MultiLocus Sequence Analysis- and Amplified Fragment Length Polymorphism-based characterization of xanthomonads associated with bacterial spot of tomato and pepper and their relatedness to Xanthomonas species.

MultiLocus Sequence Analysis (MLSA) and Amplified Fragment Length Polymorphism (AFLP) were used to measure the genetic relatedness of a comprehensive collection of xanthomonads pathogenic to solaneous hosts to Xanthomonas species. The MLSA scheme was based on partial sequences of four housekeeping genes (atpD, dnaK, efp and gyrB). Globally, MLSA data unambiguously identified strains causing bacterial spot of tomato and pepper at the species level and was consistent with AFLP data. Genetic distances derived from both techniques showed a close relatedness of (i) X. euvesicatoria, X. perforans and X. alfalfae and (ii) X. gardneri and X. cynarae. Maximum likelihood tree topologies derived from each gene portion and the concatenated data set for species in the X. campestris 16S rRNA core (i.e. the species cluster comprising all strains causing bacterial spot of tomato and pepper) were not congruent, consistent with the detection of several putative recombination events in our data sets by several recombination search algorithms. One recombinant region in atpD was identified in most strains of X. euvesicatoria including the type strain.

Insertion sequence- and tandem repeat-based genotyping techniques for Xanthomonas citri pv. mangiferaeindicae.

Molecular fingerprinting techniques that have the potential to identify or subtype bacteria at the strain level are needed for improving diagnosis and understanding of the epidemiology of pathogens such as Xanthomonas citri pv. mangiferaeindicae, which causes mango bacterial canker disease. We developed a ligation-mediated polymerase chain reaction targeting the IS1595 insertion sequence as a means to differentiate pv. mangiferaeindicae from the closely related pv. anacardii (responsible for cashew bacterial spot), which has the potential to infect mango but not to cause significant disease. This technique produced weakly polymorphic fingerprints composed of ≈70 amplified fragments per strain for a worldwide collection of X. citri pv. mangiferaeindicae but produced no or very weak amplification for pv. anacardii strains. Together, 12 tandem repeat markers were able to subtype X. citri pv. mangiferaeindicae at the strain level, distinguishing 231 haplotypes from a worldwide collection of 299 strains. Multilocus variable number of tandem repeats analysis (MLVA), IS1595-ligation-mediated polymerase chain reaction, and amplified fragment length polymorphism showed differences in discriminatory power and were congruent in describing the diversity of this strain collection, suggesting low levels of recombination. The potential of the MLVA scheme for molecular epidemiology studies of X. citri pv. mangiferaeindicae is discussed.

Pseudomonas aeruginosa outbreak linked to mineral water bottles in a neonatal intensive care unit: fast typing by use of high-resolution melting analysis of a variable-number tandem-repeat locus.

Pseudomonas aeruginosa is an opportunistic pathogen that causes nosocomial infections in intensive care units. Determining a system of typing that is discriminatory is essential for epidemiological surveillance of P. aeruginosa. We developed a method for the typing of Pseudomonas aeruginosa, namely, multiple-locus variable-number tandem-repeat (VNTR) typing with high-resolution melting analysis (HRMA). The technology was used to genotype a collection of 43 environmental and clinical strains isolated during an outbreak in a neonatal intensive care unit (NICU) that we report. Nineteen strains isolated in other departments or outside the hospital were also tested. The genetic diversity of this collection was determined using VNTR-HRMA, with amplified fragment length polymorphism (AFLP) analysis as a reference. Twenty-five and 28 genotypes were identified, respectively, and both techniques produced congruent data. VNTR-HRMA established clonal relationships between the strains of P. aeruginosa isolated during the outbreak in the NICU and proved, for the first time, the role of mineral water as the inoculum source. VNTR typing with one primer pair in association with HRMA is highly reproducible and discriminative, easily portable among laboratories, fast, and inexpensive, and it demonstrated excellent typeability in this study. VNTR-HRMA represents a promising tool for the molecular surveillance of P. aeruginosa and perhaps for molecular epidemiologic analysis of other hospital infections.

First Report of Xanthomonas hortorum pv. carotae Causing Bacterial Leaf Blight of Carrot in Mauritius.

Bacterial blight of carrot (Daucus carota) is caused by Xanthomonas hortorum pv. carotae (4). The pathogen is seed transmitted and carrot seeds can be an important source of primary inoculum (2). A 2008-2009 outbreak of a disease resembling bacterial blight was observed in Mauritius in 10 ha of carrot crops, primarily in humid areas of the island, at an estimated incidence of 10%. Carrot leaves with angular, water-soaked leaf spots that turned necrotic were collected at Plaine Sophie, Mauritius in December 2008. Yellow, Xanthomonas-like colonies were isolated onto KC agar medium (3). MultiLocus sequence analysis (MLSA) with four genes (atpD, dnaK, efp, and gyrB) was performed as described previously (1) on five carrot strains together with two reference strains of X. hortorum pv. carotae (LMG 8643 and LMG 8644). The reference strains were identical. Of the five Mauritius strains, two (LG1-1 and LG1-4) were identical, and most closely related to, but distinct from, the reference strains (genetic distance of 0.02). The other three strains represented two sequence types identified as Xanthomonas sp. based on a phylogenetic tree derived from concatenated sequences, but were not related to any type strain. PCR assays with a 3S primer pair specific for X. hortorum pv. carotae (2) produced an amplicon of approximately 350 bp from isolates LG1-1, LG1-4, and each of the reference strains. A PCR assay with a 9B primer pair (2) yielded an amplicon of 0.9 kb for strains LG1-1, LG1-4, and LMG 8644, whereas LMG 8643 yielded an amplicon of approximately 2.0 kb (2). Foliage of 4-week-old plants (36 plants per strain) of the carrot cv. Senator F1 were spray inoculated with a suspension of each strain using an 18-h culture in sterile 0.01 M tris buffer (pH 7.2) with approximately 1 × 108 CFU/ml. Plants sprayed with tris buffer were used as a negative control treatment. Plants were incubated in a growth chamber at 26 ± 1°C at a relative humidity of 95 ± 5% and a photoperiod of 16 h. Water-soaked lesions that developed into necrotic areas were observed 12 to 15 days after inoculation of LG1-1, LG1-4, and the two reference strains. Bacteria were recovered from lesions onto KC medium (3) 3 weeks after inoculation with mean Xanthomonas populations of at least 1 × 107 CFU/lesion. Colonies with morphology typical of Xanthomonas were recovered and typed using atpD sequencing to fulfill Koch's postulates. Although Xanthomonas-like bacteria were isolated from symptomatic carrot leaves in Mauritius in 1989, the results of that study were not published. To our knowledge, this is the first report of molecular and pathological characterization of this pathogen in carrot crops in Mauritius. References: (1) L. Bui Thi Ngoc et al. Int. J. Syst. Evol. Microbiol. 60:515, 2010. (2) X. Q. Meng et al. Plant Dis. 88:1226, 2004. (3) O. Pruvost et al. J. Appl. Microbiol. 99:803, 2005. (4) L. Vauterin et al. Int. J. Syst. Bacteriol. 45:472, 1995.

Genetic and Pathological Diversity Among Xanthomonas Strains Responsible for Bacterial Spot on Tomato and Pepper in the Southwest Indian Ocean Region.

Bacterial spot of tomato and pepper, a major problem in tropical climates, can be caused by several Xanthomonas genospecies. We examined the genetic and pathological diversity of a collection of 72 strains from the southwest Indian Ocean region as part of a regional research and development program to update inventories of agricultural pests and pathogens. Xanthomonas euvesicatoria, X. perforans, X. gardneri, and X. vesicatoria were identified in our strain collection. The identification of strains at the species level was consistently achieved by amplified fragment length polymorphism (AFLP) and multilocus sequence analysis (MLSA). Overall, X. euvesicatoria was the species recovered prevalently. MLSA data based on four housekeeping genes identified two to three sequence types per genospecies. It suggested that sequence variations primarily consisted of synonymous mutations, although a recombination event spanning several hundred nucleotides was detected for some strains of X. euvesicatoria on the atpD gene coding for the F1-F0-ATPase ß subunit. The pathogenicity of strains was consistent with data found in the literature. Some pathological variations were primarily observed among strains identified as X. euvesicatoria. This study provides the first ever comprehensive description of the status of Xanthomonas species that cause bacterial spot of tomato and pepper in the southwest Indian Ocean region.

A New Type of Strain of Xanthomonas euvesicatoria Causing Bacterial Spot of Tomato and Pepper in Grenada.

Bacterial spot of tomato and pepper (BSTP) can be caused by several Xanthomonas genospecies (2). BSTP is a major disease in Grenada where A and B phenotypic groups (Xanthomonas euvesicatoria and X. vesicatoria, respectively, [2]) have been reported (3). There is no previous report of group A strains, which are strongly amylolytic and pectolytic, in Grenada. In March 2007, tomato and pepper leaves with lesions typical of BSTP were collected in Saint David and Saint Andrew parishes of Grenada. Bacterial isolations were performed on KC semiselective agar medium (4), resulting in isolation of five yellow-pigmented, Xanthomonas-like strains. Three strains isolated from tomato or pepper in Saint David were negative for starch hydrolysis and pectate degradation, two tests that were found useful for strain identification in the 1990s (2). Two strains isolated from pepper in Saint David were strongly amylolytic and degraded pectate. Amplified fragment length polymorphism (AFLP) and multilocus sequence analysis (MLSA) assays targeting atpD, dnaK, efp, and gyrB were performed on the five strains from Grenada together with a type strain of each of X. euvesicatoria, X. perforans, X. gardneri, and X. vesicatoria as well as other reference strains of X. euvesicatoria and X. perforans as described previously (1). All strains from Grenada were identified as X. euvesicatoria regardless of the typing technique. On the basis of AFLP assays, the two strains with phenotypic features not reported in Grenada were closely related (distances of ≤0.002 nucleotide substitutions per site [1]) to a group of strains from India (ICMP 3381, LMG 907, LMG 908, and LMG 918). These two strains were also identical to the Indian strains based on MLSA, but differed from the X. euvesicatoria type strain by at least one nucleotide substitution in all loci examined. The three strains from Grenada that were negative for starch hydrolysis and pectate degradation had sequences identical to that of the type strain. Young leaves of tomato plants of cv. Marmande and pepper plants of cvs. Yolo Wonder and Aiguille were infiltrated (six inoculation sites per leaf, three replicate plants per cultivar per experiment, and the experiment was replicated once) using inoculum of each of the five strains from Grenada made from suspensions in Tris buffer containing approximately 1 × 105 CFU/ml. Two reference strains of X. euvesicatoria (NCPPB 2968 and LMG 922) were also inoculated as positive control treatments. Negative control treatments consisted of leaves infiltrated with sterile Tris buffer. Typical water-soaked lesions that developed into necrotic spots were observed 3 to 8 days after inoculation (dai) for all strains on all cultivars, except NCPPB 2968, which was not pathogenic on pepper cv. Aiguille. Xanthomonas population sizes from lesions plated onto KC agar medium (4) 25 dai ranged from 3 × 106 to 5 × 107, 8 × 107 to 2 × 108, and 9 × 106 to 2 × 108 CFU/lesion on tomato cv. Marmande and pepper cvs. Yolo Wonder and Aiguille, respectively. The epidemiological importance of this previously unreported group of X. euvesicatoria strains in Grenada needs to be assessed. References: (1) L. Bui Thi Ngoc et al. Int. J. Syst. Evol. Microbiol. 60:515, 2010. (2) J. B. Jones et al. Syst. Appl. Microbiol. 27:755, 2004. (3) L. W. O'Garro. Plant Dis. 82:864, 1998. (4) O. Pruvost et al. J. Appl. Microbiol. 99:803, 2005.

A new semi-selective medium for the isolation of Xanthomonas axonopodis pv. dieffenbachiae, the etiological agent of anthurium bacterial blight.

AIMS: Xanthomonas axonopodis pv. dieffenbachiae causes anthurium blight, which is regarded as the most threatening disease for the anthurium industry worldwide. The bacterium is listed as a quarantine pathogen in several regions, including Europe. We evaluated the use of Neomycin-Cephalexin-Trimethoprime-pirMecillinam 4 (NCTM4) medium for its isolation. METHODS AND RESULTS: A total of 104 bacterial strains were inoculated onto NCTM4 and on the previously published Cellobiose-Starch (CS) and Esculin-Trehalose (ET) media. The strain collection included: the anthurium blight pathogen, Xanthomonas strains, for which false positive results are known to occur using serological identification-tests; other bacterial pathogens of anthurium; and representatives of bacteria that are commonly present in the anthurium phyllosphere. Media were evaluated following the ISO 16140 protocol for the validation of alternative methods. CONCLUSION: Growth of the anthurium blight pathogen was better on NCTM4 and ET media than on CS. NCTM4 provided a better repeatability. It also displayed a lower rate of false positive and false negative results when the pathogen was isolated from plant extracts. SIGNIFICANCE AND IMPACT OF THE STUDY: This study will lead to improved isolation protocols of the anthurium blight in official procedures. NCTM4 medium could also favourably be used in studies, which aim to further understanding of the biology and epidemiology of this pathogen.

Polyphasic characterization of xanthomonads pathogenic to members of the Anacardiaceae and their relatedness to species of Xanthomonas.

We have used amplified fragment length polymorphism (AFLP), multilocus sequence analysis (MLSA) and DNA-DNA hybridization for genotypic classification of Xanthomonas pathovars associated with the plant family Anacardiaceae. AFLP and MLSA results showed congruent phylogenetic relationships of the pathovar mangiferaeindicae (responsible for mango bacterial canker) with strains of Xanthomonas axonopodis subgroup 9.5. This subgroup includes X. axonopodis pv. citri (synonym Xanthomonas citri). Similarly, the pathovar anacardii, which causes cashew bacterial spot in Brazil, was included in X. axonopodis subgroup 9.6 (synonym Xanthomonas fuscans). Based on the thermal stability of DNA reassociation, consistent with the AFLP and MLSA data, the two pathovars share a level of similarity consistent with their being members of the same species. The recent proposal to elevate X. axonopodis pv. citri to species level as X. citri is supported by our data. Therefore, the causal agents of mango bacterial canker and cashew bacterial spot should be classified as pathovars of X. citri, namely X. citri pv. mangiferaeindicae (pathotype strain CFBP 1716) and X. citri pv. anacardii (pathotype strain CFBP 2913), respectively. Xanthomonas fuscans should be considered to be a later heterotypic synonym of Xanthomonas citri.

First Report of Xanthomonas cucurbitae Causing Bacterial Leaf Spot of Watermelon in the Seychelles.

Bacterial leaf spot of cucurbits caused by Xanthomonas cucurbitae (4) can be a harmful disease of several cucurbit species in tropical environments, mainly within the Cucumis, Cucurbita, and Citrullus genera. The bacterium induces angular, water-soaked leaf spots, which sometimes become necrotic and have a chlorotic halo. Scab-like lesions on fruit can also be observed (2). Water-soaked, angular leaf lesions were collected from approximately 15 watermelon plants (Citrullus lanatus) in a production field located in Mahé, Seychelles in 2003. Yellow-pigmented Xanthomonas-like bacterial colonies were isolated on KC semiselective medium (yeast extract 7 g, peptone 7 g, glucose 7 g, agar 18 g, distilled water 1,000 ml, propiconazole 20 µg ml-1, cephalexin 40 mg liter-1, and kasugamycin 20 mg liter-1) from all isolation attempts (3). Amplified fragment length polymorphism (AFLP) analysis was performed on four watermelon strains together with reference strains of Xanthomonas cucurbitae (LMG 690 [type strain] and LMG 8663) and the type strain of all other valid Xanthomonas species using SacI/MspI and four primer pairs (unlabeled MspI + 1 [A, C, T, or G] primers and 5'-labeled - SacI + C primer for the selective amplification step) (1). The four strains from watermelon showed identical fingerprints and were most closely related to X. cucurbitae. One strain from diseased watermelon (JZ88-1) was further analyzed by MultiLocus Sequence Analysis (MLSA) using portions of three housekeeping genes (atpD, dnaK, and gyrB) as described previously (1). This strain displayed a very high relatedness (99.8 and 98.9% with strain LMG 690 and LMG 8663, respectively) to the two reference strains of X. cucurbitae. AFLP and MLSA were useful for identifying strains at the species level that were consistent with previous results (1). Bottle-gourd (Lagenaria siceraria), pumpkin (Cucurbita maxima), squash cv. Aurore (Cucurbita pepo), cucumber cv. L-04 (Cucumis sativus), cantaloupe melon cv. Cezanne (Cucumis melo), and watermelon cv. Fou-nan (C. lanatus) leaves were infiltrated (10 inoculation sites per leaf and three replicates) with bacterial suspensions (JZ88-1, LMG 690 and LMG 8663) containing approximately 1 × 105 CFU ml-1 (approximately 1 × 102 CFU per inoculation site). Typical water-soaked lesions that developed into necrotic spots were observed 6 to 8 days after inoculation for all inoculated strains on all inoculated plant species. One month after inoculation, Xanthomonas was recovered from lesions and population sizes determined on KC semiselective medium (3) ranging from 1 × 106 to 9 × 106 CFU per lesion were typical of a compatible interaction. Bacterial leaf spot has appeared sporadically in Mahé, Seychelles since 2003, most often with limited incidence. However, growers need to be aware of the potential negative effect of this disease in tropical environments. References: (1) N. Ah-You et al. Int. J. Syst. Evol. Microbiol. 59:306, 2009. (2) J. F. Bradbury. Page 309 in: Guide to Plant Pathogenic Bacteria. CAB International, Slough, UK, 1986. (3) O. Pruvost et al. J. Appl. Microbiol. 99:803, 2005. (4) L. Vauterin et al. Int. J. Syst. Bacteriol. 45:472, 1995.

First Report in the Seychelles of Xanthomonas axonopodis Genetic Cluster 9.2 Causing Bacterial Leaf Spot of Avocado.

Small, black, angular leaf lesions, which sometimes coalesced, were collected from avocado (Persea americana Miller) leaves in a government nursery located at Grand Anse, Mahé, Seychelles archipelago in 2003. Patterns of diseased plants were highly clustered, suggesting local dispersal in the nursery. Yellow-pigmented Xanthomonas-like bacterial colonies were isolated on KC semiselective medium (3). Amplified fragment length polymorphism (AFLP) analysis was performed on two avocado strains together with reference strains of the genetic clusters of Xanthomonas axonopodis (4) and the type strain of all other valid Xanthomonas species using SacI/MspI and four primer pairs (unlabeled MspI + 1 [A, C, T, or G] primers and 5'-labeled - SacI + C primer for the selective amplification step) (1). The two avocado strains showed identical fingerprints and were closely related to X. axonopodis genetic cluster 9.2 (4). One strain, JZ103-1, was further analyzed by MultiLocus Sequence Analysis (MLSA) using portions of three housekeeping genes (atpD, dnaK, and gyrB) as described previously (1). MLSA data confirmed that the xanthomonad associated with avocado was most closely related to X. axonopodis genetic cluster 9.2. No other strain in this genetic cluster shared an identical sequence type. Avocado cv. Grand collet leaves from the youngest growth flush were infiltrated with a needleless syringe (10 inoculation sites per leaf and three replicates) with bacterial suspensions. Typical, water-soaked lesions that developed into black necrotic spots appeared 6 to 8 days after infiltration on all inoculated leaves when suspensions containing ~1 × 106 CFU ml-1 were used (i.e., ~7 × 102 CFU per inoculation site), while no lesions developed on leaves inoculated with Tris buffer or with suspensions containing ~1 × 104 CFU ml-1. One month after inoculation, mean Xanthomonas population sizes determined on KC semiselective medium (3) from ~1 cm2 leaf fragments showing black lesions ranged from 2 × 106 to 4 × 106 CFU per lesion, typical of a compatible interaction. A few colonies that recovered from lesions obtained after inoculation were typed by AFLP and were identical to the inoculated strain. An extensive branch and trunk canker of avocado caused by a Xanthomonas sp. has been reported in California (2). This bacterium did not cause lesions of avocado leaves or fruit after inoculation (2). This appears to be the sole previous report of a xanthomonad being pathogenic to avocado and the symptoms observed in the Seychelles appear therefore very different from the ones reported in California. No major outbreak of bacterial leaf spot of avocado has been reported in the Seychelles archipelago since 2003. References: (1) N. Ah-You et al. Int. J. Syst. Evol. Microbiol. 59:306, 2009. (2) D. A. Cooksey et al. Plant Dis. 77:95, 1993. (3) O. Pruvost et al. J. Appl. Microbiol. 99:803, 2005. (4) J. Rademaker et al. Phytopathology 95:1098, 2005.

First Report of Xanthomonas cucurbitae Causing Bacterial Leaf Spot of Pumpkin on Réunion Island.

Bacterial leaf spot of cucurbits caused by Xanthomonas cucurbitae (3) can be of economic importance in tropical and subtropical production areas, most often within the Cucumis, Cucurbita, and Citrullus genera. The bacterium induces angular, water-soaked leaf spots, which sometimes turn necrotic with a chlorotic halo. Scab-like lesions on fruit may also be observed (1). During 2000, water-soaked, angular leaf lesions were collected from pumpkin (Cucurbita pepo) in a production field located at Petit Serré, Réunion Island. Yellow-pigmented Xanthomonas-like bacterial colonies were isolated on yeast peptone glucose agar. Amplified fragment length polymorphism analysis was performed on four pumpkin isolates together with reference strains of X. cucurbitae (LMG 690 [type strain] and LMG 8663) and the type strain of all other valid Xanthomonas species using SacI/MspI and four primer pairs (unlabeled MspI + 1 [A, C, T, or G] primers and 5'-labeled - SacI + C primer for the selective amplification step) (N. Ah-You, L. Gagnevin, P. A. D. Grimont, S. Brisse, X. Nesme, F. Chiroleu, L. Bui Thi Ngoc, E. Jouen, P. Lefeuvre, C. Verniére, and O. Pruvost, personal communication). The four isolates from pumpkin showed identical fingerprints and were most closely related to X. cucurbitae, with evolutionary genome divergences ≤0.05 (N. Ah-You et al., personal communication). One strain from diseased pumpkin (JW210-1) was further analyzed by multilocus sequence analysis using three housekeeping gene portions (atpD, dnaK, and gyrB) as described previously (N. Ah-You et al., personal communication). Although not fully identical, this strain displayed a similarity of >99% to the two reference strains of X. cucurbitae. Pumpkin and bottle-gourd (C. maxima), squash cv. aurore (C. pepo), cucumber cv. L-04 (Cucumis sativus), melon cv. cezanne (Cucumis melo), and watermelon cv. fou-nan (Citrullus lanatus) leaves were infiltrated (10 inoculation sites per leaf; three replicates) with bacterial suspensions prepared from strains JW210-1, LMG 690, and LMG 8663 and containing approximately 1 × 105 CFU ml-1. Negative controls consisted of leaves infiltrated with sterile tris buffer. Typical, water-soaked lesions that developed into necrotic spots were observed 6 to 8 days after inoculation for all inoculated plant species-strain combinations, but not for negative controls. One month after inoculation, mean Xanthomonas population sizes recovered from leaf lesions on KC semiselective medium (2) ranged from 1 × 107 to 1 × 108 CFU per lesion, typical of a compatible interaction. The reported outbreak was circumscribed to a single field and did not affect the local industry. No major outbreak of bacterial leaf spot of cucurbits has been reported on Réunion Island since 2000 on any host species of X. cucurbitae. References: (1) J. F. Bradbury. Page 309 in: Guide to Plant Pathogenic Bacteria. CAB International, Slough, UK, 1986. (2) O. Pruvost et al. J. Appl. Microbiol. 99:803, 2005. (3) L. Vauterin et al. Int. J. Syst. Bacteriol. 45:472, 1995.

First Report in Mauritius of Bacterial Leaf Blight of Syngonium Caused by Xanthomonas campestris pv. syngonii.

In November of 2006, necrotic leaf lesions with water-soaked margins were observed on Syngonium podophyllum in Floréal, Forest Side, and Réduit, Mauritius. Although not an economically important crop, the disease was of concern because syngonium is a host for Xanthomonas axonopodis pv. dieffenbachiae and the anthurium industry is of major economic importance in Mauritius. X. campestris pv. syngonii, described as the causal agent of bacterial leaf blight of syngonium (2), is genetically closely related to group 9.4 X. axonopodis pv. dieffenbachiae strains (3). In contrast to X. axonopodis pv. dieffenbachiae, X. campestris pv. syngonii strains are highly virulent on syngonium but are not pathogenic on anthurium or other Araceae, but both react similarly to the Xcd108 monoclonal antibody (Mab) (Agdia Inc., Elkhart, IN) and to a nested PCR assay designed for X. axonopodis pv. dieffenbachiae (4). X. axonopodis pv. dieffenbachiae and X. campestris pv. syngonii strains can be distinguished on the basis of restriction analysis of the amplicon of this PCR assay. Four pure cultures isolated from S. podophyllum were gram negative, yellow pigmented, and produced mucoid colonies on yeast peptone glucose agar (YPGA). One positive control strain of X. campestris pv. syngonii (LMG 9055 from the United States) and X. axonopodis pv. dieffenbachiae (LMG 695 from Brazil) were also used for all tests. All strains reacted positively with the Xcd108 MAb using indirect ELISA. DNA from all strains was amplified by the nested PCR assay, and the HincII restriction pattern of the amplicons identified strains from Mauritius as X. campestris pv. syngonii. Pathogenicity tests were performed on 8-month-old plants of Anthurium andreanum cv. Florida, Dieffenbachia maculata cv. Tropic Marianne, and S. podophyllum cv. Robusta by infiltrating suspensions containing ~1 × 105 CFU ml¯1 of each strain prepared from YPGA plates. Each strain was inoculated onto three young leaves (four inoculation sites per leaf) on two plants. Negative control plants received sterile Tris buffer solution (10 mM, pH 7.2). Plants were maintained in a growth chamber with day and night temperatures at 30 ± 1°C and 26 ± 1°C, respectively, 95 ± 5% relative humidity, 30 µmol·m¯2·s¯1 light intensity, and a photoperiod of 12 h (4). All strains caused typical water-soaked lesions 14 days after inoculation (dai) on syngonium. Lesions turned necrotic with chlorotic margins 27 to 34 dai. Typical bacterial blight lesions were observed on anthurium leaves inoculated with X. axonopodis pv. dieffenbachiae strain LMG 695, but no symptoms were observed 60 dai when strains from Mauritius and LMG 9055 were used. Amplified fragment length polymorphism analysis of four strains from Mauritius and additional reference, X. axonopodis pv. dieffenbachiae and X. campestris pv. syngonii strains, using SacI/MspI and four primer pairs (unlabeled MspI+1 [A, C, T, or G] primers and 5'-labeled-SacI+C primer for the selective amplification step) (1), showed that the strains from Mauritius could be distinguished from X. axonopodis pv. dieffenbachiae but were identical to X. campestris pv. syngonii strains from the United States and Réunion Island. References: (1) N. Ah-You et al. Phytopathology 97:1568, 2007. (2) R. S. Dickey and C. H. Zumoff. Phytopathology 77:1257, 1987. (3) J. L. W. Rademaker et al. Phytopathology 95:1098, 2005. (4) I. Robene-Soustrade et al. Appl. Environ. Microbiol. 72:1072, 2006.

Pathological Variations Within Xanthomonas campestris pv. mangiferaeindicae Support Its Separation Into Three Distinct Pathovars that Can Be Distinguished by Amplified Fragment Length Polymorphism.

ABSTRACT Bacterial black spot, caused by Xanthomonas campestris pv. mangiferaeindicae, is an important disease of mango (Mangifera indica). Several other plant genera of the family Anacardiaceae were described as host species for xanthomonads. We studied pathological variations among strains in a worldwide collection from several Anacardiaceae genera. Strains were classified into three pathogenicity groups. Group I strains (from the Old World) multiplied markedly in leaf tissue of mango and cashew (Anacardium occidentale). Group II strains (from Brazil) multiplied markedly in cashew leaf tissue, but not in mango. Moreover, mango leaves inoculated with group I and group II strains exhibited lesions with different morphologies, consistent with variations in symptomology previously reported on mango under field conditions. Group I strains produced black, raised lesions, consistent with the original description of the pathovar, whereas group II strains produced brownish, flat lesions. Group III strains produced a unique syndrome on ambarella (Spondias dulcis) and mombin (Spondias mombin). Based on evolutionary genome divergence derived from amplified fragment length polymorphism (AFLP) data, the three groups were genetically distinct and were related to groups 9.5, 9.6, and 9.4 of X. axonopodis identified by Rademaker, respectively. As each group was characterized by unique symptomology and/or host range, we propose that X. campestris pv. mangiferaeindicae be split into three pathovars of X. axonopodis: X. axonopodis pv. mangiferaeindicae, X. axonopodis pv. anacardii, and X. axonopodis pv. spondiae. Within pv. mangiferaeindicae sensu novo, AFLP data were consistent with that previously published for restriction fragment length polymorphism groups and suggested long-distance movement of the pathogen, likely through propagative material.

First Report in New Caledonia of Bacterial Blight of Anthurium Caused by Xanthomonas axonopodis pv. dieffenbachiae.

Xanthomonas axonopodis pv. dieffenbachiae, the causal agent of bacterial blight of aroids (BBA), has been reported in many regions and has been isolated on several host genera (1). During February 2004, in a nursery (Mont Dore) in New Caledonia, suspect symptoms have been observed on anthurium and dieffenbachia plants. A survey carried out on the entire island revealed that X. axonopodis pv. dieffenbachiae was present in 41 of the 89 nurseries inspected. During hot and humid weather, marginal or interveinal water-soaked spots surrounded by chlorotic or necrotic areas were observed, usually followed by a systemic phase (stem rotting and death of the plant). During the cold and dry season, only water-soaked spots were observed. Seventy pure cultures isolated from anthurium and dieffenbachia were gram negative, yellow pigmented, and had a mucoid aspect when grown on rich media. All strains responded positively to the Xcd108 monoclonal antibody (Agdia Inc., Elkhart, IN) raised against X. axonopodis pv. dieffenbachiae using indirect ELISA. A set of 18 strains (isolated from 15 anthurium and 3 dieffenbachia plants located in different sites) were further characterized by molecular and pathogenicity tests. All strains reacted positively using a specific nested PCR assay (1). Pathogenicity tests were performed on 8-month-old plants of Anthurium andreanum 'Carré', Dieffenbachia maculata 'Tropic Marianne', and Syngonium podophyllum 'Robusta' by syringue infiltration of a suspension containing approximately 105 CFU mL-1. Each strain was inoculated onto three young leaves (four inoculation sites per leaf) on two plants. Control plants received sterile Tris buffer solution (10 mM, pH 7.2). Plants were maintained in a growth chamber with day and night temperatures of 30 ± 1°C and 26 ± 1°C, respectively, 95 ± 5% relative humidity, 30 µmol m-2·s-1 light intensity and a photoperiod of 12 h (1). On all plants, all strains caused typical water-soaked symptoms within 10 days, evolving into chlorotic then necrotic areas after 20 to 24 days. Amplified fragment length polymorphism (AFLP) markers revealed three haplotypes among these strains, which suggests that several introduction events may have occurred. These AFLP fingerprints were compared with other Xanthomonas spp. pathovars, including most of X. axonopodis pv. dieffenbachiae strains obtained from international culture collections, and were found to belong to the same genomic group as all the X. axonopodis pv. dieffenbachiae strains pathogenic on anthurium. Importation in New Caledonia of aroids from countries in which X. axonopodis pv. dieffenbachiae is present (Hawaii, French Polynesia, the Netherlands, and Australia) occurred before 2004. The wide distribution of BBA is very likely due to the plant material movements occurring in New Caledonia and suggests that the pathogen may have been present on the territory some years before the first official case. Reference: (1) I. Robene-Soustrade et al. Appl. Environ. Microbiol. 72:1072, 2006.

[Discrepancies between the results of phlebography and Doppler ultrasonography in the diagnosis of asymptotic venous thrombosis after total hip prosthesis. False negatives of phlebography or false positives of Doppler ultrasonography]. / Discordances de résultats entre phlébographie et écho-Doppler dans le dépistage de thromboses veineuses asymptomatiques après prothèse totale de hanche. Faux-négatifs de la phlébographie ou faux-positifs de l'écho-Doppler?

OBJECTIVES: Assess the diagnostic performance of phlebography and discrepancies with duplex ultrasonography in screening for asymptomatic deep vein thrombosis after total hip arthroplasty. Search for arguments which would favor contributing false-negatives or false-positives to one of the two exploration methods. PATIENTS AND METHODS: The study included 24 patients who underwent the 2 explorations independently between day 7 and day 14 in a multicentric therapeutic trial of a new heparin. Discrepancies between the two techniques were recorded. Diagnostic performance of phlebography was calculated from contingency tables. The phlebograms were then reviewed with knowledge of the duplex ultrasonographic findings. The course of the venous thrombus after treatment was monitored with duplex ultrasonography. RESULTS: Phlebography allowed the diagnosis of thrombus formation in 9 patients. Ultrasonography provided the diagnosis in 14 cases out of 24. Sensitivity of phlebography compared with duplex ultrasonography was 64% (95% CI = 35.1-87.2) (9/14) and specificity was 100% (95% CI = 69.1-100) (10/10). Several localizations were not visualized with phlebography: 3 partially obstructive thrombi near the femoral junction, and 38 distal thrombi including 31 soleus thrombi (fig. 5). When the phlebograms were reviewed again, defects compatible with a partial thrombus were found for the 3 proximal localizations and for 2 of the distal localizations (fig. 1, 2, 4). None of the soleus localizations were visualized. Duplex ultrasonographic follow-up concerned 12 of the 14 patients with a thrombus identified by duplex ultrasonography involving 44 localizations. In five case, the operator was different from the operator for the initial duplex ultrasonography. Follow-up revealed: 1 new localization, 37 narrowings and 6 repermeabilizations. DISCUSSION: The coherence of the duplex ultrasonographic follow-up and the presence of images compatible with partial thrombus on the revised phlebograms which had been initially interpreted as wash out flow constitute a group of arguments suggesting that the discrepancies observed should be considered as phlebography false-negatives. This defect in the sensitivity of phlebography has been reported by others in the literature both for distal and proximal localizations.