Genetic Structure and TALome Analysis Highlight a High Level of Diversity in Burkinabe Xanthomonas Oryzae pv. oryzae Populations.

Bacterial Leaf Blight of rice (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is a major threat for food security in many rice growing countries including Burkina Faso, where the disease was first reported in the 1980's. In line with the intensification of rice cultivation in West-Africa, BLB incidence has been rising for the last 15 years. West-African strains of Xoo differ from their Asian counterparts as they (i) are genetically distant, (ii) belong to new races and, (iii) contain reduced repertoires of Transcription Activator Like (TAL) effector genes. In order to investigate the evolutionary dynamics of Xoo populations in Burkina Faso, 177 strains were collected from 2003 to 2018 in three regions where BLB is occurring. Multilocus VNTR Analysis (MLVA-14) targeting 10 polymorphic loci discriminated 24 haplotypes and showed that Xoo populations were structured according to their geographical localization and year of collection. Considering their major role in Xoo pathogenicity, we assessed the TAL effector repertoires of the 177 strains upon RFLP-based profiling. Surprisingly, an important diversity was revealed with up to eight different RFLP patterns. Finally, comparing neutral vs. tal effector gene diversity allowed to suggest scenarios underlying the evolutionary dynamics of Xoo populations in Burkina Faso, which is key to rationally guide the deployment of durably resistant rice varieties against BLB in the country.

High-Quality Draft Genome Sequences of Two Xanthomonas Pathotype Strains Infecting Aroid Plants.

We present here the draft genome sequences of bacterial pathogens of the Araceae family, Xanthomonas axonopodis pv. dieffenbachiae LMG 695 and Xanthomonas campestris pv. syngonii LMG 9055, differing in host range. A comparison between genome sequences will help understand the mechanisms involved in tissue specificity and adaptation to host plants.

New multilocus variable-number tandem-repeat analysis tool for surveillance and local epidemiology of bacterial leaf blight and bacterial leaf streak of rice caused by Xanthomonas oryzae.

Multilocus variable-number tandem-repeat analysis (MLVA) is efficient for routine typing and for investigating the genetic structures of natural microbial populations. Two distinct pathovars of Xanthomonas oryzae can cause significant crop losses in tropical and temperate rice-growing countries. Bacterial leaf streak is caused by X. oryzae pv. oryzicola, and bacterial leaf blight is caused by X. oryzae pv. oryzae. For the latter, two genetic lineages have been described in the literature. We developed a universal MLVA typing tool both for the identification of the three X. oryzae genetic lineages and for epidemiological analyses. Sixteen candidate variable-number tandem-repeat (VNTR) loci were selected according to their presence and polymorphism in 10 draft or complete genome sequences of the three X. oryzae lineages and by VNTR sequencing of a subset of loci of interest in 20 strains per lineage. The MLVA-16 scheme was then applied to 338 strains of X. oryzae representing different pathovars and geographical locations. Linkage disequilibrium between MLVA loci was calculated by index association on different scales, and the 16 loci showed linear Mantel correlation with MLSA data on 56 X. oryzae strains, suggesting that they provide a good phylogenetic signal. Furthermore, analyses of sets of strains for different lineages indicated the possibility of using the scheme for deeper epidemiological investigation on small spatial scales.

Draft Genome Sequence of the Flagellated Xanthomonas fuscans subsp. fuscans Strain CFBP 4884.

We report the draft genome sequence of the flagellated strain CFBP 4884 of Xanthomonas fuscans subsp. fuscans, which was isolated in an outbreak of common bacterial blight of beans along with non-flagellated strains. Comparative genomics will allow one to decipher the genomic diversity of strains cohabiting in epidemics.

Draft Genome Sequence of Xanthomonas axonopodis pv. allii Strain CFBP 6369.

We report here the draft genome sequence of Xanthomonas axonopodis pv. allii strain CFBP 6369, the causal agent of bacterial blight of onion. The draft genome has a size of 5,425,942 bp and a G+C content of 64.4%.

First Report of Xanthomonas citri pv. mangiferaeindicae Causing Mango Bacterial Canker on Mangifera indica in Ivory Coast.

Xanthomonas citri pv. mangiferaeindicae causing bacterial canker (or black spot) is a major mango (Mangifera indica L.) pathogen in tropical and subtropical areas (3). The bacterium infects a wide range of mango cultivars, and induces raised, angular, black leaf lesions, sometimes with a yellow chlorotic halo. Fruit symptoms first appear as small water-soaked spots on the lenticels turning into star-shaped, erumpent lesions, which exude an infectious gum, yielding tear-stain patterns. Severe infections cause severe defoliation and/or premature fruit drop. Twig cankers are potential sources of inoculum and weaken branch resistance to winds. Drastic yield losses have been reported at grove scale for susceptible cultivars (3). Mango leaves showing typical angular, black, raised leaf lesions were first observed and collected in April 2014 from trees cv. Kent in five localities of the Korhogo province of Ivory Coast (i.e., the major commercial mango-growing area in this country). Non-pigmented Xanthomonas-like colonies were isolated on KC semi-selective medium (4). Five strains (LL60-1, LL61-1, LL62-1, LL63-1, and LL64-1), one from each locality, were compared by multilocus sequence analysis (MLSA) to the type strain of X. citri and the pathotype strain of several X. citri pathovars, including pvs. anacardii and mangiferaeindicae. This assay targeted the atpD, dnaK, efp, and gyrB genes, as described previously (2). Nucleotide sequences were 100% identical to those of the pathotype strain of X. citri pv. mangiferaeindicae whatever the gene assayed, but differed from any other assayed X. citri pathovar. Leaves of mango cv. Maison Rouge from the youngest vegetative flush were infiltrated (10 inoculation sites/leaf for three replicate leaves on different plants/bacterial strain) as detailed previously (1) with the same five strains. Bacterial suspensions (~1 × 105 cfu/ml) were prepared in 10 mM Tris buffer (pH 7.2) from 16-h-old cultures on YPGA (7 g yeast, 7 g peptone, 7 g glucose, and 18 g agar/liter, pH 7.2). The negative control treatment consisted of three leaves infiltrated with sterile Tris buffer (10 sites/leaf). Plants were incubated in a growth chamber at 30 ± 1°C by day and 26 ± 1°C by night (12-h day/night cycle) at 80 ± 5% RH. All leaves inoculated with the strains from Ivory Coast showed typical symptoms of bacterial canker a week after inoculation. No lesions were recorded from the negative controls. The pathogen was recovered at high population densities (>1 × 106 cfu/lesion) from leaf lesions, typical of a compatible interaction (1) and isolated colonies were identified as the target by atpD sequencing (2). Koch's postulates have therefore been fully verified. This is the first report of the disease in Ivory Coast, a country which has been an internationally significant mango exporter (up to 15,000 tons per year) over the last two decades. A high disease incidence and severity were observed, outlining the need for implementing integrated pest management in mango groves and the production of disease-free nursery stock. This report further expands the distribution of the pathogen in West Africa after its first description from Ghana in 2011 (5) and subsequently in other neighboring countries. References: (1) N. Ah-You et al. Phytopathology 97:1568, 2007. (2) L. Bui Thi Ngoc et al. Int. J. Syst. Evol. Microbiol. 60:515, 2010. (3) L. Gagnevin and O. Pruvost. Plant Dis. 85:928, 2001. (4) O. Pruvost et al. J. Appl. Microbiol. 99:803, 2005. (5) O. Pruvost et al. Plant Dis. 95:774, 2011.

High-Quality Draft Genome Sequence of Xanthomonas alfalfae subsp. alfalfae Strain CFBP 3836.

We report the high-quality draft genome sequence of Xanthomonas alfalfae subsp. alfalfae strain CFBP 3836, the causal agent of bacterial leaf and stem spot in lucerne (Medicago sativa). Comparative genomics will help to decipher the mechanisms provoking disease and triggering the defense responses of this pathogen of the model legume Medicago truncatula.

High-Quality Draft Genome Sequences of Xanthomonas axonopodis pv. glycines Strains CFBP 2526 and CFBP 7119.

We report here the high-quality draft genome sequences of two strains of Xanthomonas axonopodis pv. glycines, the causal agent of bacterial pustule on soybeans. Comparison of these genomes with those of phylogenetically closely related pathovars of Xanthomonas spp. will help to understand the mechanisms involved in host specificity and adaptation to host plants.

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 in Mali of Xanthomonas citri pv. mangiferaeindicae Causing Mango Bacterial Canker on Mangifera indica.

Bacterial canker (or black spot) of mango caused by Xanthomonas citri pv. mangiferaeindicae is an important disease in tropical and subtropical areas (1). X. citri pv. mangiferaeindicae can cause severe infection in a wide range of mango cultivars and induces raised, angular, black leaf lesions, sometimes with a chlorotic halo. Severe leaf infection may result in abscission. Fruit symptoms appear as small, water-soaked spots on the lenticels that later become star shaped, erumpent, and exude an infectious gum. Often, a "tear stain" infection pattern is observed on the fruit. Severe fruit infections cause premature drop. Twig cankers are potential sources of inoculum and weaken branch resistance to winds. Yield loss up to 85% has been reported at grove scale for susceptible cultivars (1). Suspected leaf lesions of bacterial canker were collected in July 2010 from mango trees in four, six, and three localities of the Koulikoro, Sikasso, and Bougouni provinces of Mali, respectively (i.e., the major mango-growing areas in this country). Nonpigmented Xanthomonas-like colonies were isolated on KC semiselective medium (3). Twenty-two strains from Mali were identified as X. citri pv. mangiferaeindicae based on IS1595-ligation-mediated PCR (4) and they produced fingerprints fully identical to that of strains isolated from Ghana and Burkina Faso. Five Malian strains (LH409, LH410, LH414, LH415-3, and LH418) were compared by multilocus sequence analysis (MLSA) to the type strain of X. citri and the pathotype strain of several X. citri pathovars, including pvs. anacardii and mangiferaeindicae. This assay targeted the atpD, dnaK, efp, and gyrB genes, as described previously (2). Nucleotide sequences were 100% identical to those of the pathotype strain of X. citri pv. mangiferaeindicae whatever the gene assayed, but differed from any other assayed X. citri pathovar. Leaves of mango cv. Maison Rouge from the youngest vegetative flush were infiltrated (10 inoculation sites per leaf for three replicate leaves on different plants per bacterial strain) with the same five strains from Mali. Bacterial suspensions (~1 × 105 CFU/ml) were prepared in 10 mM Tris buffer (pH 7.2) from 16-h-old cultures on YPGA (7 g of yeast, 7 g of peptone, 7 g of glucose, and 18 g of agar/liter, pH 7.2). The negative control treatment consisted of three leaves infiltrated with sterile Tris buffer (10 sites per leaf). Plants were incubated in a growth chamber at 30 ± 1°C by day and 26 ± 1°C by night (12-h/12-h day/night cycle) at 80 ± 5% relative humidity. All leaves inoculated with the Malian strains showed typical symptoms of bacterial canker a week after inoculation. No lesions were recorded from the negative controls. One month after inoculation, mean X. citri pv. mangiferaeindicae population sizes ranging from 5 × 106 to 1 × 107 CFU/lesion were recovered from leaf lesions, typical of a compatible interaction (1). To our knowledge, this is the first report of the disease in Mali. Investigations from local growers suggest that the disease may have been present for some years in Mali but likely less than a decade. A high disease incidence and severity were observed, suggesting the suitability of environmental conditions in this region for the development of mango bacterial canker. References: (1) N. Ah-You et al. Phytopathology 97:1568, 2007. (2) L. Bui Thi Ngoc et al. Int. J. Syst. Evol. Microbiol. 60:515, 2010. (3) O. Pruvost et al. J. Appl. Microbiol. 99:803, 2005. (4) O. Pruvost et al. Phytopathology 101:887, 2011.

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.

First Report in Burkina Faso of Xanthomonas citri pv. mangiferaeindicae Causing Bacterial Canker on Mangifera indica.

Bacterial canker of mango (or bacterial black spot) caused by Xanthomonas citri pv. mangiferaeindicae, is an economically important disease in tropical and subtropical areas (1). X. citri pv. mangiferaeindicae can cause severe infection on a wide range of mango cultivars and induces raised, angular, black leaf lesions, sometimes with a chlorotic halo. Fruit symptoms are black, star shaped, erumpent, and exude an infectious gum. A survey was conducted in Burkina Faso in May 2010 because budwood putatively associated with an outbreak of bacterial canker in Ghana had originated from Burkina Faso (3). Leaves and twigs with suspected bacterial canker lesions were collected from mango trees of the cvs. Amélie, Brooks, and Kent and from seedlings at five localities in Comoe and Houet provinces. Severe infections were observed on the sampled trees in Burkina Faso and leaf symptoms were typical of bacterial canker. Leaves were surface sterilized for 15 to 30 s with 70% ethanol, and nonpigmented, Xanthomonas-like bacterial colonies were isolated on KC semiselective agar medium (1). On the basis of an IS1595-ligation mediated PCR assay, 18 strains from Burkina Faso produced identical fingerprints and were identified as X. citri pv. mangiferaeindicae (4). The haplotype for strains from Burkina Faso was identical to that reported from Ghana (3). Three strains from Burkina Faso (LH127-2, LH130-1, and LH131-1) were compared by multilocus sequence analysis (MLSA) with the type strain of X. citri and the pathotype strain of several X. citri pathovars, including pvs. anacardii and mangiferaeindicae, targeting the atpD, dnaK, efp, and gyrB genes (2). Nucleotide sequences were 100% identical to those of the pathotype strain of X. citri pv. mangiferaeindicae, regardless of the gene assayed, but differed from any other X. citri pathovar assayed. Leaves of mango cv. Maison Rouge, taken from the youngest vegetative flush, were infiltrated (10 inoculation sites per leaf for three replicate leaves on different plants per bacterial strain) with the same three strains from Burkina Faso. Bacterial suspensions (approximately 1 × 105 CFU/ml) were prepared in 10 mM Tris buffer (pH 7.2) from 16-h-old solid cultures on YPG agar (7 g of yeast, 7 g of peptone, 7 g of glucose, and 18 g of agar per liter, pH 7.2). The negative control treatment consisted of three leaves infiltrated with sterile Tris buffer (10 sites per leaf). Plants were incubated in a growth chamber at 30 ± 1°C by day and 26 ± 1°C by night (12-h/12-h day/night cycle) at 80 ± 5% relative humidity. Typical symptoms of bacterial canker were observed for all assayed strains 1 week after inoculation; no symptoms were observed from negative control leaves. One month after inoculation, mean X. citri pv. mangiferaeindicae populations ranging from 2 × 107 to 8 × 107 CFU/leaf lesion were recovered, which was typical of a compatible interaction (1). The origin of inoculum associated with the bacterial canker outbreak in Burkina Faso is unknown. This report documents severe infections in Burkina Faso (including premature fruit drop due to severe fruit infections) and confirms the presence of bacterial canker in western Africa. A more extensive survey for the disease should be conducted in this region. References: (1) N. Ah-You et al. Phytopathology 97:1568, 2007. (2) L. Bui Thi Ngoc et al. Int. J. Syst. Evol. Microbiol. 60:515, 2010. (3) O. Pruvost et al. Plant Dis. 95:774, 2011. (4) O. Pruvost et al. Phytopathology 101:887, 2011.

First Report in Ghana of Xanthomonas citri pv. mangiferaeindicae Causing Mango Bacterial Canker on Mangifera indica.

Bacterial canker of mango (or bacterial black spot), caused by Xanthomonas citri pv. mangiferaeindicae, is an economically important disease in tropical and subtropical producing areas (1). X. citri pv. mangiferaeindicae can cause severe infection in a wide range of mango cultivars and induces raised, angular, black leaf lesions, sometimes with a chlorotic halo. Several months after infection, leaf lesions dry and turn light brown or ash gray. Severe leaf infection may result in abscission. Fruit symptoms appear as small water-soaked spots on the lenticels. These spots later become star shaped, erumpent, and exude an infectious gum. Often, a "tear stain" infection pattern is observed on the fruit. Severe fruit infections will cause premature fruit drop. Twig cankers are potential sources of inoculum and weaken resistance of branches to wind damage. Leaf lesions with suspected bacterial canker were collected in January 2010 from mango trees cv. Keitt in several blocks at the Integrated Tamale Fruit Company, Ghana. Non-pigmented Xanthomonas-like bacterial colonies were isolated on Kasugamycin-Cephalexin semiselective agar medium (3). On the basis of IS1595-Ligation Mediated-PCR data, 16 strains from Ghana produced identical fingerprints and were identified as X. citri pv. mangiferaeindicae (4). The haplotype corresponding to the Ghanaian strains had not been previously reported. On the basis of multidimensional scaling (4), this haplotype clustered together with a group of strains from multiple origins and the analysis was not informative as an aid for tracing back the outbreak. Five Ghanaian strains (LH2-3, LH2-6, LH2-8, LH2-11, and LH2-15) were compared by multilocus sequence analysis to the type strain of X. citri and the pathotype strain of several X. citri pathovars, including pvs. anacardii and mangiferaeindicae. This assay targeted the atpD, dnaK, efp, and gyrB genes as described previously (2). Nucleotide sequences were 100% identical to those of the pathotype strain of X. citri pv. mangiferaeindicae whatever the gene assayed, but differed from any other assayed X. citri pathovar. Mango cv. Maison Rouge leaves from the youngest vegetative flush were infiltrated (10 inoculation sites per leaf, three replicate plants) using inoculum of each of the same five Ghanaian strains made from suspensions in Tris buffer containing ~1 × 105 CFU/ml. Negative control treatments consisted of leaves infiltrated with sterile Tris buffer. Typical symptoms of bacterial canker were observed for all assayed strains a week after inoculation. No lesions were recorded from the negative control. One month after inoculation, mean X. citri pv. mangiferaeindicae population sizes ranging from 4 × 107 to 1 × 108 CFU/lesion were recovered from leaf lesions, typical of a compatible interaction (1). High disease prevalence was observed in Ghana, indicating the suitability of environmental conditions in this region for the development of mango bacterial canker. The budwood for these blocks was imported from Burkina Faso in 2002 and symptoms were observed in these blocks shortly after establishment. To our knowledge, this is the first report of mango bacterial canker in Western Africa. References: (1) N. Ah-You et al. Phytopathology 97:1568, 2007. (2) L. Bui Thi Ngoc et al. Int. J. Syst. Evol. Microbiol. 60:515, 2010. (3) O. Pruvost et al. J. Appl. Microbiol. 99:803, 2005. (4) O. Pruvost et al. Phytopathology. Online publication. DOI:10.1094/PHYTO-11-10-0304, 2011.

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.

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 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.

Development of 14 minisatellite markers for the citrus canker bacterium, Xanthomonas citri pv. citri.

We screened the genome of Xanthomonas citri pv. citri strain 306 for tandem repeats. A multiplex polymerase chain reaction protocol was used to assess the genetic diversity of 239 strains of X. citri pv. citri from Asia. The total number of alleles per locus ranged from three to 20. Using pooled data sets, 223 different haplotypes were identified. Successful amplifications were obtained at most loci for seven other X. citri pathovars. This typing scheme is expected to be useful at different spatial scales for population studies of pathovars of X. citri, several of which cause plant diseases of economic importance.

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 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.