Pathogenicity assays restrict the species Xanthomonas campestris into three pathovars and reveal nine races within X. campestris pv. campestris

The species Xanthomonas campestris (Vauterin) groups bacteria associated with cruciferous plants. In order to clarify and refine the pathovar and race structures within X . campestris , 47 representative strains of six pathovars were characterized for their pathogenicity on a large host range of Brassicaceae, including all original hosts. Three diseases were observed on tested plants: (i) black rot disease on cruciferous plants; it was proposed that all strains causing black rot on at least one cruciferous plant be grouped in the single pathovar X . c . pv. campestris ; (ii) leaf spot disease caused by X . c . pv. raphani on hosts belonging to the Brassicaceae and Solanaceae; the sequenced strain 756C identified as X . c . pv. armoraciae was included in this pathovar and the existence of another leaf spot disease caused by X . c . pv. armoraciae was not supported; and (iii) bacterial blight of garden stocks caused by X . c . pv. incanae . No plants susceptible to X . c . pv. barbareae were found. Strains that did not induce any symptom on cruciferous plants tested, including their original hosts, were removed from the pathovar scheme and were named X . campestris only. Three new races were described in addition to the six races previously described within X . c . pv. campestris . The sequenced strains ATCC 33913 (CFBP 5241) and Xcc 8004 (CFBP 6650) belonged to race 3 and to race 9 (one of the new races described), respectively.     

Molecular characterization of the incitant of cowpea bacterial blight and pustule, Xanthomonas campestris pv. vignicola

Strains of Xanthomonas campestris pv. vignicola (Xcv), isolated from cowpea leaves with blight or minute pustules and collected from various geographic areas, were selected on the basis of pathological and physiological features. All strains were analyzed for genotypic markers by two methods: ribotyping with EcoRI endonuclease, and RFLP analysis with a plasmid probe (pthB) containing a gene required for pathogenicity from Xanthomonas campestris pv. manihotis. Ribotyping revealed a unique pattern for all the strains that corresponded to the previously described ribotype rRNA7. Based on polymorphism detected by pthB among Xcv strains, nine haplotypes were defined. The observed genetic variation was independent of the geographic origin of the strains and of pathogenic variation. Some haplotypes were widely distributed, whereas others were localized. In some cases, we could differentiate strains isolated from blight symptoms and pustules according to haplotypic composition. However, in most cases, no significant differences were observed. Our results and the previous pathogenic and biochemical characterizations suggest that the strains isolated from leaves with blight symptoms or minute pustules belong to the same pathovar. We provide information on pathogen diversity that can be used to identify and characterize resistant germplasm.     

木薯细菌性萎蔫病菌的检疫方法研究

本文对木薯细菌性萎蔫病菌的致病性测定、细菌的分离、细菌的培养条件和培养基选择、细菌的生理生化测定、分子生物学鉴定方法等方面进行了系统研究，确定了该病菌的菌落鉴定特征，建立了从木薯繁殖材料上进行病原菌检测的快速、灵敏、准确的PCR检测方法。     

Identification and Origin of Xanthomonas campestris pv. campestris Races and Related Pathovars

ABSTRACT One hundred sixty-four isolates of Xanthomonas campestris pv. campestris and other X. campestris pathovars known to infect cruciferous hosts (X. campestris pvs. aberrans, raphani, armoraciae, and incanae) were inoculated onto a differential series of Brassica spp. to determine both pathogenicity to brassicas and race. Of these, 144 isolates were identified as X. campestris pv. campestris and grouped into six races, with races 1 (62%) and 4 (32%) being predominant. Other races were rare. The remaining 20 isolates from brassicas and other cruciferous hosts were either nonpathogenic or very weakly pathogenic on the differential series and could not be race-typed. Five of these isolates, from the ornamental crucifers wallflower (Cheiranthus cheiri), stock (Matthiola incana) and candytuft (Iberis sp.), showed clear evidence of pathovar-like specificity to the hosts of origin. A gene-for-gene model based on the interaction of four avirulence genes in X. campestris pv. campestris races and four matching resistance genes in the differential hosts is proposed. Knowledge of the race structure and worldwide distribution of races is fundamental to the search for sources of resistance and for the establishment of successful resistance breeding programs.     

Assessment of genetic diversity of Xanthomonas campestris pv. campestris isolates from Israel by various DNA fingerprinting techniques

Three molecular typing methods were used to investigate genetic diversity among Xanthomonas campestris pv. campestris isolates obtained in Israel and others previously obtained from different geographical locations (collection isolates). Using pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP) and repetitive sequence-based PCR (rep-PCR), 22 different isolates were divided into 11, 12 and 13 differentiated genotypes, respectively. All collection isolates yielded different genotypes and, among the isolates from Israel, several new genotypes were found. These findings not only support the observed heterogeneity within X. campestris pv. campestris , but also suggest that variability at the genomic level in this pathovar is higher than previously estimated. Moreover, while previous studies suggested that PCR patterns obtained with integron-specific primers are conserved in most X. campestris pathovars, PCR patterns of this element yielded four different types among the X. campestris pv. campestris isolates tested, thus supporting the relatively high diversity in this pathovar. Although differences in pathogenicity were observed among isolates, assays using cauliflower and radish did not indicate a correlation between pathogenicity and genotype.     

Use of phages for identifying the citrus canker bacterium Xanthomonas campestris pv. citri in Taiwan

Phages CP115 and CP122, which were isolated from canker lesions on grapefruit and Liucheng sweet orange, respectively, showed a high degree of specificity with respect to lysis of test bacterial strains. When used jointly, they lysed 135 (97·8%) out of 138 Xanthomonas campestris pv. citri strains isolated from the canker lesions on leaves, twigs, and fruits of various citrus species, cultivars, and hybrids grown throughout Taiwan, but they did not lyse other X. campestris pathovars and other phytopathogenic bacteria, nor other bacteria isolated from soil, clinical or environmental samples. Of 252 CP115/CP122-sensitive and 78 CP115/CP122-resistant bacterial strains with colony characteristics typical of or similar to those of X. campestris pv. citri , isolated from canker lesions of various citrus plants in diverse growing regions in Taiwan, 250 (99·2%) and 76 (97·4%) strains were pathogenic and non-pathogenic, respectively, when inoculated into Liucheng sweet orange or Mexican lime. Thus, phages CP115 and CP122, when used jointly, appear to be applicable for identifying X. campestris pv. citri in Taiwan.     

Genetic Diversity and Pathogenic Variation of Common Blight Bacteria (Xanthomonas campestris pv. phaseoli and X. campestris pv. phaseoli var. fuscans) Suggests Pathogen Coevolution with the Common Bean

ABSTRACT Common bacterial blight (CBB), caused by Xanthomonas campestris pv. phaseoli and X. campestris pv. phaseoli var. fuscans, is one of the most important diseases of common bean (Phaseolus vulgaris) in East Africa and other bean-growing regions. Xanthomonad-like bacteria associated with CBB in Malawi and Tanzania, East Africa, and in Wisconsin, U.S., were characterized based on brown pigment production, pathogenicity on common bean, detection with an X. campestris pv. phaseoli- or X. campestris pv. phaseoli var. fuscans-specific PCR primer pair, and repetitive element polymerase chain reaction (rep-PCR) and restriction fragment length polymorphism (RFLP) analyses. The common bean gene pool (Andean or Middle American) from which each strain was isolated also was determined. In Malawi, X. campestris pv. phaseoli and X. campestris pv. phaseoli var. fuscans were isolated predominantly from Andean or Middle American beans, respectively. In Tanzania, X. campestris pv. phaseoli var. fuscans was most commonly isolated, irrespective of gene pool; whereas, in Wisconsin, only X. campestris pv. phaseoli was isolated from Andean red kidney beans. Three rep-PCR fingerprints were obtained for X. campestris pv. phaseoli strains; two were unique to East African strains, whereas the other was associated with strains collected from all other (mostly New World) locations. RFLP analyses with repetitive DNA probes revealed the same genetic diversity among X. campestris pv. phaseoli strains as did rep-PCR. These probes hybridized with only one or two fragments in the East African strains, but with multiple fragments in the other X. campestris pv. phaseoli strains. East African X. campestris pv. phaseoli strains were highly pathogenic on Andean beans, but were significantly less pathogenic on Middle American beans. In contrast, X. campestris pv. phaseoli strains from New World locations were highly pathogenic on beans of both gene pools. Together, these results indicate the existence of genetically and geographically distinct X. campestris pv. phaseoli genotypes. The rep-PCR fingerprints of X. campestris pv. phaseoli var. fuscans strains from East African and New World locations were indistinguishable, and were readily distinguished from those of X. campestris pv. phaseoli strains. Genetic diversity among X. campestris pv. phaseoli var. fuscans strains was revealed by RFLP analyses. East African and New World X. campestris pv. phaseoli var. fuscans strains were highly pathogenic on Andean and Middle American beans. Breeding for CBB resistance in East African beans should utilize X. campestris pv. phaseoli var. fuscans and New World X. campestris pv. phaseoli strains in order to identify germ plasm with the highest levels of resistance.     

Sources and Origin of Resistance to Xanthomonas campestris pv. campestris in Brassica Genomes

ABSTRACT Two hundred and seventy-six accessions of mainly Brassica spp. were screened for resistance to Xanthomonas campestris pv. campestris races. In Brassica oleracea (C genome), the majority of accessions were susceptible to all races, but 43% showed resistance to one or more of the rare races (2, 3, 5, and 6) and a single accession showed partial resistance to races 1, 3, 5, and 6. Further searches for resistance to races 1 and 4, currently the most important races worldwide, and race 6, the race with the widest host range, were made in accessions representing the A and B genomes. Strong resistance to race 4 was frequent in B. rapa (A genome) and B. napus (AC genome), indicating an A genome origin. Resistance to races 1 and 4 was present in a high proportion of B. nigra (B genome) and B. carinata (BC genome) accessions, indicating a B genome origin. B. juncea (AB genome) was the most resistant species, showing either strong resistance to races 1 and 4 or quantitative resistance to all races. Potentially race-nonspecific resistance was also found, but at a lower frequency, in B. rapa, B. nigra, and B. carinata. The combination of race-specific and race-nonspecific resistance could provide durable control of black rot of crucifers.     

The incidence of bacterial wilt caused by Xanthomonas campestris pv graminis in pasture grasses in the West of Scotland

Temporary hay and silage fields with a high proportion of perennial or Italian ryegrass ( Loliumperenne and L. multiflorum ) in Ayrshire, Dumfriesshire, Lanarkshire, West Perthshire and Stirlingshire were surveyed in mid-summer in 1979 and 1980 for the presence of bacterial wilt. The preliminary survey in 1979 showed that the disease was present in approximately half of the fields in the area. A quantitative survey of 24 fields in 1980 revealed the disease in 71% of these fields at densities between 100 and 380,000 symptom-bearing tillers per hectare. Most disease was found in ryegrass, but occasionally diseased plants of cocksfoot ( Dactylis glomerata ) and timothy ( Phleum pratense ) were found. Tall and meadow fescue ( Festuca arundinacea and F. pratensis ) were also susceptible to infection in the laboratory (cut leaf and root dip inoculations). Ryegrass cultivars varied in their susceptibility to laboratory inoculation. Cultures of Xanthomanas campestris pv graminis showed no loss of virulence following freeze-drying and revival.     

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.     

Modelling the spread of Xanthomonas campestris pv. campestris in module-raised brassica transplants

A series of experiments was performed to quantify the rate of dispersal of Xanthomonas campestris pv. campestris in module-raised brassica transplants, in a simulated commercial plant-raising system. Seeds were sown in '308' module seed trays and set out in blocks in the glasshouse. Primary inoculum was introduced as inoculated seeds sown in one or more cells. Trays were watered via an overhead-gantry irrigation system, hand-watered or capillary-watered. Disease symptoms were monitored visually and the presence of the pathogen on samples of plants was monitored by leaf washing, dilution and plating on selective medium. Spread of symptoms was greatest in the gantry-watered trays, was very limited in hand-watered trays and was almost non-existent in capillary-watered trays. Dispersal of bacteria followed a similar pattern, but the proportion of plants contaminated was much greater than the proportion showing symptoms, and approached 100% after six weeks in the gantry-watered trays within 50 plants distance from a single primary infector. Models relating the proportion of plants with symptoms, or contaminated, to the distance from primary infector and time since sowing were fitted to the data. Predictions of the proportions of plants contaminated in commercial-scale blocks of transplants suggested that high levels of disease in the field could be explained by rapid rates of pathogen spread during plant-raising, and that the widely-used tolerance standard for seed health testing (0·01%) should be revised to 0·004%. In addition to seed health testing, control should focus on raising transplants under conditions that minimise the rates of disease spread and pathogen dispersal.     

Genetic Diversity of Xanthomonas campestris pv. vitians, the Causal Agent of Bacterial Leafspot of Lettuce

ABSTRACT Bacterial leafspot of lettuce (BLS), caused by Xanthomonas campes-tris pv. vitians, has become more prevalent in many lettuce-growing areas of the world over the past decade. To gain insight into the nature of these outbreaks, the genetic variation in X. campestris pv. vitians strains from different geographical locations was examined. All strains were first tested for pathogenicity on lettuce plants, and then genetic diversity was assessed using (i) gas-chromatographic analysis of bacterial fatty acids, (ii) polymerase chain reaction analysis of repetitive DNA sequences (rep-PCR), (iii) DNA sequence analysis of the internal transcribed spacer region 1 (ITS1) of the ribosomal RNA, (iv) restriction fragment length polymorphism (RFLP) analysis of total genomic DNA with a repetitive DNA probe, and (v) detection and partial characterization of plasmid DNA. Fatty acid analysis identified all pathogenic strains as X. campestris, but did not consistently identify all the strains as X. campestris pv. vitians. The rep-PCR fingerprints and ITS1 sequences of all pathogenic X. campestris pv. vitians strains examined were identical, and distinct from those of the other X. campestris pathovars. Thus, these characteristics did not reveal genetic diversity among X. campestris pv. vitians strains, but did allow for differentiation of X. campestris pathovars. Genetic diversity among X. campestris pv. vitians strains was revealed by RFLP analysis with a repetitive DNA probe and by characterization of plasmid DNA. This diversity was greatest among strains from different geographical regions, although diversity among strains from the same location also was detected. The results of this study suggest that these X. campestris pv. vitians strains are not clonal, but comprise a relatively homogeneous group.     

十字花科黑腐病菌hrp基因致病功能分析

通过对Xcc 8004菌株的hrp基因进行逐一敲除,系统研究单个hrp基因对Xcc致病性的贡献。结果表明,9个hrc(hypersensitive response and conserved)基因单独突变后在满身红萝卜上的致病性和在辣椒ECW-10R上激发HR的能力完全丧失;9个hrp基因中,hrpW突变后在辣椒ECW-10R上仍能产生HR,满身红萝卜上致病性减弱,其余hrp基因突变后致病性和过敏反应均丧失;4个hpa(hrp associated protein)基因突变后,hpaA和hpaB突变体完全丧失在满身红萝卜上的毒性也不能在辣椒ECW-10R上引起HR,hpa1和hpaP突变后致病性和HR显著减弱。另外,通过RT-PCR对Xcc 8004 hrp基因簇的每个基因受hrpX和hrpG的调控情况进行分析,结果表明所有hrp基因在不同程度上都受到hrpG、hrpX的正向调控。     

Movement of Xanthomonas campestris pv. vitians in the stems of lettuce and seed contamination

Xanthomonas campestris pv. vitians , the causal agent of bacterial leaf spot of lettuce (BLS), can be seedborne, but the mechanism by which the bacteria contaminates and/or infects lettuce seed is not known. In this study, the capacity of X. campestris pv. vitians to enter and translocate within the vascular system of lettuce plants was examined. The stems of 8- to 11-week-old lettuce plants were stab-inoculated, and movement of X. campestris pv. vitians was monitored at various intervals. At 4, 8, 12 and 16 h post-inoculation (hpi), X. campestris pv. vitians was recovered from 2 to 10 cm above (depending on stem length) and 2 cm below the inoculation site. Xanthomonas campestris pv. vitians was also recovered from surface-disinfested stem sections of spray-inoculated plants. Together, these results are consistent with X. campestris pv. vitians invading and moving systemically within the vascular system of lettuce plants. To investigate the mechanism of seed contamination, lettuce plants at the vegetative stage of growth were spray-inoculated with X. campestris pv. vitians and allowed to develop BLS. Seed collected from these plants had a 2% incidence of X. campestris pv. vitians external colonization, but no bacteria were recovered from within the seed.     

Genetic Diversity in Populations of Xanthomonas campestris pv. campestris in Cruciferous Weeds in Central Coastal California

ABSTRACT Xanthomonas campestris pv. campestris (X. campestris) infects a large number of cruciferous plants, including weeds. California has one of the largest and most diverse populations of wild cruciferous plants in the world. Although considerable information is available on the genetic diversity of X. campestris in commercial crop plants, nothing is known about the diversity in strains infecting weeds. To assess the genetic diversity among strains of X. campestris in weeds in noncultivated and cultivated areas, strains of the pathogen were isolated from populations of cruciferous weeds growing in coastal valley crop-production sites and from remote nonproduction sites along the California central coast. Results of fingerprinting over 68 strains using amplified fragment length polymorphism along with representative strains by sequence analysis showed the presence of seven genotypes. Genotypes A and B were limited to coastal sites; genotypes C, D, and E were from inland cultivated sites; and genotypes F and G were present in both coastal noncultivated and inland cultivated sites. Crop strains were grouped outside any weed strain group and were separated from the weed strains and other pathovars of X. campestris. These results revealed, for the first time, that strains of X. campestris present in noncultivated coastal weed populations generally were unique to a site and genetically distinct from strains present in populations of weeds in crop-production areas located nearby.     

Inheritance of Race-Specific Resistance to Xanthomonas campestris pv. campestris in Brassica Genomes

ABSTRACT The inheritance of resistance to three Xanthomonas campestris pv. campestris races was studied in crosses between resistant and susceptible lines of Brassica oleracea (C genome), B. carinata (BC genome), and B. napus (AC genome). Resistance to race 3 in the B. oleracea doubled haploid line BOH 85c and in PI 436606 was controlled by a single dominant locus (Xca3). Resistance to races 1 and 3 in the B. oleracea line Badger Inbred-16 was quantitative and recessive. Strong resistance to races 1 and 4 was controlled by a single dominant locus (Xca1) in the B. carinata line PI 199947. This resistance probably originates from the B genome. Resistance to race 4 in three B. napus lines, cv. Cobra, the rapid cycling line CrGC5, and the doubled haploid line N-o-1, was controlled by a single dominant locus (Xca4). A set of doubled haploid lines, selected from a population used previously to develop a restriction fragment length polymorphism map, was used to map this locus. Xca4 was positioned on linkage group N5 of the B. napus A genome, indicating that this resistance originated from B. rapa. Xca4 is the first major locus to be mapped that controls race-specific resistance to X. campestris pv. campestris in Brassica spp.     

Heterogeneity of Xanthomonas campestris pv. hederae Strains from Araliaceous Hosts

ABSTRACT Xanthomonas campestris pv. hederae (synonym X. hortorum pv. hederae) strains (59 total) were collected from plants in the araliaceae family. Strains were isolated from Hedera helix, Schefflera arboricola, Brassaia actinophylla, and Polyscias spp. from Florida, California, Hawaii, and New Zealand. All strains produced yellow mucoid growth; hydrolyzed esculin, starch, casein and gelatin; were pectolytic; produced urease; and grew on minimal media containing asparagine. All bacterial strains were pathogenic on H. helix (English ivy), B. actinophylla (dwarf schefflera), and Polyscias fruticosa (ming aralia). No differences in symptomatology were detected among strains; however, severity of symptoms usually was greatest on the host of origin. In planta growth rates of representative strains isolated from H. helix, B. actinophylla, and Polyscias spp. also were compared among these three hosts. In all cases, populations grew more rapidly when strains were inoculated to their original host species. All 59 bacterial strains were compared by 95-carbon source GN microplate, fatty acid methyl ester (FAME), and restriction fragment-length polymorphisms (RFLP), with the pulse-field gel electrophoresis method, analyses. All three analyses grouped strains into two distinct groups that correlated with the host of origin. Using metabolic profiles, 75% of the H. helix strains were separated from strains isolated from Brassaia and Schefflera and 95% of the Polyscias strains. FAME analysis separated strains into two distinct groups, with 96% of the H. helix strains placed in one group. RFLP analysis placed all of the H. helix and Schefflera strains in one group, as well as 33% of the Brassaia strains, whereas the other group contained all of the Polyscias strains and the remainder of the Brassaia strains. It is apparent that the pathovar hederae is made up of heterogeneous populations that can be separated by biochemical, pathological, genetic, and physiological analyses into two groups that are closely associated with the host of origin.     

低浓度Cu和Cd复合污染对小白菜生理生化特性的影响

采用盆栽试验,研究了不同质量浓度Cu(0,25,50,100,200 mg/kg)和Cd(0,1.0,10.0 mg/kg)及其复合污染对小白菜生理生化指标的影响。以不添加Cu和Cd为对照组。结果表明,小白菜外观正常,没有出现Cd中毒症状,但是Cd-Cu复合处理组小白菜的生物量、叶绿素a、叶绿素总量、叶绿素a/b值都低于对照及单一处理组,Cu、Cd复合处理对叶绿素a的影响程度大于叶绿素b;复合处理组的细胞膜透性明显低于单一处理组,硝酸还原酶(NR)活性显著高于对照及单一处理组,过氧化物酶(POD)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)活性高于相同浓度Cd单一处理组。     

The dynamics of homologous and heterologous interactions between rice and strains of Xanthomonas campestris

Lesion development, bacterial multiplication and spread were measured in leaves of cultivars of rice containing different Xa (resistance) genes, following inoculation with different races of the bacterial leaf blight pathogen. Xanthomonas campestris pv. oryzae. Both compatible and incompatible races possessed the ability to colonize rice plants. The difference between compatible and incompatible host pathogen combinations appeared to be mainly in symptom production since multiplication rates and spread were very similar until after the onset of symptoms. No form of HR (hypersensitive response) was observed. The ability of incompatible races to modify host reaction in dual-inoculation was dependent on the genotype of the host plant. The heterologous non-pathogen of rice X. campestris pv. campestris produced few symptoms, failed either to multiply or spread within rice leaves and was unable to induce any marked cross-protection against homologous pv. oryzae strains in dual-inoculation experiments.     

Resistance to Xanthomonas campestris pv. campestris (Pammel) Dowson in cabbage Brassica oleracea L

Journal of Plant Diseases and Protection - Xanthomonas campestris pv. campestris (Xcc) is one of the most important pathogens of Brassica oleracea vegetables. To develop resistant basic breeding...