MultiLocus Variable number of tandem repeat Analysis (MLVA) has been extensively used to examine epidemiological and evolutionary issues on monomorphic human pathogenic bacteria, but not on bacterial plant pathogens of agricultural importance albeit such tools would improve our understanding of their epidemiology, as well as of the history of epidemics on a global scale. Xanthomonas citri pv. citri is a quarantine organism in several countries and a major threat for the citrus industry worldwide. We screened the genomes of Xanthomonas citri pv. citri strain IAPAR 306 and of phylogenetically related xanthomonads for tandem repeats. From these in silico data, an optimized MLVA scheme was developed to assess the global diversity of this monomorphic bacterium. Thirty-one minisatellite loci (MLVA-31) were selected to assess the genetic structure of 129 strains representative of the worldwide pathological and genetic diversity of X. citri pv. citri. Based on Discriminant Analysis of Principal Components (DAPC), four pathotype-specific clusters were defined. DAPC cluster 1 comprised strains that were implicated in the major geographical expansion of X. citri pv. citri during the 20th century. A subset of 12 loci (MLVA-12) resolved 89% of the total diversity and matched the genetic structure revealed by MLVA-31. MLVA-12 is proposed for routine epidemiological identification of X. citri pv. citri, whereas MLVA-31 is proposed for phylogenetic and population genetics studies. MLVA-31 represents an opportunity for international X. citri pv. citri genotyping and data sharing. The MLVA-31 data generated in this study was deposited in the Xanthomonas citri genotyping database (http://www.biopred.net/MLVA/).

Molecular epidemiology studies further our understanding of migrations of phytopathogenic bacteria, the major determining factor in their emergence. Asiatic citrus canker, caused by Xanthomonas citri pv. citri, was recently reported in Mali and Burkina Faso, a region remote from other contaminated areas. To identify the origin and pathways of these emergences, we used two sets of markers, minisatellites and microsatellites, for investigating different evolutionary scales. Minisatellite typing suggested the introduction of two groups of strains in Mali (DAPC 1 and DAPC 2), consistent with microsatellite typing. DAPC 2 was restricted to Bamako district, whereas DAPC 1 strains were found much more invasive. The latter strains formed a major clonal complex based on microsatellite data with the primary and secondary founders detected in commercial citrus nurseries and orchards. This suggests that human activities played a major role in the spread of DAPC 1 strains via the movement of contaminated propagative material, further supported by the frequent lack of differentiation between populations from geographically distant nurseries and orchards. Approximate Bayesian Computation analyses supported the hypothesis that strains from Burkina Faso resulted from a bridgehead invasion from Mali. Multi-locus variable number of tandem repeat analysis and Approximate Bayesian Computation are useful for understanding invasion routes and pathways of monomorphic bacterial pathogens.

Citrus canker, caused by Xanthomonas citri pv. citri, is a bacterial disease of economic importance in tropical and sub-tropical citrus-producing areas (EPPO-PQR online database). X. citri pv. citri causes severe infection in a wide range of citrus species, and induces erumpent, callus-like lesions with water-soaked margins leading to premature fruit drop and twig dieback. It has consequently been subjected to eradication efforts and international regulations. It was first described on the African continent in South Africa at the beginning of the 20th century, from which it was eventually eradicated. Since 2006, several outbreaks caused by phylogenetically diverse strains of X. citri pv. citri have been reported from several African countries (Ethiopia, Mali, Senegal, and Somalia). In July 2011, citrus canker in Burkina Faso was suspected in the area adjacent to the Sikassso Province of Mali where X. citri pv. citri has been confirmed. In November and December 2012, leaves of clementine (Citrus clementina), lemon (C. limon), Volkamer lemon (C. volkameriana), sweet orange (C. sinensis), tangelo (C. paradisi× C. reticulata), and mandarin (C. reticulata) were collected from orchards with trees showing symptoms of citrus canker in the Comoé, Houet, and Kénédougou provinces of Burkina Faso. Isolations performed using KC semi-selective medium (4) recovered 45 Xanthomonas-like strains. All Xanthomonas-like strains were tentatively identified as X. citri pv. citri by PCR (4/7 primers) using IAPAR 306 and sterile distilled water as the positive and negative controls, respectively (3). Among these, two strains (LK4-4 and LK4-5) produced a ‘fuscans’-like brown diffusible pigment, a phenotype never reported previously for X. citri pv. citri. MultiLocus Sequence Analysis targeting six housekeeping genes (atpD, dnaK, efp, gltA, gyrB, and lepA) (1,2) fully identified seven strains from Burkina Faso (LJ301-1, LJ303-1, LK1-1, LK2-6, LK4-3, LK4-4, and LK4-5) as X. citri pv. citri (and not to any other Xanthomonas pathovars pathogenic to citrus or host range-restricted pathotypes of pathovar citri), and more specifically as sequence type ST2 which is composed mostly of pathotype A strains of X. citri pv. citri (2). The same seven strains were inoculated to at least four leaves of each of grapefruit cv. Henderson, Mexican lime SRA 140 (C. aurantifolia), Tahiti lime SRA 58 (C. latifolia), and sweet orange cv. Washington Navel, using a detached leaf assay (2). All strains developed typical erumpent, callus-like tissue at wound sites on all citrus species inoculated. No lesions developed on the negative control (sterile 10 mM tris buffer). Koch's postulate was fulfilled after reisolation of Xanthomonas-like yellow colonies from symptoms on Mexican lime produced by the seven strains. Boiled bacterial suspensions were assayed by PCR with 4/7 primers (3) and produced the expected 468-bp amplicon in contrast with the PCR negative control. To our knowledge, this is the first report of X. citri pv. citri in Burkina Faso. Citrus canker-free nurseries and grove sanitation should be implemented for reducing the prevalence of Asiatic canker in Burkina Faso and a thorough survey of citrus nurseries and groves in the region should be conducted. References: (1) N. F. Almeida et al. Phytopathology 100:208, 2010. (2) L. Bui Thi Ngoc et al. Int. J. Syst. Evol. Microbiol. 60:515, 2010. (3) J. S. Hartung et al. Phytopathology 86:95, 1996. (4) O. Pruvost et al. J. Appl. Microbiol. 99:803, 2005.

Noroviruses (NoV) are responsible for many shellfish outbreaks. Purification processes may be applied to oysters before marketing to decrease potential fecal pollution. This step is rapidly highly effective in reducing Escherichia coli; nevertheless, the elimination of virus genomes has been described to be much slower. It is therefore important to identify (i) the purification conditions that optimize virus removal and (ii) the mechanism involved. To this end, the effects of oyster stress, nutrients, and the presence of a potential competitor to NoV adhesion during purification were investigated using naturally contaminated oysters. Concentrations of NoV (genomes) and of the viral indicator F-specific RNA bacteriophage (FRNAPH; genomes and infectious particles) were regularly monitored. No significant differences were observed under the test conditions. The decrease kinetics of both virus genomes were similar, again showing the potential of FRNAPH as an indicator of NoV behavior during purification. The T90 (time to reduce 90% of the initial titer) values were 47.8 days for the genogroup I NoV genome, 26.7 days for the genogroup II NoV genome, and 43.9 days for the FRNAPH-II genome. Conversely, monitoring of the viral genomes could not be used to determine the behavior of infectious viruses because the T90 values were more than two times lower for infectious FRNAPH (20.6 days) compared to their genomes (43.9 days). Finally, this study highlighted that viruses are primarily inactivated in oysters rather than released in the water during purification processes. IMPORTANCE This study provides new data about the behavior of viruses in oysters under purification processes and about their elimination mechanism. First, a high correlation has been observed between F-specific RNA bacteriophages of subgroup II (FRNAPH-II) and norovirus (NoV) in oysters impacted by fecal contamination when both are detected using molecular approaches. Second, when using reverse transcription-quantitative PCR and culture to detect FRNAPH-II genomes and infectious FRNAPH in oysters, respectively, it appears that genome detection provides limited information about the presence of infectious particles. The comparison of both genomes and infectious particles highlights that the main mechanism of virus elimination in oysters is inactivation. Finally, this study shows that none of the conditions tested modify virus removal.