Variation in Albicidin Biosynthesis Genes and in Pathogenicity of Xanthomonas albilineans, the Sugarcane Leaf Scald Pathogen

ABSTRACT Total genomic DNA from 137 strains of Xanthomonas albilineans from worldwide locations was hybridized with two DNA probes that together harbor the entire 49-kb albicidin biosynthesis gene cluster and two additional 3-kb genomic regions required for albicidin production. Fourteen haplotypes and two major genetic groups (albicidin [ALB]-restriction fragment length polymorphism [RFLP] A and ALB-RFLP B) were identified, and strains that were isolated after recent outbreaks of leaf scald disease belonged to group ALB-RFLP B. Albicidin genetic diversity was very similar to the previously described genetic diversity of the pathogen based on the whole genome. No relationship was found between variability of albicidin biosynthesis genes and the amount of albicidin produced in vitro by X. albilineans. Leaf scald-susceptible sugarcane cv. H70-144 was inoculated with 20 strains of the pathogen belonging to different ALB-RFLP haplotypes. Among them, 10 strains from Guadeloupe belonged to the same ALB-RFLP group but differed in the amount of albicidin produced in vitro. Strains were distributed in at least three different pathogenicity groups based on symptom severity and pathogen population density in the stalk. These two pathogenicity factors varied concurrently; however, no relationship between variation in albicidin biosynthesis genes, variation in the amount of albicidin produced in vitro, and variation in pathogenicity of X. albilineans was found. Further investigation is necessary to identify other genes involved in pathogenicity of X. albilineans.     

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.     

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.     

Serological variability in Xanthomonas albilineans, causal agent of leaf scald disease of sugarcane

The serological characteristics of 215 strains of Xanthomonas albilineans from 28 locations throughout the world were investigated. Three serovars were identified by using three sera prepared against strains from different locations. The same variation among strains was observed with different serological assays: immunofluorescence. dot-blot enzyme immunoassay and agglutination. Serovar I is the largest group and consists of strains from various geographic locations such as Australia. USA (Florida), Guadeloupe. India. Mauritius. Réunion and including South Africa. Serovar II contains only strains from Africa: Burkina Faso. Cameroon. Kenya. Ivory Coast. Serovar III is the smallest group and contains strains from Caribbean islands (Guadeloupe. Saint Kitts. Martinique). Oceania (Fiji) and Asia (Sri Lanka). This serological variability should be considered when serological techniques are applied for the diagnosis of leaf-scald disease of sugarcane.     

植物病原菌黄单胞菌的分类研究进展

黄单胞菌是一类重要的植物病原细菌。由于其经济重要性,对该属细菌进行了大量的研究,方法包括DNA杂交、基于代表性序列的多聚酶链式反应技术和扩增片段长度多态性(AFLP)基因组指纹图谱技术等。本文主要介绍了黄单胞菌属的分类研究进展。     

Synopsis on the taxonomy of the genus xanthomonas

ABSTRACT The genus Xanthomonas exhibits a high phytopathogenic diversity in contrast to a phenotypic uniformity, which has hampered the genesis of a stable classification for a long time. In past decades, a large number of Xanthomonas strains have been characterized by a variety of phenotypic and genotypic methods in a multitude of studies. Extensive DNA hybridization studies and repetitive sequence-based polymerase chain reaction and amplified fragment length polymorphism genomic fingerprinting have clearly revealed the genomic diversity and relationships within the genus. A review of the current classification of the genus Xanthomonas based on the synopsis of these studies is given here.     

Polyphasic characterization of Xanthomonas axonopodis pv. allii associated with outbreaks of bacterial blight on three Allium species in the Mascarene archipelago

Based on the number of new reports during the last two decades, bacterial blight of onion (Allium cepa) is considered an emerging disease. The causal agent, Xanthomonas axonopodis pv. allii, is pathogenic to several Allium species after inoculation, but outbreaks worldwide have been primarily reported on onion. We describe a unique epidemiological situation in Réunion Island, France, with concomitant outbreaks on three Allium species, onion, leek (A. porrum), and garlic (A. sativum). There was no host specialization within Allium spp. among strains associated with the three host species. Based on amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism, strains associated with these outbreaks in Réunion Island were highly related genetically to strains isolated from diseased plant samples and contaminated seed lots in the neighboring island of Mauritius, where the disease has occurred since 1984. All AFLP haplotypes were identified as X. axonopodis pv. allii based on polymerase chain reaction analysis using specific primers, biochemical tests, and/or pathogenicity tests. Two genetically related groups of strains (A and B) that can be distinguished by AFLP, differential utilization of three carbon sources, and xanthomonadin pigment production were detected initially after establishment of the pathogen. In less than 10 years after the establishment of the pathogen there was nearly an extinction of group A strains in Réunion Island, suggesting differences in fitness between strains in the two groups.     

Distribution of Xanthomonas oryzae pv. oryzae Strains Virulent to Xa21 in Korea

ABSTRACT Strains of Xanthomonas oryzae pv. oryzae that are virulent to rice lines carrying the Xa21 resistance gene were widely distributed in Korea. A total of 105 strains collected during 1987 to 1996 in Korea was characterized by pathogenicity tests and restriction fragment length polymorphism analysis of the XorII methyltransferase (xorIIM) and avrXa10 genes. Although the lesion lengths on rice line IRBB21, which carries Xa21, decreased as plant age increased, resistance and susceptibility of the plants to 31 strains were clearly differentiated at the seedling (14, 21, and 28 days old), maximum tillering, and flag leaf stages. The resistance or susceptibility of seedlings was correlated with bacterial populations within an inoculated leaf. There was a significant change in the population structure of X. oryzae pv. oryzae with regard to virulence to Xa21 over the last 10 years; this change in population was confirmed by genome analysis. Lineage I, which is avirulent to Xa21 and does not have a genomic xorIIM homolog, was the predominant lineage found between 1987 and 1989, while lineage II, which is virulent to Xa21 and contains the xorIIM homolog, was predominant in strains collected between 1994 and 1995. Our results demonstrate that introduction of Xa21 into commercial rice should be based on the regional structure of X. oryzae pv. oryzae populations and suggest that Xa21 will not be useful in Korea.     

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.     

Chromosomal Gene Transfer by Conjugation in the Plant Pathogen Xanthomonas axonopodis pv. vesicatoria

ABSTRACT Genes for copper resistance, located on the chromosome of strain XvP26 of Xanthomonas axonopodis pv. vesicatoria, were transferred by conjugation to a recipient strain of the bacterium. The chromosomal gene transfer was verified by analyses of the genomes of donor, recipient, and putative transconjugants for plasmid profiles, by polymorphism of DNA bands obtained by digesting total genomic DNA by a rare-cutting endonuclease and pulsed-field gel electrophoresis, and by Southern hybridization with a probe containing the copper genes. Transfer of kanamycin resistance to a recipient strain, associated with Tn5 insertion into the chromosome of another strain of the bacterial spot pathogen, was also verified. The frequency of kanamycin resistance transfer to recipient was more than 75 times greater in pepper leaves than in vitro. The transfer of chromosomal sequences containing the hypersensitive reaction and pathogenicity (hrp) genes and pigmentation (pig) genes was linked with transfer of kanamycin resistance (Tn5). Horizontal transfer in planta of the chromosomal genes (i.e., cop, pig, hrp, and Tn5 sequences) among strains of X. axonopodis pv. vesicatoria means that horizontal chromosomal gene transfer is possible in nature. This type of gene transfer may explain the presence of great diversity among strains of the bacterial spot pathogen in terms of DNA polymorphism and may also explain the apparent horizontal transfer of hrp sequences among pathovars of Xanthomonas.     

Differentiation of three species of Xanthomonas and Stenotrophomonas maltophilia using cellular fatty acid analyses

Five hundred eighty-eight strains, representing Xanthomonas albilineans, X. fragariae, ten pathovars of X. campestris, and Stenotrophomonas maltophilia from ornamentals, were subjected to fatty acid methyl ester (FAME) analyses. Quantitative variance among FAME profiles enabled identification of the four species with 100% accuracy. Dendrogram cluster analysis placed strains of X. albilineans remotely from those of the other two Xanthomonas species and S. maltophilia. Whereas some profiles of pathovars of X. campestris were distinct, strains within X. albilineans, X. fragariae, and S. maltophilia were homogeneous by their conserved FAME ratios. Pathovars of X. campestris that had conserved profiles were fittonia, hederae, malvacearum, pelargonii, and zinniae. FAME profiles of X. campestris pathovars begoniae, dieffenbachiae, fici, maculifoliigardeniae, and poinsettiicola were, however, quantitatively diverse. These pathovars did not form discrete subgroups, and intercalated randomly with one another on the dendrogram. Certain species or pathovars of X. campestris which have homogeneous FAME profiles can easily be identified with fatty acid analysis; however, pathovars of X. campestris with heterogeneous profiles are not readily identified by fatty acid analysis.     

Differentiation of Xanthomonas albilineans strains with monoclonal antibody reaction patterns and DNA fingerprints

Thirty-eight strains of the sugarcane leaf scald pathogen, Xanthomonas albilineans , were compared using a series of seven species-specific monoclonal antibodies as well as genomic DNA fingerprint patterns. The strains, which were obtained from 13 countries and states in the USA, were placed in three major groups with approximately eight subgroups based on similarity of their serological reactions. The serological groupings correlated strongly with groupings based on DNA fingerprint patterns. The groups of strains that were genetically and serologically related did not necessarily come from nearby locations but were usually from widely separated regions. Thus, it appears that disease transmission occurred by means other than natural spread. The methods used in this study are useful for comparing relationships among strains of X. albilineans . A combination of the monoclonal antibodies could be used in a serodiagnostic-based method to test for the presence of leaf scald disease.     

Classification and Identification of Xanthomonas translucens Isolates, Including Those Pathogenic to Ornamental Asparagus

ABSTRACT In order to confirm and refine the current classification scheme of Xanthomonas translucens and to identify novel strains from ornamental asparagus, a collection of field and reference strains was analyzed. Rep-polymerase chain reaction (PCR) genomic fingerprint profiles were generated from 33 isolates pathogenic to asparagus as well as 61 X. trans-lucens reference strains pathogenic to cereals and grasses. Amplified ribo-somal gene restriction analysis profiles were obtained from most of these and 29 additional Xanthomonas reference strains. Rep-PCR genomic fingerprint profiles of all strains were compared with those in a large Xanthomonas database using computer-assisted analysis. Rep-PCR ge-nomic fingerprinting facilitated the characterization and discrimination of X. translucens, including the pathovars arrhenatheri, graminis, phlei, phleipratensis, and poae, as well as a number of strains received as X. translucens pv. cerealis. Strains received as pathovars hordei, secalis, translucens, undulosa, and other cerealis strains were grouped in two subclusters that correspond to the recently redefined pathovars X. trans-lucens pvs. undulosa and translucens. All 33 novel isolates from ornamental asparagus (tree fern; Asparagus virgatus) were identified as X. translucens pv. undulosa. Moreover, a unique amplified small subunit ribosomal gene MspI/AluI restriction profile specific for all X. translucens strains tested, including those pathogenic to asparagus, allowed discrimination from all other Xanthomonas spp. Although phage tests were inconclusive, the classification of the asparagus strains within the X. translucens complex was supported by pathogenicity assays in which all the isolates from ornamental asparagus induced watersoaking on wheat. Surprisingly, several X. translucens reference strains affected asparagus tree fern as well. That the novel asparagus isolates belong to X. translucens pv. undulosa is extraordinary because all hosts of X. translucens pathovars described to date belong only to the families Gramineae and Poaceae, whereas asparagus belongs to the phylogenetically distant family Liliaceae.     

Xanthomonas translucens from Small Grains: Diversity and Phytopathological Relevance

ABSTRACT Sixty-eight presumptive Xanthomonas translucens strains isolated from 15 small grains or grass species were studied by pathogenicity tests on barley, bread wheat, oat, and bromegrass species, and also by AFLP, analysis of fatty acid methyl esters (FAME), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of protein extracts. The X. translucens strains were divided into three pathogenicity types based on differences observed on barley and bread wheat. Two unspeciated strains producing atypical symptoms formed a fourth pathogenicity type. Pathogenicity on oat and bromegrass species varied within these types. Clusterings observed by AFLP analysis and, to a lesser extent, by FAME analysis were consistent with these pathogenicity groupings. The current results, as well as those of previous restriction fragment length polymorphism analyses of the same strains, support the recent reclassification of X. translucens pv. translucens and X. translucens pv. hordei as true synonyms. X. translucens pv. cerealis, X. translucens pv. translucens, and X. translucens pv. undulosa cluster in different groups by AFLP and FAME analyses. Even though distinction by simple biochemical tests is not clear-cut, the data indicate that the pathovars cerealis, translucens, and undulosa correspond to true biological entities.     

Multiphasic analysis of xanthomonads causing bacterial spot disease on tomato and pepper in the Caribbean and central america: evidence for common lineages within and between countries

ABSTRACT Four hundred thirty-three xanthomonad strains isolated from tomato or pepper plants from 32 different fields in four Caribbean and Central American countries were screened for the ability to hydrolyze starch and sodium polypectate and for resistance to copper and streptomycin. Of these, 95 representative strains were further characterized by various phnetic tests, and 63 of these strains were then analyzed by genomic fingerprinting. Most of the strains (>90%) were tolerant to copper. However, there was much more variability in sensitivity to streptomycin. All strains in Guadeloupe and 93% of the strains in Barbados were sensitive to streptomycin. The majority of strains were typical Xanthomonas campestris pv. vesicatoria group A strains. In Barbados, however, a unique group of strains was identified that was serologically similar to group A strains but was amylolytic. These strains were designated A1. The occurrence of X. campestris pv. vesicatoria group B strains in Central America was found to be limited to two fields in Costa Rica and one in Guatemala. No group B strains were identified in the Caribbean, in contrast to common occurrence in the central United States and in South America. T3 strains were not found in this study, despite the recent increase of such strains in Florida and Mexico. Unique strains from Costa Rica belonging to the X. gardneri group were identified. Little linkage was found among phenotypic and rep-polymerase chain reaction (rep-PCR) genomic fingerprinting profiles of the pathogens except at the species/pathovar level; strains displaying virtually identical fingerprint profiles were found to correspond to distinct races and vice versa. The rep-PCR genomic fingerprinting analyses suggest that certain lineages may have evolved or predominated in specific regions or specific countries.     

Comparison of PCR, BIO-PCR, DIA, ELISA and isolation on semiselective medium for detection of Xanthomonas albilineans, the causal agent of leaf scald of sugarcane

Polymerase chain reaction (PCR) and newly designed primers, XAF1/XAR1, were tested for selective detection of the causal agent of leaf scald of sugarcane, Xanthomonas albilineans . The efficiency and reliability of PCR were compared with dot immunobinding assay (DIA), ELISA and classical isolation techniques for detecting X. albilineans in suspensions of pure cells and extracts of field-collected stalk and leaf samples of sugarcane. In addition, classical PCR and BIO-PCR (biological amplification followed by PCR) were compared with isolation on a semiselective agar medium. Classical PCR and BIO-PCR techniques had the advantage of not requiring pathogenicity tests to confirm the identity of colonies tentatively identified as X. albilineans on modified semiselective XAM agar medium. The m-XAM medium and BIO-PCR techniques were the most sensitive; however, the former required seven days whereas the latter required only four days. The BIO-PCR technique was as sensitive as the semiselective medium technique and eliminated the need to conduct any additional tests to confirm the identification.     

Structure and Origin of Xanthomonas arboricola pv. pruni Populations Causing Bacterial Spot of Stone Fruit Trees in Western Europe

ABSTRACT Xanthomonas arboricola pv. pruni, the causal agent of bacterial spot on stone fruit, was found in 1995 in several orchards in southeastern France. We studied population genetics of this emerging pathogen in comparison with populations from the United States, where the disease was first described, and from Italy, where the disease has occurred since 1920. Four housekeeping genes (atpD, dnaK, efp, and glnA) and the intergenic transcribed spacer region were sequenced from a total of 3.9 kb of sequences, and fluorescent amplified fragment length polymorphism (FAFLP) analysis was performed. A collection of 64 X. arboricola pv. pruni strains, including 23 strains from France, was analyzed. The X. arboricola pv. pruni population had a low diversity because no sequence polymorphisms were observed. Population diversity revealed by FAFLP was lower for the West European population than for the American population. The same bacterial genotype was detected from five countries on three continents, a geographic distribution that can be explained by human-aided migration of bacteria. Our data support the hypothesis that the pathogen originated in the United States and subsequently has been disseminated to other stone-fruit-growing regions of the world. In France, emergence of this disease was due to a recent introduction of the most prevalent genotype of the bacterium found worldwide.     

Polyphasic characterization of xanthomonas strains from onion

ABSTRACT Xanthomonas leaf blight has become an increasingly important disease of onion, but the diversity among Xanthomonas strains isolated from onion is unknown, as is their relationship to other species and pathovars of Xanthomonas. Forty-nine Xanthomonas strains isolated from onion over 27 years from 10 diverse geographic regions were characterized by pathogenicity to onion and dry bean, fatty acid profiles, substrate utilization patterns (Biolog), bactericide resistance, repetitive sequence-based polymerase chain reaction fingerprinting, rDNA internally transcribed spacer (ITS) region, and hrp b6 gene sequencing. Multiplication of onion Xanthomonas strain R-O177 was not different from X. axonopodis pv. phaseoli in dry bean, but typical common bacterial blight disease symptoms were absent in dry bean. Populations from each geographical region were uniformly sensitive to 100 mug of CuSO(4), 100 mug of ZnSO(4), and 100 mug of streptomycin sulfate per ml. Biolog substrate utilization and fatty acid profiles revealed close phenoltypic relatedness between onion strains of Xanthomonas and X. axonopodis pv. dieffenbachiae (57% of strains) and X. arboricola pv. poinsettiicola (37% of strains), respectively. A logistic regression model based on fatty acid composition and substrate utilization classified 69% of strains into their geographical region of origin. Sequencing of a portion of the hrp B6 gene from 24 strains and ITS region from 25 strains revealed greater than 97% sequence similarity among strains. DNA fingerprinting revealed five genotype groups within onion strains of Xanthomonas and a high degree of genetic diversity among geographical regions of origin. Based on pathogenicity to onion, carbon substrate utilization, fatty acid profiles, rDNA genetic diversity, and genomic fingerprints, we conclude that the strains examined in this study are pathovar X. axonopodis pv. allii. Implications of genetic and phenotypic diversity within X. axonopodis pv. allii are discussed in relation to an integrated pest management program.     

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.