Claudine Boyer

Multilocus Sequence Analysis of Xanthomonads Causing Bacterial Spot of Tomato and Pepper Plants Reveals Strains Generated by Recombination among Species and Recent Global Spread of Xanthomonas gardneri

ABSTRACT Four Xanthomonas species are known to cause bacterial spot of tomato and pepper, but the global distribution and genetic diversity of these species are not well understood. A collection of bacterial spot-causing strains from the Americas, Africa, Southeast Asia, and New Zealand were characterized for genetic diversity and phylogenetic relationships using multilocus sequence analysis of six housekeeping genes. By examining strains from different continents, we found unexpected phylogeographic patterns, including the global distribution of a single multilocus haplotype of X. gardneri, possible regional differentiation in X. vesicatoria, and high species diversity on tomato in Africa. In addition, we found evidence of multiple recombination events between X. euvesicatoria and X. perforans. Our results indicate that there have been shifts in the species composition of bacterial spot pathogen populations due to the global spread of dominant genotypes and that recombination between species has generated genetic diversity in these populations.

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.

Revisiting the Specificity of PCR Primers for Diagnostics of Xanthomonas citri pv. citri by Experimental and In Silico Analyses

Asiatic citrus canker disease, caused by Xanthomonas citri pv. citri, seriously impacts citrus production worldwide. Two pathogenic variants, A and A*/Aw, have been described within this pathovar. Two additional pathovars of X. citri with a limited geographic distribution and reduced pathogenicity, namely X. citri pvs. aurantifolii and bilvae, are also pathogenic to citrus and some rutaceous species. Rapid and reliable identification is required for these citrus pathogens, which are classified as a quarantine organism in citrus-producing countries. The specificity of nine polymerase chain reaction primers previously designed for the identification of X. citri pv. citri or citrus bacterial canker strains (both pvs. citri and aurantifolii) was assayed on a large strain collection (n = 87), including the two pathotypes of X. citri pv. citri, other genetic related or unrelated pathogenic xanthomonads, and saprophytic xanthomonads. This study gave congruent results with the original articles when testing the same strains or pathovars but the use of a broad inclusivity and exclusivity panel of strains highlighted new findings. Particularly, primers 2/3, 4/7, and KingF/R failed to provide amplification for three strains from the pathotype A*/Aw. Moreover, all pairs of primers detected at least one non-target strain. These data were supported by in silico analysis of the DNA sequences available from National Center for Biotechnology Information databases.

Adaptation of genetically monomorphic bacteria: evolution of copper resistance through multiple horizontal gene transfers of complex and versatile mobile genetic elements

Copper‐based antimicrobial compounds are widely used to control plant bacterial pathogens. Pathogens have adapted in response to this selective pressure. Xanthomonas citri pv. citri, a major citrus pathogen causing Asiatic citrus canker, was first reported to carry plasmid‐encoded copper resistance in Argentina. This phenotype was conferred by the copLAB gene system. The emergence of resistant strains has since been reported in Réunion and Martinique. Using microsatellite‐based genotyping and copLAB PCR, we demonstrated that the genetic structure of the copper‐resistant strains from these three regions was made up of two distant clusters and varied for the detection of copLAB amplicons. In order to investigate this pattern more closely, we sequenced six copper‐resistant X. citri pv. citri strains from Argentina, Martinique and Réunion, together with reference copper‐resistant Xanthomonas and Stenotrophomonas strains using long‐read sequencing technology. Genes involved in copper resistance were found to be strain dependent with the novel identification in X. citri pv. citri of copABCD and a cus heavy metal efflux resistance–nodulation–division system. The genes providing the adaptive trait were part of a mobile genetic element similar to Tn3‐like transposons and included in a conjugative plasmid. This indicates the systems great versatility. The mining of all available bacterial genomes suggested that, within the bacterial community, the spread of copper resistance associated with mobile elements and their plasmid environments was primarily restricted to the Xanthomonadaceae family.

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

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). Kochs 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.

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.

Complete Genome Sequences of Six Copper-Resistant Xanthomonas Strains Causing Bacterial Spot of Solaneous Plants, Belonging to X. gardneri, X. euvesicatoria, and X. vesicatoria, Using Long-Read Technology

ABSTRACT Xanthomonas vesicatoria, Xanthomonas euvesicatoria, and Xanthomonas gardneri cause bacterial spot disease. Copper has been applied since the 1920s as part of integrated management programs. The first copper-resistant strains were reported some decades later. Here, we fully sequenced six Xanthomonas strains pathogenic to tomato and/or pepper and having a copper-resistant phenotype.

Complete Genome Sequences of Six Copper-Resistant Xanthomonas citri pv. citri Strains Causing Asiatic Citrus Canker, Obtained Using Long-Read Technology

ABSTRACT The gammaproteobacterium Xanthomonas citri pv. citri causes Asiatic citrus canker. Pathotype A strains have a broad host range, which includes most commercial citrus species, and they cause important economic losses worldwide. Control often relies on frequent copper sprays. We present here the complete genomes of six X. citri pv. citri copper-resistant strains.

Natural Infection of Cashew (Anacardium occidentale) by Xanthomonas citri pv. mangiferaeindicae in Burkina Faso

Xanthomonas citri pv. mangiferaeindicae is the causal agent of bacterial canker of mango (Mangifera indica, Anacardiaceae), a disease of international importance. Since the original description of the bacterium in the 1940s, the status of cashew (Anacardium occidentale, Anacardiaceae) as a host species has been unclear. Here, we report the first outbreak of a cashew bacterial disease in Burkina Faso (Western Africa) where X. citri pv. mangiferaeindicae recently emerged on mango. A comprehensive molecular characterization, based on multilocus sequence analysis, supplemented with pathogenicity assays of isolates obtained during the outbreak, indicated that the causal agent on cashew in Burkina Faso is X. citri pv. mangiferaeindicae and not X. citri pv. anacardii, which was previously reported as the causal agent of a cashew bacterial leaf spot in Brazil. Pathogenicity data supported by population biology in Burkina Faso suggest a lack of host specialization. Therefore, the inoculum from each crop is potentially harmful to both host species. Symptoms induced on cashew leaves and fruit by X. citri pv. mangiferaeindicae and nonpigmented strains of X. citri pv. anacardii are similar, although the causative bacteria are genetically different. Thus, xanthomonads pathogenic on cashew may represent a new example of pathological convergence in this bacterial genus.

First Report of Xanthomonas citri pv. citri Pathotype A Causing Asiatic Citrus Canker in Martinique, France

Asiatic canker, caused by Xanthomonas citri pv. citri, is a major threat to worldwide citriculture. Three pathotypes differing in host range and hypersensitive reactions toward citrus species have been defined. Whereas pathotypes Aw and A* have a restricted host range, X. citri pv. citri pathotype A infects a broader range including most commercial citrus species and hybrids and can cause important economic losses in tropical and subtropical areas. Pathotype A strains, especially those assigned to lineage 1, were implicated in the major geographical expansion of X. citri pv. citri during the 20th century from their native area, Asia (Pruvost et al. 2014). X. citri pv. citri is listed as a quarantine pathogen in the European Union (EU) – Directive 2000/29/EC annex II A1. Martinique (France) is an outermost region of the EU in the eastern Caribbean Sea. Canker lesions were first observed at Morne Rouge, Martinique in June 2014 on grapefruit (Citrus paradisi), mandarin (C. reticulata), Tahiti lime (C. latifolia), and Valencia and Washington Navel oranges (C. sinensis). Official diagnostics, including bacterial isolations on YPGA or KC semiselective medium (Pruvost et al. 2005), PCR-based identification with 4/7 primers (Hartung et al. 1993), and pathogenicity tests, were performed following the EPPO standard PM7/44 (www.eppo.int) and identified isolates as X. citri pv. citri. Three strains isolated in Martinique in 2014 from grapefruit or Tahiti lime were further characterized (LL074-4, LL077-2, and LL079). Multilocus sequence analysis (MLSA) targeting six housekeeping genes (atpD, dnaK, efp, gltA, gyrB, and lepA) (Almeida et al. 2010; Bui Thi Ngoc et al. 2010) identified Martinique strains as X. citri pv. citri with 100% sequence identity to the type strain LMG 9322. Using MLVA-31 targeting 31 minisatellites, Martinique strains were assigned to lineage 1 composed of pathotype A strains (Pruvost et al. 2014). All strains were inoculated by a detached leaf assay onto Mexican lime SRA 140 (C. aurantifolia), sweet orange Washington Navel SRA 102, and grapefruit Henderson SRA 336 (Bui Thi Ngoc et al. 2010). All inoculated leaves produced typical erumpent, callus-like tissue at wound sites. Xanthomonas-like colonies were reisolated from lesions that had developed. Boiled suspensions were assayed by PCR with 4/7 primers and produced the expected amplicon, fulfilling Kochs postulates. No lesions developed on the negative control consisting of sterile 0.01M tris buffer pH 7.2. This is the first report of X. citri pv. citri in Martinique and to our knowledge in the Caribbean region. Surprisingly, all strains collected to date in Martinique grew on YPGA supplemented with 300 mg liter−1 copper sulfate even when no extensive copper spray programs have been used, suggesting that copper-resistant strains may have been introduced. Disease is contained by tree removal and burning and the situation is presently under apparent control although positive trees were sporadically detected in 2015 in backyards or small orchards at Le Lorrain and Saint Pierre. An extensive surveillance program is currently implemented in Martinique for quarantine pathogens of citrus. (Texte integral)