量测序技术分析小檗碱对水稻细菌性条斑病菌菌体基因表达的影响。结果表明：经15 μg/mL小檗碱处理后,GX13菌株侵染水稻品种玉丝占后引起水稻细菌性条斑病的病情指数为6.70,低于对照组;每1×10⁸ CFU菌体胞外多糖产量为1.58 μg,显著低于对照的2.42 μg;纤维素酶、蛋白酶、淀粉酶等胞外酶活性均低于对照组;与能量代谢相关的苹果酸脱氢酶（MDH）活性为1.42 U/g prot,低于对照组,而丙酮酸和三磷酸腺苷含量升高,分别为1.71 μmol/mL和379.49 μmol/g prot,均高于对照组。同时,细菌的生物膜形成受到抑制,细胞结构受到破坏。转录反应分析发现,与II型分泌系统相关的xpsD、xpsF和xpsM等基因、与III型分泌系统相关的hrcC、hrcJ、hrcN和hrcQ等基因、与胞外多糖分泌相关的gumF和gumI基因,以及与细胞结构与功能相关的murD、lpxA、lpxH、lpxL、fabG和fabZ等基因均下调表达。此外,与丙酮酸代谢途径相关的ppc、aceF和lpdA等基因也下调表达,但与氧化磷酸化途径相关的NADH脱氢酶和ATP合成酶编码基因以及与趋化性相关的编码基因均显著上调表达。表明小檗碱对GX13菌株的抑菌作用与其影响病菌的细胞结构和能量代谢相关,小檗碱通过影响病菌II型分泌系统和III型分泌系统的功能、生物膜的形成以及胞外多糖的合成,从而影响病菌的致病力。与此同时,水稻细菌性条斑病菌借助强化氧化磷酸化功能,启动与趋化性相关的基因上调表达来抵御小檗碱对自身的损害。