Pathogenic and Genetic Relatedness Among Xanthomonas axonopodis pv. allii and Other Pathovars of X. axonopodis

ABSTRACT Xanthomonas axonopodis pv. allii is phenotypically and genetically diverse and its relationship to other X. axonopodis pathovars within DNA homology group 9.2 is unknown. In growth chamber experiments, disease symptoms were produced on onion only by inoculation with X. axonopodis pv. allii. Citrus bacterial spot symptoms were induced by X. axonopodis pvs. alfalfae, itrumelo, and allii on Duncan grapefruit and key lime. X. axonopodis pv. allii multiplication and persistence in Duncan grapefruit were equal to those of an aggressive strain of X. axonopodis pv. citrumelo, but populations of X. axonopodis pvs. alfalfae, betlicola, citrumelo, phaseoli, and vesicatoria were 1.3 to 4.0 log units less than X. axonopodis pv. allii in onion. Genomic fingerprinting by repetitive sequence- based polymerase chain reaction demonstrated that X. axonopodis pvs. allii, alfalfae, and citrumelo are distinct from other Xanthomonas species and X. axonopodis pathovars, but these pathovars were indistinguishable from each other. Three genotype groups were apparent among DNA homology group 9.2 strains, and generally correspond to the aggressiveness and genotype groups previously described for X. axonopodis pv. citrumelo. X. axonopodis pvs. allii, alfalfae, and citrumelo appear to have recently diverged from a common ancestral strain.     

红掌细菌性疫病病原菌的PCR特异性检测

 红掌细菌性疫病(Xanthomonas axonopodis pv. dieffenbachiae,简称Xad)是红掌等天南星科花卉毁灭性病害,该病通过带菌种苗调运不断在我国扩散蔓延,国内尚未有检测方法。通过筛选和重新设计引物,建立了Xad 的PCR 检测方法,结果表明,利用引物Xad-F / Xad-R 进行PCR,能扩增出检测Xad 的特异性DNA 片断,其灵敏度可达1 × 102 CFU / mL,DNA 的最低检出限为0. 44 ng / μL,可用于红掌苗的带菌检测和红掌细菌病害的鉴定。     

Genetic and pathogenic variation of Xanthomonas axonopodis pv. manihotis in Venezuela

DNA polymorphism and variation in virulence of Xanthomonas axonopodis pv . manihotis (Xam), the causal agent of cassava bacterial blight, were studied within a pathogen population from Venezuela. Collections were made in several fields at different sites within an edaphoclimatic zone where cassava is a major crop. DNA polymorphism was assessed by RFLP analysis, using an Xam plasmidic DNA sequence ( pth B) as a probe to determine the relatedness of 91 Venezuelan isolates. A high degree of polymorphism existed among the isolates, whether collected from the same or different fields. Based on a multiple correspondence analysis, the Xam population was distributed into eight clusters and no correlation was observed between genetic diversity and geographic origin. One set of haplotype strains representing the range of variability detected in Venezuela was further characterized by another RFLP analysis using two repetitive genomic probes (pBS6 and pBS8) to establish the usefulness of these probes and their complementarity with the pth B probe. Variation for virulence was observed in the Xam Venezuelan collection by inoculating a set of cassava cultivars with 28 isolates of the pathogen, each representing a haplotype. Understanding the genetic and pathogenic variation in the pathogen population is useful for designing cassava bacterial blight management strategies.     

利用PCR-DHPLC技术检测柑橘溃疡病菌

柑桔溃疡病是由柑桔溃疡病菌Xanthomonas axonopodis pv.citri(Xac)引起的重要的国际国内检疫性病害.柑桔溃疡病菌属黄单胞菌属,由于该属的种类及变种繁多,对Xac的生化鉴定十分繁杂和困难.     

PCR-based detection of Xanthomonas campestris pathovars in Brassica seed

Xanthomonas campestris is a seedborne bacterium that causes black rot of crucifers. Substantial crop losses may result from the rapid spread of the bacteria under favourable conditions, especially those occurring during seedling production. A PCR-based method has been developed for the rapid and sensitive detection of the pathovars of X. campestris that affect crucifers. Primers were designed to specifically amplify a 619 bp fragment of the hrpF gene from X. campestris . Amplification products were not detected from other Xanthomonas species, or from other pathogenic or epiphytic bacteria occurring on these plants. To avoid false-negative results arising from the presence of amplification inhibitors in plant extracts, primers targeting a 360 bp section of the internal transcribed spacer (ITS) region from Brassica spp. were included in a multiplex PCR. The assay readily detected X. campestris pv. campestris infections in diseased plants and from bacterial colonies isolated on growth media, and was more sensitive and specific than traditional plating methods and a commercially available ELISA. A seed-washing protocol was optimized to allow the detection of a single artificially infected seed among 10 000 healthy seeds using the multiplex PCR.     

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.     

Comparison of extraction procedures for detection of Xanthomonas axonopodis pv. phaseoli in common bean seed

Xanthomonas axonopodis pv. phaseoli (Xap) is an important seedborne pathogen of Phaseolus vulgaris. Accurate seed health testing methods are critical to protect seed quality and meet phytosanitary requirements. Currently employed selective media‐based methods include several variations in extraction procedures. In order to optimize pathogen extraction from seeds, the influence of different extraction steps on the sensitivity of Xap detection was assessed. Seeds were inoculated by vacuum infiltration with Xap to achieve inoculum levels from 10¹ to 10⁵ CFU per seed; one contaminated seed was mixed into 1000‐seed subsamples of uncontaminated P. vulgaris seeds. Thirty subsamples of 1000 seeds were tested using each different extraction procedure. These included soaking whole seeds in sterilized saline phosphate buffer, either overnight at 4°C or for 3 h at room temperature, with or without vacuum extraction, and either with or without concentrating the seed extract by centrifuging. Seed extract dilutions were cultured on semiselective agar media MT and XCP1. The percentages of positive subsamples were compared to measure the effects of each extraction step on detection sensitivity. Vacuum extraction and centrifugation of seed extracts increased sensitivity; the highest sensitivity was obtained with the 3 h vacuum extraction followed by centrifugation. These results were confirmed with naturally infested seeds; Xap was detected in 48 of 70 samples using the 3 h vacuum extraction with centrifugation, whereas only 35 of 70 field samples tested positive using overnight soaking, a significant difference. The results suggest that these steps would be valuable modifications to the current method approved by the International Seed Testing Association (ISTA).     

木薯细菌性萎蔫病菌的LAMP快速检测方法

建立了一种木薯细菌性萎蔫病菌的环介导恒温扩增快速检测方法,为木薯细菌性萎蔫病的快速检测提供有力的技术支持。针对木薯细菌性萎蔫病菌TAL效应器蛋白质(pthBXam)靶序列的6个位点设计4条特异性引物,并对反应温度和内引物浓度等参数进行了优化,设计的引物与试验中提供的其他黄单胞近缘种都没有扩增反应,表现了较好的特异性。LAMP方法对木薯细菌性萎蔫病菌菌株DNA的检测下限为1pg/μL,比常规PCR灵敏度高100倍。该方法采用SYBR Green I染料法对扩增产物闭管检测,裸眼观察颜色变化判断反应结果,能快速、准确地对田间样品进行检测,没有出现假阳性和假阴性。与其他检测方法相比,LAMP方法检测时间短,效率高,降低了设备投入,易于操作,适合木薯细菌性萎蔫病菌的现场检疫和大规模监测。     

Measuring the Genetic Diversity of Xanthomonas axonopodis pv. manihotis Within Different Fields in Colombia

ABSTRACT Cassava bacterial blight, caused by Xanthomonas axonopodis pv. manihotis, is a widespread disease that affects cassava (Manihot esculenta). We collected 238 X. axonopodis pv. manihotis strains by intensively sampling single fields in four edaphoclimatic zones (ECZs) in Colombia. DNA polymorphism of different X. axonopodis pv. manihotis populations was assessed by restriction fragment length polymorphism (RFLP) analyses, repetitive sequence-based polymerase chain reaction (rep-PCR), and amplified fragment length polymorphism (AFLP) assays. Genetic diversity, phenetic relationships among strains, and the coefficient of genetic differentiation were determined. All strains were tested for aggressiveness on the susceptible cassava cv. MCOL 1522. Strains were also tested for virulence on cassava differentials adapted to the strains' respective ECZs. Our study showed that the Colombian X. axonopodis pv. manihotis population has a high degree of genetic diversity. The hierarchical analysis of diversity showed genotypic differentiation at all levels, among ECZs, among fields within ECZs, and among strains within fields planted to several cassava genotypes. New RFLP haplotypes were detected, leading to the characterization of a new pathotype. Dendrograms from AFLP were more robust than those from RFLP data. A close association between the strains' geographical origin and DNA polymorphism was obtained using RFLP and AFLP data. We suggest that the host played a role in causing pathogen differentiation.     

Genetic basis of resistance to systemic infection by Xanthomonas axonopodis pv. dieffenbachiae in Anthurium

The genetic basis of systemic resistance to bacterial blight disease (blight) of anthurium (Anthurium andraeanum) caused by Xanthomonas axonopodis pv. dieffenbachiae was investigated in progenies of 53 crosses involving 31 parent cultivars using segregation analysis. Inoculation of parents and progenies was achieved by injecting the petiole base of the most recent fully expanded leaf with 100 microl of 10(9) colony forming units per ml of the blight pathogen (strain X4gfp) transformed with the green fluorescent protein (GFP) gene. The time to death and the presence or absence of GFP fluorescence on newly emerging leaves was monitored over a period of 30 weeks after inoculation (WAI), on an individual plant basis. The expected resistance to susceptible ratios based on a digenic model involving two dominant genes, designated A and B, interacting according to a duplicate recessive epistasis model fitted the observed segregation ratios in the crosses. Based on the segregation ratios obtained, the parental cultivars were assigned plausible genotypes. There were significant differences (P < 0.001) in time to death following inoculation between the various genotypic designations. Cultivars with genotypes AABB, AABb, AaBB, and AaBb died within 10 WAI and designated as susceptible; AAbb and aaBB died from 18.8 to 25.6 WAI and were designated as moderately resistant; and Aabb, aaBb, and aabb produced resistant phenotypes. There was also some evidence for dosage effect especially in the highly resistant category. Hence, (AABb = AaBB = AaBb) < (aaBB = AAbb) < Aabb = aaBb = aabb). An approach to fixing resistance to blight in anthurium is discussed.     

Development of a real-time PCR-based method for detection of Xylophilus ampelinus

A real-time PCR MGB-probe-based detection method specific to Xylophilus ampelinus , the cause of grapevine bacterial blight, was developed. Used in combination with the DNeasy plant mini kit, the sensitivity of X. ampelinus detection was approximately 100 cells from tissue extracts, surpassing the sensitivity of an existing nested PCR method at least tenfold. In field samples a high correlation was observed between real-time PCR cycle threshold (Ct) values obtained and X. ampelinus isolation on artificial media. Isolation was successful from samples with Ct values below 25. Lower concentrations of X. ampelinus , with Ct values up to 36, could also be reliably detected in real-time PCR. The newly developed method offers a reliable and sensitive test for X. ampelinus, suitable as a screening test, complementary to isolation on media or other methods, and could also be used for fast and specific identification of isolated colonies and for relative quantification of X. ampelinus bacteria.     

Comparative and collaborative studies for the validation of a nested PCR for the detection of Xanthomonas axonopodis pv. dieffenbachiae from Anthurium samples

Efficient control of Xanthomonas axonopodis pv. dieffenbachiae, the causal agent of anthurium bacterial blight, requires sensitive and reliable diagnostic tools. The European standard EN ISO 16140:2003 has been followed to compare a nested PCR assay (N‐PCR) to a reference method (isolation and serological identification of bacterial colonies) and to other alternative serological detection methods. The evaluation was performed in two steps: a comparative study and a collaborative study involving 15 European laboratories. Although inclusivity was maximal (100%) for all methods, a maximal exclusivity was obtained only with N‐PCR followed by an enzymatic restriction digestion of the amplicons. Exclusivity indices of 90·6, 88·7 and 47·2% were found for indirect ELISA, immunofluorescence and double antibody sandwich ELISA, respectively. An exclusivity of 92·5% was obtained with the reference method, further increased to 100% if pathogenicity tests were performed as a supplemental assay. The best level of sensitivity (relative detection level) was obtained with the reference method followed by the N‐PCR assay. The N‐PCR performance in terms of relative accuracy, accordance and concordance was very similar to that of the reference method. Moreover, N‐PCR had undeniable advantages compared to the reference method (less labour‐intensive and less time‐consuming). In addition, post‐test probabilities of infection were calculated to select the most appropriate detection scheme related to the prevalence of the pathogen. The N‐PCR assay has since been included in a revised version of the EPPO detection protocol.     

广东水稻白叶枯病菌遗传多样性和小种分化研究

 通过IS-PCR和rep-PCR指纹技术,分析了广东水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae)群体遗传多样性。用2对特异性引物J3和ERIC对114个菌株基因组DNA进行了PCR扩增,分别呈现89和40种谱型,以彼此间的带位相似率达70%为界,J3扩增的谱型被分为11簇,ERIC的谱型被分为8簇。J3的簇1包含89个菌株,占总数的78.07%,ERIC的簇1包含52个菌株,占总数的45.61%,均为优势簇群。群体遗传多样性值J3为0.8919,ERIC为0.8278。上述结果表明,广东水稻白叶枯病菌的遗传多样性较高。全部参试菌株接种于含有不同抗性基因近等基因系及高感品种金刚30共6个鉴别品种,被划分为6个小种(X-gd1,X-gd2,X-gd3,X-gd4,X-gd5和X-gd6),X-gd4出现频率最高,为广东省的优势小种。     

A history of passion fruit woodiness disease with emphasis on the current situation in Brazil and prospects for Brazilian passion fruit cultivation

Plants of the Passiflora genus are widely cultivated for the production of fruit, cosmetics and pharmacological products and for floriculture. Here, we review the history of the identification of viral pathogens involved in passion fruit woodiness disease, which can affect different species of Passiflora. Additionally, we discuss prospects for research and development related to this pathosystem and applications to plant breeding. We discuss the different potyviruses identified as potential causal agents of passion fruit woodiness in Africa, Asia, Australia and Brazil. We focus on the status of the cultivation of different Passiflora species in Brazil and place this problem in a global context. Both classical and molecular strategies for obtaining virus-resistant Passiflora plants are discussed in this work, thus providing insight into this pathosystem and useful information for breeders of Passiflora species.     

A green fluorescent protein-based screening method for identification of resistance in anthurium to systemic infection by Xanthomonas axonopodis pv. dieffenbachiae

Resistance of cultivars of Anthurium andraeanum to systemic infection by Xanthomonas axonopodis pv. dieffenbachiae , the causal organism of bacterial blight disease of anthurium, was investigated using a bioengineered bacterial strain containing p519ngfp plasmid. Successful infection establishment in anthurium was found to be cultivar and inoculum density dependent, but independent of plant age. Injection of cut petioles (stage-2 leaf) with 100  µ L inoculum (10⁹ CFU mL⁻¹) resulted in 100% infection establishment in susceptible cultivars on a repeatable basis, and differentiated between various levels of observed field resistance. Time to death (weeks) and proportion of dead plants best differentiated between levels of resistance and cultivars were placed in four groups based on these criteria. The susceptible group (32 cultivars) rapidly declined within 6–12 weeks of inoculation (WAI) and resulted in 100% plant death; the moderately resistant group (10 cultivars) declined within 12 WAI, but resulted in less than 100% plant death; the resistant category had less than 100% plant death with a slow decline taking over 20 weeks; and the highly resistant category (15 cultivars) showed 0% infection. The correlation coefficient between green fluorescent protein (GFP)-fluorescence and eventual death of plants was 0·90, indicating that the final death of individual plants can be reasonably well predicted based on GFP-fluorescence data at 5 WAI. Hence GFP data at 5 WAI can be used for early detection of latently infected plants and may assist screening for resistance in segregating populations of anthurium.     

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。     

HrpG regulates type II secretory proteins in Xanthomonas axonopodis pv. citri

HrpG, a two-component response regulator-like protein, is a key regulator of the type III secretion system (T3SS) in Xanthomonas spp. In X. campestris pv. vesicatoria, HrpG with a single amino acid substitution (HrpG*) gains the ability to induce the expression of T3SS-related genes even under nutrient-rich conditions. In this study, we investigated the role of HrpG in the synthesis of the secretory protein using HrpG* in strain NA-1 of X. axonopodis pv. citri (Xac NA-1), a causal agent of citrus canker. Eleven proteins secreted via a type II secretion system (T2SS) were induced by HrpG*. In proteomic analyses, six of the 11 proteins were identified as extracellular enzymes, and the others as a fimbrial biogenesis-related protein, a type IV-related protein, two hypothetical proteins, and a conserved hypothetical protein. Further analysis of these proteins revealed that the genes coding all 11 proteins were upregulated by HrpG*, even though they had different expression patterns for HrpXct-dependency. The data indicated that HrpG, a key regulator of T3SS, also acts as a positive regulator of certain proteins secreted via a T2SS in Xac NA-1.     

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。