柑橘溃疡病菌抗铜相关基因copA和 copB的克隆及原核表达

 以柑橘溃疡病菌DNA为模板,对抗铜相关基因copA和copB进行了PCR扩增和克隆,获得了大小分别为1 782 bp 和1 095 bp的目标片段。构建了这2个基因的原核表达载体 (pET-copA和pET-copB),并在大肠杆菌 [Escherichia coli BL21(DE3)] 中成功诱导表达。利用原核表达的融合蛋白免疫大耳白兔,制备了抗copA和copB原核表达蛋白的多克隆抗体。用间接ELISA法测定了所制备的多克隆抗体的效价均为1∶6 400。用所制备的抗体分别对copA和copB的原核表达产物进行Western blot分析的结果显示,在相应位置产生了较强的免疫反应条带,表明所制备抗体能特异性地与相应的抗原发生免疫反应。     

木质部次生细胞壁增厚参与疣粒野生稻对黄单胞杆菌水稻变种的抗性

 黄单胞杆菌水稻变种（Xanthomonas oryzae pv.oryzae,Xoo）引起的水稻白叶枯病是一个世界性的严重病害。疣粒野生稻（Oryzae meyeriana）对Xoo具有高度抗性,但其抗性机制仍不清楚。本文以抗病的疣粒野生稻和感病的水稻品种大粒香为材料,研究了Xoo侵染对叶片病斑、叶绿体超微结构、光合系统活性和木质部超微结构的影响。结果表明,多种Xoo生理小种导致的疣粒野生稻叶片病斑长度都明显短于大粒香叶片的病斑长度。Xoo病菌侵染显著破坏了大粒香的叶绿体结构,明显抑制了其光合活性,而疣粒野生稻中的变化要轻得多。通过电镜切片,发现疣粒野生稻叶片导管内存在大量的Xoo病菌,这表明Xoo能够侵染疣粒野生稻且能够在叶片内增殖。病菌的侵染诱导了疣粒野生稻木质部次生细胞壁的增厚,抑制了病菌通过导管纹孔向邻近细胞的进一步侵染,这种反应可能参与了疣粒野生稻对Xoo的抗性。     

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。     

Characterization of pathotypes among isolates of Xanthomonas axonopodis pv. manihotis in Colombia

Cassava bacterial blight, caused by Xanthomonas axonopodis pv. manihotis ( Xam ) is a destructive disease occurring in most cassava growing-areas. Although Colombian isolates of Xam differ in DNA polymorphism and pathogenicity, no suitable host differentials have been identified to demonstrate physiological specialization. A set of 26 Xam isolates from three edaphoclimatic zones (ECZs) in Colombia was selected for inoculation on a set of 17 potential cassava differentials. Leaf inoculation and stem puncture were used in order to detect possible specific interactions between cultivars and isolates. Cultivar × isolate interaction was highly significant ( P  < 0·001) after stem inoculation, but not after leaf inoculation. The stem inoculation technique was selected as a method for resistance screening of cassava cultivars for bacterial blight resistance. A highly significant interaction was also detected when cultivar behaviour was rated as area under the disease progress curve (AUDPC) after stem inoculation. Different pathotypes were defined among the 26 isolates and differential cultivars were proposed to define the pathotypic composition of Xam populations in three ECZs in Colombia. The results should help to improve selection of sources of resistance to cassava bacterial blight.     

Cloning of a Genomic DNA Encoding Caffeoyl-coenzyme A 3-<Emphasis Type="Italic">O</Emphasis>-methyltransferase of Citrus

In recent studies, we found that Apll (a PthA homologue) bound to three citrus proteins. Amino acid sequence analysis revealed that one of the target proteins was homologous to that of S-adenosyl-l-methionine : trans-caffeoyl-coenzyme A 3-O-methyltransferase (CCoAMT), an enzyme which is specific for the substrate trans-caffeoyl-CoA and catalyzes the synthesis of trans-feruloyl-CoA. From the consensus nucleotide sequences of CCoAMT genes, primers were chosen for PCR amplification of this gene from citrus total DNA. Two selected DNA fragments of 1.0 kb and 2.0 kb were obtained. These fragments were used as the probe to screen a citrus library. One clone, pCCl00, contained a 1.0-kb SalI fragment that hybridized to the probes. The nucleotide sequence of this fragment was determined in both directions. In this fragment, there was an open reading frame of 232 amino acids interrupted by an intron of 106 nt, and the deduced amino acid sequence had 95.9% homology to tobacco CCoAMT. Southern blot analysis of total citrus DNA showed that four EcoRI fragments hybridized to the probes, suggesting the presence of more than one copy of CCoAMT in the citrus DNA. Received 4 November 1999/ Accepted in revised form 26 November 1999     

安徽省四地市水稻细菌性条斑病对链霉素的抗药性监测

于2010年采集安徽省滁州、安庆、宣城和巢湖四个城市水稻细菌性条斑病菌108株,以及2011年水稻细菌性条斑病菌168株,采用区分剂量法,测定其对链霉素的抗药性。能够在含10 μg/ml链霉素的 NA平板上生长,为抗药性菌株,不能生长的为敏感菌株。抗药性监测结果表明,安徽省四个城市的细菌性条斑病菌对链霉素还比较敏感,未发现抗药性菌株。对于该病菌对链霉素的抗药性仍需要连年进行。     

不同立地条件下“核桃黑”的发生情况调查

为了了解影响核桃产量和质量的“核桃黑”发生情况以及造成“核桃黑”的主要原因。笔者在商洛市山阳县核桃园中根据不同海拔高度确定3种试验地,在每种试验地里随机选择10棵10~15年的核桃树,调查温湿度、虫果率、引起“核桃黑”的病虫害。结果表明,“核桃黑”的发生情况与立地条件有直接关系,“核桃黑”发生率最大的是深山区,浅山区次之,平原区发生的最小;深山区造成“核桃黑”的主要是黑斑病,然后是核桃举肢蛾,而浅山区和平原区的“核桃黑”主要是核桃举肢蛾造成,其次才是黑斑病。调查结果表明：对于“核桃黑”的防治应该在7月13日后开始进行,防治的重点主要集中在防治核桃黑斑病和核桃举肢蛾上。     

十字花科黑腐病菌IV型分泌系统的诱导表达与抗逆功能分析

 细菌通过IV型分泌系统(Type IV Secretion System, T4SS)的接合系统、效应物转运系统和释放/吸收系统3个子家族进行DNA、蛋白质及毒素的分泌和转运。十字花科黑腐病菌(Xanthomonas campestris pv. campestris, Xcc)是一种重要的植物病原菌,也是研究植物病原细菌与植物相互作用机理的模式细菌之一。本研究通过检测Xcc 8004野生型菌株和T4SS突变体在不同条件下的生长、诱导情况及对过敏反应的影响发现:T4SS不影响在培养基中的生长,与野生型菌株相比,T4SS突变体在非寄主辣椒ECW-10R上的过敏反应减弱;T4SS相关基因受基本培养基MMX诱导表达,且与Ni²⁺、H₂O₂、Phenol等抗逆相关。推测T4SS相关基因在该病原菌的接触、识别阶段起作用。     

小麦矮缩病毒NASH快速检测方法的建立及应用

以非放射性物质地高辛为标记物,采用PCR法制备了特异性强、灵敏度高的DNA探针。通过优化反应体系,建立了小麦矮缩病毒(Wheat dwarf virus,WDV)的核酸斑点杂交(nucleic acid spot hybridization,NASH)快速检测技术体系。该方法诊断准确率高,操作简单,周期短,整个检测过程仅需5h左右。利用建立的NASH技术开展WDV流行学调查,发现近年来WDV在我国陕西韩城、山西太原和河北石家庄等地区点片发生,没有大面积暴发成灾。     

The genetic characterization of Xanthomonas arboricola pv. juglandis,the causal agent of walnut blight in Poland

Leaves and fruits of walnut trees exhibiting symptoms of bacterial blight were collected from six locations in Poland. Isolations on agar media resulted in 18 bacterial isolates with colony morphology resembling that of the Xanthomonas genus. PCR using X1 and X2 primers specific for Xanthomonas confirmed that all isolates belonged to this genus. In pathogenicity tests on unripe walnut fruits, all isolates caused typical black necrotic lesions covering almost the entire pericarp. Results of selected phenotypic tests indicated that characteristics of all isolates were the same as described for the type strain of Xanthomonas arboricola pv. juglandis. Genetic analyses (PCR MP, ERIC‐, BOX‐PCR and MLSA) showed similarities between the studied isolates and the reference strain of X. arboricola pv. juglandis CFBP 7179 originating from France. However, reference strains I‐391 from Portugal and LMG 746 from the UK were different. MLSA analysis of partial sequences of the fyuA, gyrB and rpoD genes of studied isolates and respective sequences from GenBank of pathotype strains of other pathovars of X. arboricola showed that the X. arboricola pv. juglandis isolates consisted of different phylogenetic lineages. An incongruence among MLSA gene phylogenies and traces of intergenic recombination events were proved. These data suggest that the sequence analysis of several housekeeping genes is necessary for proper identification of X. arboricola pathovars.