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.     

Pathogenic and genetic diversity of Xanthomonas translucens pv. undulosa in North Dakota

Bacterial leaf streak (BLS), caused by Xanthomonas translucens pv. undulosa, has become more prevalent recently in North Dakota and neighboring states. From five locations in North Dakota, 226 strains of X. translucens pv. undulosa were collected and evaluated for pathogenicity and then selected strains were inoculated on a set of 12 wheat cultivars and other cereal hosts. The genetic diversity of all strains was determined using repetitive sequence-based polymerase chain reaction (rep-PCR) and insertion sequence-based (IS)-PCR. Bacterial strains were pathogenic on wheat and barley but symptom severity was greatest on wheat. Strains varied greatly in aggressiveness, and wheat cultivars also showed differential responses to several strains. The 16S ribosomal DNA sequences of the strains were identical, and distinct from those of the other Xanthomonas pathovars. Combined rep-PCR and IS-PCR data produced 213 haplotypes. Similar haplotypes were detected in more than one location. Although diversity was greatest (≈92%) among individuals within a location, statistically significant (P ≤ 0.001 or 0.05) genetic differentiation among locations was estimated, indicating geographic differentiation between pathogen populations. The results of this study provide information on the pathogen diversity in North Dakota, which will be useful to better identify and characterize resistant germplasm.     

Protein electrophoresis and DNA:DNA hybridizations of xanthomonads from grasses and cereals1

More than 120 Xanthomonas campestris strains pathogenic for grasses and cereals were compared by polyacrylamide gel electrophoresis (SDS-PAGE) of their whole-cell proteins. Genotypic relationships between representative strains of the electrophoretic groups were determined by DNA:DNA hybridizations. Two major groups of bacteria were delineated. The first included X. campestris pv. graminis, pv. arrhenatheri and some isolates from Bromus, which could be differentiated from each other by their protein fingerprints, and also the following pathovars which it was impossible to differentiate by SDS-PAGE: cerealis, hordei, poae, secalis, translucens and undulosa. DNA:DNA hybridizations indicated that significant degrees of DNA-binding (>60% D) exist between all these pathovars. In the second group, strains of X. campestris pv. holcicola, pv. vasculorum and pv. oryzae were related at 40–45% DNA-binding, while strains of pv. oryzae and pv. oryzicola were genotypically highly related (85% D). All the pathovars of this second group could be differentiated from each other by their protein electrophoretic fingerprints.     

Detection of Xanthomonas campestris pv. undulosa infested wheat seeds by combined liquid medium enrichment and ELISA

An enzyme-linked immunosorbent assay (ELISA) was standardized for detecting Xanthomonas campestris pv. undulosa (Xcu) in plant tissues. Antiserum prepared against somatic antigens of Xcu reacted with cells of pathovars undulosa, cerealis, translucens and phleipratensis , but not with other bacterial species belonging to the genera Xanthomonas, Pseudomonas, Agrobacterium, Clavibacter , and Erwinia. The lower limit of detection of pure cultures was 5 × 10³ cfu/ml. A semi-selective enrichment broth (SSEB) improved the recovery of Xcu in cultures mixed with contaminating bacteria commonly found on wheat seeds. In ELISA tests the enriched samples gave two- to three-fold increases in A_405nm readings when viable cells of Xcu were present. By enrichment, X. campestris pathovars undulosa, cerealis, translucens and phleipratensis were detected in samples that originally had less than 5 × 10² cfu/ml. Semi-selective enrichment combined with ELISA (SSEB-ELISA) allowed for determination of the percentages of infestation of wheat seed lots. Potential seedling infection (PSI) of naturally infested wheat seed lots was obtained by growing seed samples in the greenhouse under conditions optimal for disease development. Three methods were evaluated for their capacity to estimate the PSI: ELISA, combined SSEB and ELISA, and direct plating onto semi-selective XTS agar. Percentages of seed infestation determined by combined SSEB and ELISA resulted in a highly significant correlation with the PSI (r = 0·87, P × 005), whereas determinations made by ELISA or direct plating onto XTS did not significantly correlate with the PSI determined in the greenhouse. This test may constitute a convenient tool for fast initial screening of wheat seed lots in wheat certification programmes.     

Grouping of Xanthomonas campestris pathovars of cereals and grasses by fatty acid profiling1

Fatty acid profiles were prepared for a range of strains representing all 14 Xanthomonas campestris pathovars from the Gramineae. Profiles were complex, containing up to 40 acids, most of which were iso- and anteiso-branched acids. Grouping of profile types generally correlated with pathovar, although pvs translucens, hordei, cerealis, secalis and undulosa could not be differentiated. Pvs graminis, arrhenatheri, poae and phlei formed another profile type although there were some differences between the pathovars. These graminis and translucens groups had similar profiles but could usually be differentiated by the ratios of 15:0 iso/16:0 iso and 15:0 iso/15:0 anteiso fatty acid methyl esters. The other pathovars each had a very different profile type. Pvs oryzae and oryzicola had profiles which were very different from all others. Pv. vasculorum comprised at least two distinct profile types. This did not correlate with host species or geographic distribution. Fatty acid profiling can be used to identify strains of X. campestris pathovars from Gramineae.     

A comprehensive species to strain taxonomic framework for xanthomonas

ABSTRACT A comprehensive classification framework was developed that refines the current Xanthomonas classification scheme and provides a detailed assessment of Xanthomonas diversity at the species, subspecies, pathovar, and subpathovar levels. Polymerase chain reaction (PCR) using primers targeting the conserved repetitive sequences BOX, enterobacterial repetitive intergenic consensus (ERIC), and repetitive extragenic palindromic (REP) (rep-PCR) was used to generate genomic fingerprints of 339 Xanthomonas strains comprising 80 pathovars, 20 DNA homology groups, and a Stenotrophomonas maltophilia reference strain. Computer-assisted pattern analysis of the rep-PCR profiles permitted the clustering of strains into distinct groups, which correspond directly to the 20 DNA-DNA homology groups(genospecies) previously identified. Group 9 strains (X. axonopodis) were an exception and did not cluster together into a coherent group but comprised six subgroups. Over 160 strains not previously characterized by DNA-DNA hybridization analysis, or not previously classified, were assigned to specific genospecies based on the classification framework developed. The rep-PCR delineated subspecific groups within X. hortorum, X. arboricola, X. axonopodis, X. oryzae, X. campestris, and X. translucens. Numerous taxonomic issues with regard to the diversity, similarity, redundancy, or misnaming were resolved. This classification framework will enable the rapid identification and classification of new, novel, or unknown Xanthomonas strains that are pathogenic or are otherwise associated with plants.     

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.     

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.     

Evaluation of proposed amended names of several pseudomonads and xanthomonads and recommendations

ABSTRACT In 1980, over 90% of all plant-pathogenic pseudomonads and xanthomonads were lumped into Pseudomonas syringae and Xanthomonas campestris, respectively, as pathovars. The term "pathovar" was created to preserve the name of plant pathogens, but has no official standing in nomenclature. Proposals to elevate and rename several pathovars of the genera Pseudomonas and Xanthomonas to the rank of species has caused great confusion in the literature. We believe the following changes have merit and expect to adopt them for publication in a future American Phytopathological Society Laboratory Guide for Identification of Plant Pathogenic Bacteria. Upon review of published data and the Rules of The International Code of Nomenclature of Bacteria, we make the following recommendations. We reject the proposal to change the name of P. syringae pvs. phaseolicola and glycinea to P. savastanoi pvs. phaseolicola and glycinea, respectively, because both pathogens are easily differentiated phenotypically from pv. savastanoi and convincing genetic data to support such a change are lacking. We accept the elevation of P. syringae pv. savastanoi to the rank of species. We accept the reinstatement of X. oryzae to the rank of species with the inclusion of X. oryzicola as a pathovar of X. oryzae and we accept the species X. populi. We agree with the elevation of the pvs. cassavae, cucurbitae, hyacinthi, pisi, and translucens to the rank of species but not pvs. melonis, theicola, and vesicatoria type B. We recommend that all type A X. vesicatoria be retained as X. campestris pv. vesicatoria and all type B X. vesicatoria be named X. exitiosa. We reject the newly proposed epithets arboricola, bromi, codiaei (poinsettiicola type B), hortorum, sacchari, and vasicola and the transfer of many pathovars of X. campestris to X. axonopodis. The proposed pathovars of X. axonopodis should be retained as pathovars of X. campestris.     

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.     

The genetic characterization of Xanthomonas arboricola pv. juglandis,the causal agent of walnut blight in Poland

Leaves and fruits of walnut trees exhibiting symptoms of bacterial blight were collected from six locations in Poland. Isolations on agar media resulted in 18 bacterial isolates with colony morphology resembling that of the Xanthomonas genus. PCR using X1 and X2 primers specific for Xanthomonas confirmed that all isolates belonged to this genus. In pathogenicity tests on unripe walnut fruits, all isolates caused typical black necrotic lesions covering almost the entire pericarp. Results of selected phenotypic tests indicated that characteristics of all isolates were the same as described for the type strain of Xanthomonas arboricola pv. juglandis. Genetic analyses (PCR MP, ERIC‐, BOX‐PCR and MLSA) showed similarities between the studied isolates and the reference strain of X. arboricola pv. juglandis CFBP 7179 originating from France. However, reference strains I‐391 from Portugal and LMG 746 from the UK were different. MLSA analysis of partial sequences of the fyuA, gyrB and rpoD genes of studied isolates and respective sequences from GenBank of pathotype strains of other pathovars of X. arboricola showed that the X. arboricola pv. juglandis isolates consisted of different phylogenetic lineages. An incongruence among MLSA gene phylogenies and traces of intergenic recombination events were proved. These data suggest that the sequence analysis of several housekeeping genes is necessary for proper identification of X. arboricola pathovars.     

Genetic relationships among strains of <Emphasis Type="Italic">Xanthomonas campestris</Emphasis>pv. <Emphasis Type="Italic">campestris</Emphasis>revealed by novel rep-PCR primers

Novel primers for rep-PCR were developed with the original software and based on `ancient diverged periodical sequences'. Rep-PCR with these primers was applied to study genetic relationships among 51 Xanthomonas campestris strains. The strains were collected from different countries including Russia, Japan, UK, Germany and Hungary. Reference strains of three X. campestrispathovars and five other Xanthomonas species were included. Based on qualitative differences in amplification profiles, the strains were divided into four major groups. Two subgroups recognised within X. campestrispopulation were similar to RFLP haplotypes. The third subgroup included strains of two other pathovariants and Japanese isolates of X. campestris pv. campestriswhile the fourth group comprised the other species of Xanthomonas. The analysis of the diversity within X. campestris resulted in the conclusion that isolates belong to distinct clonal populations (subgroups). The differences between the subgroups of X. campestris were only slightly smaller than between species of Xanthomonas. A PCR fragment about 600 bp amplified by primer KRPN2 was found in nearly all tested strains of X. campestris.SCAR primers designed for this marker produced a single specific band for strains of X. campestris, but not for other Xanthomonas, Pseudomonas and Erwiniastrains tested. Application of the new primer set for rep-PCR offers a rapid, simple and reproducible method for identification of bacterial strains. The X. campestris-specific SCAR primers may be used in diagnostics of this important plant pathogen.     

Identification of Isolates that Cause a Leaf Spot Disease of Brassicas as Xanthomonas campestris pv. raphani and Pathogenic and Genetic Comparison with Related Pathovars

ABSTRACT Twenty-five Xanthomonas isolates, including some isolates received as either X. campestris pv. armoraciae or pv. raphani, caused discrete leaf spot symptoms when spray-inoculated onto at least one Brassica oleracea cultivar. Twelve of these isolates and four other Xanthomonas isolates were spray- and pin-inoculated onto 21 different plant species/cultivars including horseradish (Armoracia rusticana), radish (Raphanus sativus), and tomato (Lycopersicon esculentum). The remaining 13 leaf spot isolates were spray-inoculated onto a subset of 10 plant species/cultivars. The leaf spot isolates were very aggressive on several Brassica spp., radish, and tomato causing leaf spots and dark sunken lesions on the middle vein, petiole, and stem. Based on the differential reactions of several Brassica spp. and radish cultivars, the leaf spot isolates were divided into three races, with races 1 and 3 predominating. A differential series was established to determine the race-type of isolates and a gene-for-gene model based on the interaction of two avirulence genes in the pathogen races and two matching resistance genes in the differential hosts is proposed. Repetitive-DNA polymerase chain reaction-based fingerprinting was used to assess the genetic diversity of the leaf spot isolates and isolates of closely related Xanthomonas pathovars. Although there was variability within each race, the leaf spot isolates were clustered separately from the X. campestris pv. campestris isolates. We propose that X. campestris isolates that cause a nonvascular leaf spot disease on Brassica spp. should be identified as pv. raphani and not pv. armoraciae. Race-type strains and a neopathotype strain for X. campestris pv. raphani are proposed.     

Relationship Between Phyllosphere Population Sizes of Xanthomonas translucens pv. translucens and Bacterial Leaf Streak Severity on Wheat Seedlings

ABSTRACT The relationship between leaf-associated population sizes of Xanthomonas translucens pv. translucens on asymptomatic leaves and subsequent bacterial leaf streak (BLS) severity was investigated. In three experiments, X. translucens pv. translucens was spray-inoculated onto 10-day-old wheat seedlings over a range of inoculum densities (10(4), 10(5), 10(6), 10(7), and 10(8) CFU/ml). Lesions developed most rapidly on plants inoculated with higher densities of X. translucens pv. translucens. Leaf-associated pathogen population sizes recovered 48 h after inoculation were highly predictive of BLS severity 7 days after inoculation (R(2) = 0.970, P < 0.0001). The relationship between pathogen population size on leaves and subsequent BLS severity was best described by the logistic model. Leaf-associated X. translucens pv. translucens population size and BLS severity from a particular pathogen inoculum density often varied among experiments; however, the disease severity level caused by a particular leaf-associated X. translucens pv. translucens population size was not significantly different among experiments. Biological and disease control implications of the X. translucens pv. translucens population size-BLS severity relationship are discussed.     

Inference of the phylogenetic diversity and population structure of Xanthomonas campestris affecting Brassicaceae using a multilocus sequence typing‐based approach

Xanthomonas campestris pathovars are widely distributed throughout the globe and have a broad host range, causing severe economic losses in the food and ornamental crucifers markets. Using an approach based on multilocus sequence typing, phylogenetic diversity and population structure of a set of 75 Portuguese and other Xanthomonas campestris isolates from several cruciferous hosts were assessed. Although this population displayed a major clonal structure, neighbour‐net phylogenetic analysis highlighted the presence of recombinational events that may have driven the ecological specialization of X. campestris with different host ranges within the Brassicaceae family. A high level of genetic diversity within and among X. campestris pathovars was also revealed, through the establishment of 46 sequence types (STs). This approach provided a snapshot of the global X. campestris population structure in cruciferous host plants, correlating the existing pathovars with three distinct genetic lineages. Phylogenetic relationships between the founder genotype and remaining isolates that constitute the X. campestris pv. campestris population were further clarified using goeBURST algorithm. Identification of an intermediate link between X. campestris pv. campestris and X. campestris pv. raphani provided new insights into the mechanisms driving the differentiation of both pathovars. Wide geographic distribution of allelic variants suggests that evolution of X. campestris as a seedborne pathogen was not shaped by natural barriers. However, as Portuguese isolates encompass 26 unique STs and this country is an important centre of domestication of Brassica oleracea crops, a strong case is made for its role as a diversification reservoir, most probably through host–pathogen coevolution.     

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.     

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.     

Genetic, phenotypic and pathogenic diversity among xanthomonads isolated from pistachio (Pistacia vera) in Australia

Repetitive extragenic palindromic polymerase chain reaction (rep-PCR), sequencing of the 16S−23S rDNA internal transcribed spacer (ITS), biochemical and physiological tests, the Biolog microplate system, polyacrylamide gel electrophoresis (PAGE) of whole-cell proteins, and pathogenicity tests were used to characterize variability among xanthomonads isolated from pistachio trees suffering from bacterial dieback in four regions of Australia. ITS sequencing and rep-PCR revealed two distinct genotypes among the strains. The ITS sequencing suggested that the pistachio strains were closely related to Xanthomonas translucens pathovars, in particular X. translucens pv . poae . Results of physiological and biochemical tests, as well as Biolog microplate analysis and protein profiling, confirmed the existence of two groups. Furthermore, pathogenicity and host-range studies indicated that the two groups were biologically different. There was an association between the two groups and the geographical origin of the strains.     

Interaction of common bacterial blight bacteria with disease resistance quantitative trait loci in common bean

Common bacterial blight (CBB) of common bean (Phaseolus vulgaris L.) is caused by Xanthomonas campestris pv. phaseoli and X. fuscans subsp. fuscans, and is the most important bacterial disease of this crop in many regions of the world. In 2005 and 2006, dark red kidney bean fields in a major bean-growing region in central Wisconsin were surveyed for CBB incidence and representative symptomatic leaves collected. Xanthomonad-like bacteria were isolated from these leaves and characterized based upon phenotypic (colony) characteristics, pathogenicity on common bean, polymerase chain reaction (PCR) with X. campestris pv. phaseoli- and X. fuscans subsp. fuscans-specific primers, and repetitive-element PCR (rep-PCR) and 16S-28S ribosomal RNA spacer region sequence analyses. Of 348 isolates that were characterized, 293 were identified as common blight bacteria (i.e., pathogenic on common bean and positive in PCR tests with the X. campestris pv. phaseoli- and X. fuscans subsp. fuscans-specific primers), whereas the other isolates were nonpathogenic xanthomonads. Most (98%) of the pathogenic xanthomonads were X. campestris pv. phaseoli, consistent with the association of this bacterium with CBB in large-seeded bean cultivars of the Andean gene pool. Two types of X. campestris pv. phaseoli were involved with CBB in this region: typical X. campestris pv. phaseoli (P) isolates with yellow mucoid colonies, no brown pigment production, and a typical X. campestris pv. phaseoli rep-PCR fingerprint (60% of strains); and a new phenotype and genotype (Px) with an X. campestris pv. phaseoli-type fingerprint and less mucoid colonies that produced brown pigment (40% of strains). In addition, a small number of X. fuscans subsp. fuscans strains, representing a new genotype (FH), were isolated from two fields in 2005. Representative P and Px X. campestris pv. phaseoli strains, an FH X. fuscans subsp. fuscans strain, plus five previously characterized X. campestris pv. phaseoli and X. fuscans subsp. fuscans genotypes were inoculated onto 28 common bean genotypes having various combinations of known CBB resistance quantitative trait loci (QTL) and associated sequence-characterized amplified region markers. Different levels of virulence were observed for X. campestris pv. phaseoli strains, whereas X. fuscans subsp. fuscans strains were similar in virulence. The typical X. campestris pv. phaseoli strain from Wisconsin was most virulent, whereas X. campestris pv. phaseoli genotypes from East Africa were the least virulent. Host genotypes having the SU91 marker-associated resistance and one or more other QTL (i.e., pyramided resistance), such as the VAX lines, were highly resistant to all genotypes of common blight bacteria tested. This information will help in the development of CBB resistance-breeding strategies for different common bean market classes in different geographical regions, as well as the identification of appropriate pathogen genotypes for screening for resistance.     

Diagnostic methods for phytopathogenic bacteria of stone fruits and nuts in COST 873

Different diagnostic methods used or developed in the EU-COST ² 873 project 'Bacterial diseases of stone fruits and nuts' are presented. The methods concern detection and identification of the plant pathogenic bacteria Xylella fastidiosa (EPPO A1 list), Xanthomonas arboricola pv corylina , X.a . pv. pruni , Pseudomonas syringae pv. persicae (A2 list pathogens), Agrobacterium tumefaciens , Brenneria nigrifluens and B. rubrifaciens , P. amygdali , P. avellanae , P.s. pv . avii , P.s. pv. morsprunorum , P.s . pv. syringae , X.a . pv. juglandis . Furthermore, a recently described xanthomonas species (proposed name X. translucens pv . pistachiae ), causing a new disease on pistachio, viz. Pistachio decline, in Australia and the recently renamed Xanthomonas citri pv. mangiferaeindicae on mango are included. The methods range from classical ones such as nutritional tests, use of (semi-)selective media, PCR, fatty acid analysis, serology and pathogenicity tests as well as (more) modern ones such as free flow capillary electrophoresis, real-time PCR, rep-PCR, fAFLP and sequencing of open reading frames (ORFs) and/or housekeeping genes such as gyrB and rpoD . The usefulness of these methods are outlined and reference made to publications where they were successfully used. Development of useful (molecular) tools are also indicated. Whole genome sequencing has been performed for Pantoea agglomerans (a relevant biocontrol agent) by the Swiss laboratory and initiated for X. a . pv. pruni by the French laboratory in cooperation with the Swiss and Italian laboratories and development of a microarray test has been initiated by the Swiss laboratory, Details of meetings and training programmes throughout the region are elaborated.