Bridgehead invasion of a monomorphic plant pathogenic bacterium: Xanthomonas citri pv. citri, an emerging citrus pathogen in Mali and Burkina Faso.

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.

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.

First Report of Xanthomonas citri pv. citri Causing Citrus Canker in Mali.

Citrus canker, caused by Xanthomonas citri pv. citri, is a bacterial disease of economic importance in tropical and subtropical citrus-producing areas. X. citri pv. citri can cause 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. Citrus canker occurs in Asia, South America, the United States, parts of Oceania, and some islands off the African continent (Comoros, Mauritius, Reunion, and Seychelles islands). It was described on the African continent, but in some cases, diagnosis errors might have occurred. The only well-documented outbreak occurred in South Africa where it was eradicated at the beginning of the twentieth century. In 2004, citrus canker symptoms on limes, sweet oranges, tangerines, and sour oranges were reported from different orchards around Bamako and in the Koulikoro Province of Mali. Isolations were performed on KC semiselective medium (2). PCR was used to check the identity of the pathogen, testing 21 Xanthomonas-like strains collected from citrus in the epidemic area. X. citri pv. citri strain CFBP 2525 from New Zealand was also used as the positive control, and the expected DNA fragment was obtained from all the isolates using primer pair 4/7 (1), whereas no fragment was observed for negative controls (distilled water as the template). Amplified fragment length polymorphism (AFLP) analysis of these X. citri pv. citri strains from Mali and other reference strains from X. citri pv. citri-A, -A*, and X. axonopodis pv. aurantifolii (3), 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), showed that the strains were closely related to X. citri pv. citri-A strains with a wide host range. On the basis of AFLP, the Mali strains were not closely related to X. axonopodis pv. aurantifolii. One week after inoculation, Duncan grapefruit and alemow citrus leaves inoculated with all strains from Mali by a detached leaf assay (3) developed erumpent, callus-like tissue at wound sites, similar to the reaction produced by X. citri pv. citri strain CFBP 2525 (positive control). A survey conducted in 2006 in nine orchards revealed disease incidences of 50, 15, 24, and 25% for lime, sweet orange, sour orange, and tangor groves, respectively. As of this report, citrus canker has spread to new citrus orchards and this might be due to the propagation and dissemination of infected material from small nurseries. To our knowledge, this represents the first outbreak of citrus canker in West Africa. Spread of the pathogen in Mali and neighboring countries should be monitored and a drastic surveillance of citrus nurseries in the region should be performed. References: (1) J. S. Hartung et al. Phytopathology 86:95, 1996. (2) O. Pruvost et al. J. Appl. Microbiol. 99:803, 2005. (3) C. Vernière et al. Eur. J. Plant Pathol. 104:477, 1998.