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.

INTRODUCTIONThe classification of some phytopathogenic bacterial species was initially based on host specialization, although this was evidently not sound taxonomically (Dowson, 1939;Stolp et al., 1965). Nevertheless, plant pathologists and phytosanitary policy regulators required names for plant-pathogenic bacteria that clearly reflected their pathogenic ability. This led to the development of the pathovar classification at an infrasubspecific level (Dye et al., 1980). Furthermore, strains with differential host ranges within a pathovar were sometimes described (e.g. xanthomonads pathogenic to citrus or cassava) and often referred to as pathotypes (Civerolo & Fan, 1982;Verdier et al., 1998).A total of four xanthomonads are pathogenic to plant species within the Rutaceae. Two pathogens that cause spot diseases of citrus (i.e. water-soaked spots turning into flat, necrotic lesions) have been described as two distinct Details of strains and primers, maximum-likelihood trees based on individual sequences and a neighbour-joining tree based on concatenated sequences are available as supplementary material with the online version of this paper.

Efficient control of Xanthomonas axonopodis pv. dieffenbachiae, the causal agent of anthurium bacterial blight, requires a sensitive and reliable diagnostic tool. A nested PCR test was developed from a sequencecharacterized amplified region marker identified by randomly amplified polymorphic DNA PCR for the detection of X. axonopodis pv. dieffenbachiae. Serological and pathogenicity tests were performed concurrently with the nested PCR test with a large collection of X. axonopodis pv. dieffenbachiae strains that were isolated worldwide and are pathogenic to anthurium and/or other aroids. The internal primer pair directed amplification of the expected product (785 bp) for all 70 X. axonopodis pv. dieffenbachiae strains pathogenic to anthurium tested and for isolates originating from syngonium and not pathogenic to anthurium. This finding is consistent with previous studies which indicated that there is a high level of relatedness between strains from anthurium and strains from syngonium. Strains originating from the two host genera can be distinguished by restriction analysis of the amplification product. No amplification product was obtained with 98 strains of unrelated phytopathogenic bacteria or saprophytic bacteria from the anthurium phyllosphere, except for a weak signal obtained for one X. axonopodis pv. allii strain. Nevertheless, restriction enzyme analysis permitted the two pathovars to be distinguished. The detection threshold obtained with pure cultures or plant extracts (10 3 CFU ml ؊1 ) allowed detection of the pathogen from symptomless contaminated plants. This test could be a useful diagnostic tool for screening propagation stock plant material and for monitoring international movement of X. axonopodis pv. dieffenbachiae.

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 β 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.

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 variablenumber 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.

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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.

On Reunion Island successful biological control of the sugarcane white grub Hoplochelus marginalis Fairmaire (Coleoptera: Melolonthidae) has been conducted for decades with strains from the entomopathogenic fungal genus Beauveria (Ascomycota: Hypocreales). A study based on morphological characters combined with a multisequence phylogenetic analysis of genes that encode the translation elongation factor 1-alpha (TEF1), RNA polymerase II largest subunit (RPB1), RNA polymerase II second largest subunit (RPB2) and the Bloc nuc intergenic region was carried out on Beauveria strains isolated on Reunion and Madagascar from H. marginalis. This study revealed that these strains, previously identified as Beauveria brongniartii, did not match that species and are closely related to but still distinct from B. malawiensis strains. Therefore we describe the Reunion Island fungus as the new species B. hoplocheli.