壳聚糖和2株芽孢杆菌对柑橘溃疡病菌的杀菌效果评价

柑橘溃疡病是重要的柑橘细菌性病害,为探明壳聚糖和枯草芽孢杆菌对柑橘溃疡病的防治潜力,本文用平板菌落计数法评价了3种粘度壳聚糖对柑橘溃疡病菌的杀菌效果,以改良的纸碟法测定2株枯草芽孢杆菌(Bacillus subtilis)发酵液对柑橘溃疡病菌的抑菌效果。综合处理0.5 h、3 h和6 h的结果显示,3种粘度壳聚糖对柑橘溃疡病的杀菌强弱依次为:粘度12粘度100粘度20。枯草芽孢杆菌菌株BS 101和54-6在YPG培养基中有氧发酵3 d产生发酵液原液的抑菌圈直径分别为3.45 cm和3.21 cm,比0.1 g/mL氨苄青霉素的抑菌圈分别大45%和35%。可见,发酵液中存在对溃疡病菌高度有效的成分。因此,认为粘度12的壳聚糖和芽孢杆菌菌株BS 101和54-6具有防治柑橘溃疡病的潜力。     

N‐acetylcysteine interferes with the biofilm formation,motility and epiphytic behaviour of Xanthomonas citri subsp. citri

Citrus canker is caused by Xanthomonas citri subsp. citri. Bacterial biofilm formation is important in the development of this disease because it is a factor in epiphytic bacterial survival on leaves and in infection. N‐acetylcysteine (NAC), in addition to having antibacterial properties, reduces biofilm formation by a variety of bacteria and was therefore tested for impairing biofilm formation by X. citri. Copper is currently the antimicrobial compound most commonly applied in agriculture to control citrus canker. Therefore, this study also evaluated a possible synergistic effect between NAC and copper to improve the strategy for controlling this phytopathogen. NAC was found to decrease biofilm formation, the production of extracellular polysaccharides and bacterial stickiness. Motility was also affected in the presence of NAC. The best combination of NAC and copper for controlling X. citri was application of NAC followed by copper 48 h later. The concentrations of 6 mg mL^?1 of NAC and 3·5 μg mL^?1 of copper were able to kill X. citri. NAC inhibited the epiphytic behaviour of X. citri on leaves, altering cell growth and the bacterial ability to form biofilms. The addition of copper to cells previously treated with NAC enhanced its bactericidal activity. In conclusion, NAC has antibacterial properties against X. citri, interfering with bacterial growth, motility and biofilm formation. Under epiphytic conditions, NAC made the cells more susceptible to copper by affecting X. citri biofilm formation. This study opens new possibilities for the use of NAC in combination with copper, possibly resulting in more sustainable management of citrus canker.     

柑橘溃疡病菌EMA-PCR快速活体检测技术的建立

传统PCR方法不能诊断柑橘溃疡病菌(Xanthomonas citri subsp.citri Gabriel)的死活状态,往往导致假阳性检测结果.本研究将特异性核酸染料叠氮溴化乙锭(ethidium monoazide bromide,EMA)与PCR技术结合,旨在建立柑橘溃疡病活菌的快速检测技术.根据柑橘溃疡病菌独有的保守蛋白基因设计特异性引物扩增出278 bp的靶带,PCR反应的检测下限为25个细胞/25 μL或2.75 pg/25 μL.EMA-PCR结果表明:当卤钨灯曝光时间1 min,EMA终浓度为1.0 mg/L时,能有效抑制1.0×108 cfu/mL死菌的扩增;当EMA的浓度小于30 mg/L时,EMA对上述相同浓度活菌靶基因的扩增没有明显的抑制.EMA-PCR对死活混合菌的扩增表明,活菌数在6.875×101～6.875×105 cfu/PCR范围时,荧光强度与混合体系中活菌的对数值有线性关系.基于以上建立的EMA-PCR活体检测技术,对疑似带病柑橘材料进行检测,结果发现能降低柑橘溃疡病菌检测过程中的假阳性,有望为柑橘溃疡病的检疫检验提供更科学的技术手段.     

Unstable green fluorescent protein for study of Xanthomonas citri subsp. citri survival on citrus

Xanthomonas citri subsp. citri (Xac) is the causal agent of citrus bacterial canker, an important disease for the citrus industry. Studies of Xac survival in environments outside of the lesion performed in the past may have underestimated the viable population because the recovery was based on the ability of the bacterium to grow on culture media. This study monitored survival of Xac that express green fluorescent protein (GFP) in two different forms: the native protein, and a protein that is unstable due to a specific oligopeptide tail targeted by proteases within the bacterium. Transformed strains of Xac were verified to be stable in their expression of GFP and to show no differences in virulence and fitness compared to wild type strains. Evaluation of protein stability confirmed that strains with unstable GFP only expressed and fluoresced in metabolically active cells, and not in dead bacteria. Fluorescence of unstable GFP strains under confocal microscopy was used to track bacterial survival and biofilm formation on leaf and fruit surfaces. After spray inoculation, aggregates of fluorescing cells of unstable GFP strains formed biofilms on leaves and fruit. Bacterial cells that aggregated on the surfaces only survived when protected from desiccation. Aggregation of viable bacteria in biofilms confirms their role in pathogen survival outside of lesions and protection from bactericide treatments in the field or in the fruit disinfection process.     

Timing of copper sprays to protect mechanical wounds against infection by Xanthomonas citri subsp. citri,causal agent of citrus canker

European Journal of Plant Pathology - Copper sprays are the most important measure for control of citrus canker. Mechanical wounds caused by cultural practices are entry points for the causal...     

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。     

A PCR-based assay for differentiating A- and A*-type strains of Xanthomonas citri subsp. citri, the causal agent of Asiatic citrus canker

By applying A- and A*-type strains of Xanthomonas citri subsp. citri (Xcc) in a repetitive sequence-based polymerase chain reaction (rep-PCR), two DNA amplicons, one unique to each strain, were evaluated as a probe against the DNA of Xcc strains. Two pairs of primers derived from these amplicons were tested in a PCR analysis. The results confirmed that primers Ms⁺/Ms^? are useful for differentiating A-type from A*-type strains of Xcc. Also, a multiplex PCR with both set of primers can be used to distinguish three groups in Xcc populations: A-type strains and two subgroups of A* strains including Iranian and Thai A* strains.     

Detection of Xanthomonas citri subsp. citri from field samples using single‐tube nested PCR

A single‐tube nested PCR was developed for detection of Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker disease. The assay targets the pthA gene of Xcc and utilizes different annealing temperatures for the two primer pairs. It reliably detected as few as 1·0 × 10² Xcc cells, and was unaffected by the presence of PCR inhibitors. It was 10‐fold and 8500‐fold more sensitive than standard PCR and ELISA, respectively. Increased sensitivity was also achieved via the use of a washing method for DNA extraction, as opposed to direct extraction from leaf tissue. When evaluated for Xcc detection in 90 samples collected from affected pomelo orchards, the single‐tube nested PCR was superior to standard PCR, detecting the pathogen in 67 vs. 54 samples. It was also able to detect Xcc from samples with and without symptoms. This assay can be used as a rapid and sensitive technique for routine Xcc detection in field samples for surveillance of citrus canker.     

两种植物病原黄单胞菌基因组中同义密码子使用的分析

 根据已释放的基因组序列,对野油菜黄单胞菌野油菜致病变种(Xanthomonas campestris pv. campestris,Xcc)和地毯草黄单胞菌柑桔致病变种(X. axonopodis pv. citri,Xac)的密码子使用进行了分析。相对同义密码子使用值(relative synonymous codon usage,RSCU)的计算表明,它们具有高度相似的密码子使用模式。2个基因组密码子第3位的GC含量(GC3s)平均达0.806±0.077(Xcc)和0.791±0.075(Xac),倾向于使用GC含量较高的密码子。对有效密码子数量和密码子适应指数的分析表明,Xcc与Xac基因组中,高表达基因具有较高的GC含量,倾向于使用少数种类的密码子,而低表达基因具有较高的AT含量,倾向于随机地使用密码子。对密码子使用绝对次数进行的对应分析也证明了上述结论。同时,计算也证明了基因在基因组中的位置不影响密码子使用的模式。因此,基因组的GC含量、基因的表达水平和基因的种类与起源是影响这2个基因组密码子使用的主要因素。     

Development of a simplified NASBA protocol for detecting viable cells of the citrus pathogen Xanthomonas citri subsp. citri under different treatments

Nucleic acid sequence based amplification (NASBA) is a method of amplifying RNA, for the detection of RNA viruses and human pathogenic bacteria. Recently, NASBA has also been employed for the detection of plant diseases caused by viruses and quarantine bacteria. A major citrus pathogen, Xanthomonas citri subsp. citri (Xcc), causal agent of citrus bacterial canker, is being studied in depth due to its economic importance, with recent focus concentrating on its viability and survival under different stress conditions and control treatments. In this work, a NASBA protocol using primers for gumD mRNA has been developed to assess the viability of this pathogen under different bacteriocidal treatments. This method is rapid, specific and sensitive, and is able to detect viable bacterial cells, using a hybridization device which allows the visualization of the results in only 30 min. The usefulness of the method has been confirmed with bacterial suspensions subjected to different heat treatments and to sodium orthophenylphenate.     

Comparative transcriptional analyzes of Xanthomonas citri subsp. citri reveal mechanisms of adaptation and bacterial virulence in the early stage of citrus canker disease

European Journal of Plant Pathology - The Allium cepa L. (onion) crop is affected by leaf blight disease caused by Stemphylium vesicarium; the shifts that S. vesicarium infection causes in the...     

松脂酸铜防治柑橘溃疡病效果研究

2009-2010连续2年田间药效试验结果表明,试验药剂松脂酸铜20%可湿性粉剂对柑橘叶片和果实溃疡病具有较好防效,400mg/kg处理的防效分别可达75.35%和70.69%,是防治柑橘溃疡病较为理想的药剂。     

Short‐distance dispersal of splashed bacteria of Xanthomonas citri subsp. citri from canker‐infected grapefruit tree canopies in turbulent wind

Citrus canker (Xanthomonas citri subsp. citri (Xcc)) can cause yield loss and trade restrictions. The pathogen is dispersed in rain splash and spread is promoted by wind. The goal of this study was to gain some insight into the properties of short‐distance splash dispersal of Xcc from ～1·5 m‐tall cankered grapefruit canopies in turbulent wind, common during rainstorms in Florida. Turbulent wind up to 19·9 m s^?1 was tested in five experiments. Bacteria flux density (BFD, bacteria cm^?2 min^?1) was quantified at heights of 30, 70, 110, 130 and 180 cm above ground, and at four horizontal points (17, 51, 85 and 119 cm) at each height across the direction of the wind 1 m downwind. BFD varied among experiments, but the lowest BFDs were consistently detected at the greatest sample height. Despite differences between experiments, the relationship between log BFD and sample height was consistently described by a linear function (P = 0·06–<0·0001, R² = 0·75–>0·99). The BFD collected at the horizontal points across the wind path was variable. BFDs collected were sometimes significantly different, but no relationship was discernible. Stronger, turbulent wind resulted in greater BFD, with a linear function describing the relationship between log BFD and wind speed (P = 0·2–0·02, R² = 0·94–0·96). Multiple regression analysis demonstrated predictability of the proportion of total bacteria collected (F = 141, P < 0·0001, d.f. = 3, R² = 0·53).     

辣椒疮痂病菌(Xanthomonas vesicatoria)和水稻细菌性条斑病菌(X. oryzae pv. oryzicola)的质粒及其与耐链霉素和耐铜性关系

 从不同省份收集到辣椒疮痂病菌Xanthomonas vesicatoria (XV)7个菌株和水稻细菌性条斑病菌X. oryzae pv. oryzicola (XOZ)14个菌株,进行了质粒微量制备。XV菌株除XV1外都检测到质粒,单个菌株拥有的质粒数为2~5个,大小在10~100kb,没有发现为所有菌株所共有的质粒,但XV2、XV3、XV4、XV5、XV6共享1个大约55 kb的质粒。14个XOZ菌株都含有1个质粒,除XOZ5外,其余的13个菌株都含1个约40 kb的质粒。所有菌株都不耐铜。XOZ菌株仅XOZ7对链霉素表现耐性,4个XV菌株即XV4、XV5、XV6和XV7耐链霉素。用XV质粒转化无质粒菌株,发现XV5的1个76 Kb质粒与耐链霉素有关。     

Genetic structure analysis of strains causing citrus canker in Iran reveals the presence of two different lineages of Xanthomonas citri pv. citri pathotype A*

West Asia has been recognized as a major centre for the diversification of Xanthomonas citri pv. citri, a citrus quarantine pathogen of considerable economic importance. However, little genotyping data is available mainly due to the paucity of microbial resources in this region. Using a comprehensive strain collection, several genotyping techniques and a pathogenicity assay, the status of strains causing Asiatic citrus canker in Iran, an internationally significant citrus‐producing country, was clarified. All strains were genetically related to X. citri pv. citri pathotype A* (i.e. strains with a host range restricted to Mexican lime and related species) but not to pathotype A (i.e. strains with a wide host range among rutaceous species). The findings were based on discriminant analysis of the principal components of MLVA‐31 data and were further confirmed by pathogenicity data. Two genetically, geographically and pathologically separate groups of strains in Iran were identified. One of the groups had never been previously reported anywhere in the world. A very strong genetic structure was found (R_ST = 0·938), consistent with their geographical isolation. Strains from these two groups also differed in terms of their type III effector repertoire. The atypical host range of one of these groups could explain why some Iranian strains had previously been mistakenly identified as pathotype A. This study suggests the absence of invasive pathotype A strains in Iran (known as DAPC 1), which account for most of the economically important outbreaks internationally.     

柑橘溃疡病菌免疫荧光和生物学检测技术研究

本研究通过对7个不同柑橘品种的离体叶片接种及致病性观察筛选出了柑橘溃疡病菌敏感寄主材料，并建立了柑橘溃疡病菌的生物学检测方法；还初次尝试建立了一种简便易行的柑橘溃疡病菌免疫荧光快速鉴定方法。柑橘离体叶片接种实验结果表明：福本、红肉脐橙、纽荷尔和枳壳对柑橘溃疡病菌表现出一定的感病性，而粗柠檬和邓肯葡萄柚未表现出明显感病迹象，反而出现了坏死性应激反应。免疫荧光试验结果显示：浓度为10^3cfu／mL的菌液在30℃下用BSA封闭2h，30℃下抗体结合1h，荧光抗体浓度为4μg,／mL，室温放置40min后镜检，就可以在荧光显微镜下清晰地看到绿色的柑橘溃疡病菌体，而用该方法检测不到水稻白叶枯病菌。用免疫荧光抗体检测柑橘溃疡病菌操作简便，仅需一台荧光显微镜，整个检测过程仅需要4h。     

菜豆种子普通细菌性疫病菌检测

 由Xanthomonas axonopodis pv. phaseoli和Xanthomonas fuscans subsp. fuscans引起的菜豆普通细菌性疫病是严重影响菜豆生产的限制因子之一,可造成严重的产量和品种损失。染菌种子是病原菌传播的主要途径。本研究对5个菜豆主要产区的60份菜豆种子样品进行普通细菌性疫病菌检测。在MT选择性培养基上有36份种子样品浸提液检测到目标病原菌, 种子样品带菌量为2.49×10²~5.20×10⁷ CFU/粒。选择36个分离物接种感病品种“英国红”植株,所有分离物均引起接种植株发病。特异PCR检测结果表明,有20个分离物为X. fuscans subsp. fuscans,16个分离物为X. axonopodis pv. phaseoli。试验结果表明,我国一些菜豆主产区商业种植和研究用种子多数污染普通细菌性疫病菌,建议建立无菌种子生产区和加强种子管理,以有效控制病害发生。     

柑橘溃疡病菌的普通LAMP及快速LAMP检测方法的建立

建立柑橘溃疡病菌的普通LAMP和快速LAMP检测方法,使其能应用于基层检验检疫部门对病害的快速检测.利用柑橘溃疡病菌基因组特有的保守区域设计LAMP引物,通过优化反应条件,建立柑橘溃疡病菌的普通LAMP检测体系;在普通LAMP引物的基础上设计一对环引物,建立柑橘溃疡病菌的快速LAMP检测体系,并以多种参比菌DNA以及健康柑橘叶片基因组DNA为模板对普通LAMP和快速LAMP检测体系的特异性进行了验证,利用柑橘溃疡病菌菌液和DNA溶液梯度稀释液对普通LAMP和快速LAMP检测体系的灵敏度进行了验证.普通LAMP检测体系菌体和DNA检测灵敏度分别达到了2.25×104 cfu和2.03×10-1 ng,快速LAMP检测体系菌体和DNA检测灵敏度分别达到了2.25 cfu和2.03×10-5ng.在特异性测试中,普通LAMP检测体系与快速LAMP检测体系均仅对柑橘溃疡病菌进行扩增,对非靶标菌和柑橘叶片基因组DNA不产生扩增,普通LAMP与快速LAMP检测体系特异性测试结果一致.快速LAMP检测体系在0.5h内就可以达到普通LAMP检测体系的扩增量,是普通LAMP检测体系反应时间的一半,大大提高了检测的效率;快速LAMP检测体系菌悬液和DNA检测灵敏度均比普通LAMP检测体系提高了10 000倍.成功地建立了柑橘溃疡病菌的普通LAMP及快速LAMP检测方法,为柑橘溃疡病菌的检测提供了一种新的简便、快速的检测手段.