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.

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.

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.

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.

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.

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.

Quantitative and molecular epidemiology of bacterial blight of onion in seed production fields.

ABSTRACT Onion, a biennial plant species, is threatened by the emerging, seed-borne, and seed-transmitted Xanthomonas axonopodis pv. allii. Bacterial blight epidemics were monitored in seed production fields over two seasons. Temporal disease progress was different between the two seasons, with final incidence ranging from 0.04 to 0.06 in 2003 and from 0.44 to 0.61 in 2004. The number of hours with temperatures above 24 degrees C was the best descriptor for predicting the number of days after inoculation for bacterial blight development on inoculated plants. Fitting the beta-binomial distribution and binary power law analysis indicated aggregated patterns of disease incidence data. The beta-binomial distribution was superior to the binomial distribution for 97% of the examined data sets. Spatial dependency ranged from 5.9 to 15.2 m, as determined by semivariance analysis. Based on amplified fragment length polymorphism (AFLP) analysis, it was concluded that plots predominantly were infected by the inoculated haplotype. A single other haplotype was identified by AFLP in all plots over the 2 years, and its detection in the field always followed wind-driven rains. X. axonopodis pv. allii-contaminated seed were detected by semiselective isolation and a nested polymerase chain reaction assay at levels up to 0.05% when final disease incidence was 0.61. Contaminated seed originated from both diseased and asymptomatic plants.