Fingerprinting of Xanthomonas campestris pv. pelargonii and Related Pathovars Using Random-Primed PCR

Polymerase chain reaction (PCR) amplification of total DNA was evaluated as a method to distinguish Xanthomonas campestris pv. pelargonii from other pathovars within this species. Two sets of highly conserved enterobacterial consensus sequences were used as targets for PCR amplification: (a) enterobacterial repetitive intergenic consensus [ERIC] and (b) repetitive extragenic palindromic [REP] sequences. Nucleic acid was extracted from a total of 37 isolates of bacteria: 19 isolates ofX campestris pv. pelargonii and 18 isolates representing 10 other pathovars of X. campestris. After PCR amplification using the ERIC primer pair the DNA fingerprints of X. campestris pv, pelargonii contained two major DNA products (estimated size 500 and 740 pp) that were conserved among all 19 isolates. With the REP primer pair, the fingerprints were more complex and major DNA products ranging from -690 to 1650 bp were detected. Using information from both ERIC- and REP-primed Imgerprints, the X. campestris pv. pelargonii fingerprints were distinguishable from the fingerprints of the other pathovars examined: pvs. citrumelo. citri, beganiae, vittans B and C. phaseoli. campestris, manihotis, juglandis, carotae and pruni.     

核桃黑斑病拮抗放线菌WMF106的筛选、鉴定及防效

【背景】核桃黑斑病是由2种病原菌引起的细菌性病害,目前缺乏有效的生物防治方法。【目的】从核桃树根际土壤中筛选对核桃黑斑病病原菌具有拮抗效果的放线菌菌株,为该病害生防菌剂的开发提供基础。【方法】采用稀释涂布法分离放线菌,并以病原菌野油菜黄单胞菌(Xanthomonas campestris pv. campestris)和成团泛菌(Pantoea agglomerans)作为指示菌,利用平板对峙法和改良牛津杯法筛选具有高拮抗活性的菌株,通过形态学特征、生理生化特性和16SrRNA基因序列分析确定其分类地位,并测定其无菌发酵液的抗菌谱和室内防效。【结果】筛选到一株对野油菜黄单胞菌和成团泛菌均有较强拮抗作用的放线菌菌株WMF106,该菌株对2种病原菌的抑菌圈直径分别为2.38 cm和1.82 cm,无菌发酵液对2种病原菌的抑菌圈直径分别为1.75 cm和1.55 cm。根据菌株形态学、生理生化特性及16SrRNA基因序列分析,将菌株WMF106鉴定为暗蓝色链霉菌(Streptomyces caeruleatus)。该菌株对尖孢镰刀菌、腐皮镰孢菌、辣椒刺盘孢菌、灰葡萄孢菌、胶孢炭疽菌5种植物病原菌及大肠杆菌、金黄色葡萄球菌、铜绿假单胞菌、白色念珠菌、黑曲霉5种指示菌均有抑制作用,抗菌性能广谱高效,其无菌发酵液原液对离体叶片上由野油菜黄单胞菌和成团泛菌造成的核桃黑斑病防效分别为77.44%和58.33%。【结论】菌株WMF106可作为防治核桃黑斑病的生防材料,具有良好的开发价值和应用前景。     

Pepper Tree (Schinus terebenthifolius Radii), a New Host Plant for Xanthomonas campestris pv. mangiferaeindicae

Apigmented bacterial colonies were obtained in Reunion Island from angular leaf lesions on Pepper tree (Schinus terebenthifolius Radii), a member of Anacardiaceae. All isolates were identified as Xanthomonas campestris, using physiological and biochemical tests. These strains were reinoculated to Pepper tree leaves, and Koch postulates were verified. Furthermore, they were inoculated to mango leaves and produced lesions identical to those induced by Xanthomonas campestris pv. mangiferaeindicae, the causal agent of bacterial black spot of mangoes. Apigmented and pigmented strains of X. c. pv. mangiferaeindicae from Mango and Ambarella were pathogenic to Pepper tree. Strains isolated from Pepper tree were compared to X. c. pv. mangiferaeindicae, by means of phenotypic features (utilization of 147 carbon sources) and using a serological assay. A high homology among the strains was observed. Thus, It is concluded that strains isolated from Pepper tree belong to pv. mangiferaeindicae, and that Pepper tree is a host species for X. c. pv. mangiferaeindicae.     

Genomic Relatedness of Xanthomonas campestris Strains Causing Diseases of Citrus

Xanthomonas campestris strains that cause disease in citrus were compared by restriction endonuclease analysis of DNA fragments separated by pulsed-field gel electrophoresis and by DNA reassociation. Strains of X. campestris pv. citrumelo, which cause citrus bacterial spot, were, on average, 88% related to each other by DNA reassociation, although these strains exhibited diverse restriction digest patterns. In contrast, strains of X. campestris pv. citri groups A and B, which cause canker A and canker B, respectively, had relatively homogeneous restriction digest patterns. The groups of strains causing these three different citrus diseases were examined by DNA reassociation and were found to be from 55 to 63% related to one another. Several pathovars of X. campestris, previously shown to cause weakly aggressive symptoms on citrus, ranged from 83 to 90% similar to X. campestris pv. citrumelo by DNA reassociation. The type strain of X. campestris pv. campestris ranged from 30 to 40% similar in DNA reassociation experiments to strains of X. campestris pv. citrumelo and X. campestris pv. citri groups A and B. Whereas DNA reassociation quantified the difference between relatively unrelated groups of bacterial strains, restriction endonuclease analysis distinguished between closely related strains.     

Isolation and characterization of bacteriophages infecting <Emphasis Type="Italic">Xanthomonas arboricola</Emphasis> pv. <Emphasis Type="Italic">juglandis</Emphasis>, the causal agent of walnut blight disease

Walnut orchards suffer from a blight caused by the bacteria Xanthomonas arboricola pv. juglandis. These bacteria can be infected by viral bacteriophages and this study was carried out to isolate and characterize bacteriophages from walnut orchards located throughout the South Island of New Zealand. Twenty six X. arboricola phages were isolated from three hundred and twenty six samples of plant material representing phyllosphere and rhizosphere ecosystems. The phage isolates were characterized by host-range, plaque and particle morphology, restriction digest and phylogenetic analysis and stability under various storage conditions. From capsid and tail dimensions the bacteriophages were considered to belong to the double-stranded DNA families Podoviridae and Siphoviridae. Of the twenty six bacteriophages, sixteen belonged to Podoviridae and were found both in the phyllosphere and rhizosphere. In contrast, Siphoviridae were present only in the rhizosphere isolates. Phage genome sizes ranged from 38.0 to 52.0 kb from a Hind III restriction digestion and had in common a 400 kb fragment that was identical at the DNA level. Despite the similar restriction patterns, maximum parsimony bootstrap analysis showed that the phage were members of different groups. Finally, we hypothesise that these phage might have use in a biocontrol strategy and therefore storage stability and efficacy was tested. Titres declined more than 50% over a 12-months storage period. Deep-freezing temperatures (−34°C) increased while chloroform decreased the stability.     

Genetic Diversity of Xanthomonas arboricola pv. fragariae Strains and Comparison with some other X. arboricola Pathovars using Repetitive PCR Genomic Fingerprinting

The genetic relationship within 26 Xanthomonas arboricola pv. fragariae strains and between this pathovar and 20 strains of X. arboricola pv. corylina, 22 strains of X. arboricola pv. juglandis and 16 strains of X. arboricola pv. pruni has been assessed by means of repetitive polymerase chain reaction (rep‐PCR) using Enterobacterial Repetitive Intergenic Consensus), BOX (BOXA subunit of the BOX element of Streptococcus pneumoniae) and repetitive extragenic palindromic primer sets. Cluster analysis was performed by means of unweighted paired group method using arithmetic average (UPGMA). Upon rep‐PCR and UPGMA cluster analysis, a relevant genetic diversity was found within the strains. The overall similarity, however, was high (i.e. 80%). The four X. arboricola pathovars showed similar but clearly different genomic patterns and clustered into four different groups, with X. arboricola pv. corylina and X. arboricola pv. juglandis more closely related to X. arboricola pv. fragariae. Representative strains of X. arboricola pv. fragariae and the putative xanthomonads isolated from strawberry leaves showing leaf blight symptoms underwent pathogenicity tests. After artificial inoculation, X. arboricola pv. fragariae induced necrotic spots accompanied, sometimes, by a chlorotic halo. The blackening of the leaf veins and peduncle was, sometimes, also observed. The four putative xanthomonads isolated from diseased strawberry leaves and not inducing symptoms after artificial inoculation, clustered apart from X. arboricola pathovars.     

Systemic Invasion of Plum Seed and Fruit by Xanthomonas campestris pv. pruni through Stalks

Many fruits on Golden King plum trees inoculated through the stalks with Xanthomonas campestris pv. pruni developed unusual lesions extending from the exocarp to the endocarp. A few uninoculated, diseased fruits had similar lesions. The pathogen was isolated from both inoculated and uninoculated stalks and from seeds inside fruits. Scanning electron microscopy of inoculated stalks and mature fruits with unusual lesions revealed that vascular channels of the stalk, seed coat, stony endo, carp, and mesocarp were filled with masses of X. campestris pv. pruni. Bacterial colonies also occurred in other tissues of these fruit parts but were apparently absent from the starchy endosperm or surface of the diseased exocarp. This is the first full report of systemic movement of X. campestris pv. pruni to seed and fruit through stalks.     

Differentiation between Xanthomonas campestris pv. graminis (ISPP List 1980), pv. phleipratensis (ISPP List 1980) emend., pv. poae Egli and Schmidt 1982 and pv. arrhenatheri Egli and Schmidt 1982, by Numerical Analysis of Phenotypic Features and Protein Gel Electrophoregrams

A numerical analysis of 257 phenotypic features of 45 bacterial isolates from grasses, revealed three phenons corresponding to (i) X. campestris pv. graminis (ISPP List 1980), (ii) X. campestris pv. phleipratensis (ISPP List 1980) and (iii) X. campestris pv. poae Egli and Schmidt 1982 and X. campestris pv. arrhenatheri Egli and Schmidt 1982. In each phenon, the strains clustered together regardless of the geographical origin of the isolates orthe year of isolation. Polyacrylamide gel electrophoresis of soluble proteins and host range studies, revealed four groups corresponding to the pathovars mentioned above. The four pathovars constitute definite biological entities that can be differentiated by phenotypic, gel electrophoretic and host range features.     

Variability of Xanthomonas Campestris pv. vasculorum From Sugarcane and Other Gramineae in Reunion Island. Characterization of a Different Xanthomonad

Strains presumed to be of Xanthomonas campestris pv. vasculorum (Cobb.) Dye, obtained from sugarcane and other gramineae in Réunion Island, were compared in terms of cultural aspects, pathogenic and physiological reactions, fatty acid profiles and restriction fragment length polymorphism (RFLP) of genomic DNA. The strains could be divided into two separate groups (G1 and G2). The G1 strains were identical to strains described as X. campestris pv. vasculorum; they showed an important variability in their cultural characteristics and in their aggressiveness. The G2 strains did not induce the usual symptoms of gumming disease on sugarcane cultivars infected under natural conditions or inoculated in the greenhouse. The G2 strains grew faster on agar medium, their colonies were more pigmented and less fluidal and had a different morphology on agar slant. Unlike the G1 strains, G2 strains hydrolyzed starch weakly and casein strongly; they utilized L-fucose and, to a lesser extent, melibioze. The fatty acid and genomic DNA profiles differed between the groups. Differences seemed large enough to support separation of G1 and G2 strains into distinct taxonomic entities, namely G1 as Xanthomonas campestris pv. vasculorum and G2 as a different pathovar of X. campestris. The taxonomic position of G2 strains is discussed.     

Variability of Xanthomonas Campestris pv. vasculorum From Sugarcane and Other Gramineae in Reunion Island. Characterization of a Different Xanthomonad

Stains presumed to be of Xanthomonas campestris pv. vasculorum (Cobb.) Dye, obtained from sugarcane and other gramineae in Réunion Island, were compared in terms of cultural aspects, pathogenic and physiological reactions, fatty acid profiles and restriction fragment length polymorphism (RFLP) of genomic DNA. The strains could be divided into two separate groups (G1 and G2). The G1 strains were identical to strains described as X. campestris pv. vasculorum; they showedan important variability in their cultural characteristics and in their aggressiveness. The G2 strains did not induce the usual symptoms of gumming disease on sugarcane cultivars infected under natural conditions or inoculated in the greenhouse. The G2 strains grew faster on agar medium, their colonies were more pigmented and less fluidal and had a different morphology on agar slant. Unlike the G1 strains, G2 strains hydrolyzed starch weakly and casein strongly; they utilized L-fucose and, to a lesser extent, melibioze. The fatty acid and genomic DNA profiles differed between the groups. Differences seemed large enough to support separation of G1 and G2 strains into distinct taxonomic entities, namely G1 as Xanthomonas campestris pv. vasculorum and G2 as a different pathovar of X. campestris. The taxonomic position of G2 strains is discussed.     

Quantitative analysis of resistance in cotton to three new isolates of the bacterial blight pathogen

Summary Genetic variability for virulence of the bacterial blight pathogen [Xanthomonas campestris pv malvacearum (Smith) Dye] on cotton (Gossypium hirsutum L.) has been shown by the identification of 19 races of the pathogen based on disease reactions of a set of ten host differentials. This study was conducted to determine the inheritance of host resistance to three recently identified isolates of X. campestris pv malvacearum, which are virulent on the entire set of differentials. True leaves of Tamcot CAMD-E, LEBOCAS-3-80, Stoneville 825, and their f₁, F₂, and backcross progenies were wound-inoculated in the field with separate bacterial suspensions of the virulent HV3, HV7, and Sudan isolates of the pathogen. LEBOCAS-3-80 was replaced with S295, a new immune cultivar, for a greenhouse study in which both cotyledons and true leaves were inoculated. Disease reactions were rated on a scale of 1–10, and genetic models were proposed utilizing generation means analysis. Dominance, when significant, was in the direction of resistance in all but one cross-isolate combination. Digenic interaction components indicated a duplicate type. Narrow-sense heritability for resistance ranged from 0.59 to 0.68; therefore, primarily additive-genetic variability among the selected cutlivars was detected, indicating that breeding for improved resistance to these isolates is a practical goal.Contribution of the Department of Soil and Crop Sciences and the Texas Agricultural Experiment Station     

Bacterial Streak Disease of Maize (Zea mays L.) in South Africa

Bacterial streak disease of maize is currently causing some concern among breeders in South Africa. The causal organism of this previously undescribed disease was successfully isolated and its pathogenicity established using KoCH's postulates. Standard physiological and biochemical tests used to identify phytopathogenic bacteria indicated that the bacterium is a Xanthomonas campestris pathovar. Comparisons between this organism and other recognized X. campestris pathovars of the Poaceae indicated that apart from some minor differences the maize streak pathogen is physiologically similar to X. campestris pv. holcicola. However, in repeated reciprocal inoculation experiments all attempts to induce disease symptoms in sorghum with the maize streak pathogen were unsuccessful. Conversely, X. campestris pv. holcicola did produce symptoms in maize leaves. In all the maize cultivars tested the symptoms produced by the maize streak pathogen were, however, always considerably more severe than those caused by X. campestris pv. holcicola. Notwithstanding its physiological similarity to X. campestris pv. holicola it would appear that on the grounds of host specificity the maize streak pathogen warrants new pathovar status. The name X. campestris pv. zeae is proposed.     

Characterization of Xanthomonas campestris Pathovars by rRNA Gene Restriction Patterns

Genomic DNA of 191 strains of the family Pseudomonadaceae, including 187 strains of the genus Xanthomonas, was cleaved by EcoRI endonuclease. After hybridization of Southern transfer blots with 2-acetylamino-fluorene-labelled Escherichia coli 16+23S rRNA probe, 27 different patterns were obtained. The strains are clearly distinguishable at the genus, species, and pathovar levels. The variability of the rRNA gene restriction patterns was determined for four pathovars of Xanthomonas campestris species. The 16 strains of X. campestris pv. begoniae analyzed gave only one pattern. The variability of rRNA gene restriction patterns of X. campestris pv. manihotis strains could be related to ecotypes. In contrast, the variability of patterns observed for X. campestris pv. malvacearum was not correlated with pathogenicity or with the geographical origins of the strains. The highest degree of variability of DNA fingerprints was observed within X. campestris pv. dieffenbachiae, which is pathogenic to several hosts of the Araceae family. In this case, variability was related to both host plant and pathogenicity.     

Systemic Migration and Establishment of Xanthomonas campestris pv. pruni in Plum Leaves and Twigs

The systemic migration of Xanthomonas campestris pv. pruni (Xcp) through vascular bundles of leaves and twigs of plum was investigated. A rifampicin-resistant strain of Xcp was inoculated into leaves located midway from the tip of new green twigs of Golden King plum trees in a glasshouse. High numbers of the pathogen were recovered 4 and 8 weeks after inoculation from sections of uninoculated and symptomless veins, petioles, and twig tissue. Symptoms of bacterial spot developedwithin 8 weeks on main and secondary veins of uninoculated leaves located as far as 13 cm from the inoculated leaf on the same twig. Weekly isolation indicated the constant presence of Xcp in apparently unaffected shields of twig tissue obtained from a naturally infected Golden King orchard. Xcp apparently enters plum twigs through veins of infected leaves and migrates systemically through twigs to leaves.     

Pathological and Molecular Characterization of Xanthomonas campestris Strains Causing Diseases of Cassava (Manihot esculenta)

Fifty-one strains representing Xanthomonas campestris pv. manihotis and cassavae and different pathovars occurring on plants of the family Euphorbiaceae were characterized by ribotyping with a 16S+23S rRNA probe of Escherichia coli and by restriction fragment length polymorphism analysis with a plasmid probe from X. campestris pv. manihotis. Pathogenicity tests were performed on cassava (Manihot esculenta). Histological comparative studies were conducted on strains of two pathovars of X. campestris (vascular and mesophyllic) that attack cassava. Our results indicated that X. campestris pv. manihotis and cassavae have different modes of action in the host and supplemented the taxonomic data on restriction fragment length polymorphism that clearly separate the two pathovars. The plasmid probe could detect multiple restriction fragment length polymorphisms among strains of the pathovar studied. Ribotyping provides a useful tool for rapid identification of X. campestris pathovars on cassava.     

First report of bacterial leaf blight of jute (Corchorus capsularis L.) caused by Xanthomonas campestris pv. capsularii in India

Xanthomonas campestris pv. capsularii causing blight on jute (Corchorus capsularis) leaves was reported for the first time in India. The symptom of the disease initially observed was appearance of small angular brown leaf spots and later as blighted areas on leaf lamina. The disease-causing pathogen was isolated and identified on the basis of its colony morphology, PCR, sequencing and subsequent BLASTn analysis.     

Cross-reactive Antigens between Xanthomonas campestris pv. citri Pathotypes and Citrus Species

Cross-reactive antigens were detected in crude and semi-purified preparations from acetone powder of Citrus aurantifolia and Citrus sinensis leaves with antisera to Xanthomonas campestris pv. citri pathotypes A and C by DAS-ELISA. Antiserum to X. campestris pv. citri pathotype C revealed an antigenic disparity between C. aurantifolia (susceptible host to pathotype C) and C. sinensis (resistant host to pathotype C) whereas antiserum to X, campestris pv. citri pathotype A did not reveal any antigenic disparity between these hosts, both susceptible to pathotype A. The occurrence of “key” cross-reactive antigens in Citrus species and X. campestris pv. citri and their possible involvement in such interaction are discussed.     

Isolation of a Plasmid from Xanthomonas campestris pv. vignicola

A modified method is described for isolating high yields of plasmids without chromosome contamination from Xanthomonas campestris pv. vignicola (X. c. pv. vignicola), the causal agent of blight disease in Vigna species and also in Phaseolus vulgaris. Applying this method a plasmid of X, c. pv. vignicola was detected representing an estimated molecular weight of 95 megadaltons. Heat curing of the strain revealed that the detected plasmid had no effecton virulence but seemed to influence colony morphology.     

Phenotypic Switching Affecting Chemotaxis, Xanthan Production, and Virulence in Xanthomonas campestris

The chemotaxis towards sucrose and yeast extract of nine strains of Xanthomonas campestris representing pathovars campestris, armoraciae, translucens, vesicatoria, and pelargonii was analyzed by using swarm plates. Unexpectedly, each of these strains formed small or reduced swarms typical of nonmotile or nonchemotactic bacteria. With time, however, chemotactic cells appeared on the swarm plates as blebs of bacteria. These cells were strongly chemotactic and were concomitantly deficient in exopolysaccharide production. The switch from the wild type (exopolysaccharide producing and nonchemotactic) to the swarmer type (exopolysaccharide deficient and chemotactic) appeared irreversible ex planta in bacteriological medium. However, in radish leaves swarmer-type strains of X. campestris pv. campestris were able to revert to the wild type. Swarmer-type derivatives of two X. campestris pv. campestris wild-type isolates showed reduced virulence and growth in the host plants cauliflower and radish. However, exocellular complementation of X. campestris pv. campestris Hrp (nonpathogenic) mutant was achieved by coinoculation with a swarmer-type strain.