Complete Genome Sequence of a Copper-Resistant Xanthomonas campestris pv. campestris Strain Isolated from Broccoli in Mauritius Suggests Adaptive Gene Gain Through Horizontal Gene Transfer.

Bacterial adaptation is facilitated by the presence of mobile genetic elements and horizontal gene transfer of genes, such as those coding for virulence factors or resistance to antimicrobial compounds. A hybrid assembly of Nanopore MinIon long-read and Illumina short-read data was produced from a copper-resistant Xanthomonas campestris pv. campestris strain isolated from symptomatic broccoli leaves in Mauritius. We obtained a 5.2-Mb high-quality chromosome and no plasmid. We found four genomic islands, three of which were characterized as integrative conjugative elements or integrative mobilizable elements. These genomic islands carried type III effectors and the copper resistance copLABMGF system involved in pathogenicity and environmental adaptation, respectively.

First report of 'Candidatus Phytoplasma palmae' (16SrIV-A subgroup) associated with palm Lethal Yellowing disease on Cocos nucifera and Pritchardia sp. in Guadeloupe, French West Indies.

Lethal Yellowing (LY) disease causes major damage to palms in Central America and the Caribbean. It has been reported as far south as Antigua (Myrie et al., 2014). LY affects over forty palm species, seriously impacts the coconut industry and alters the landscapes on islands with a tourist-based economy. In March 2021, the presence of LY disease was regularly monitored in Guadeloupe. Two palm species (Cocos nucifera and Pritchardia sp.) died on a private property in Saint-Anne, Grande Terre. Yellowing of lower fronds and necrosis of inflorescences were reported on some neighboring palms. One symptomatic Cocos nucifera (GP21-007) and four symptomatic Pritchardia sp. (GP21-005, GP21-006, GP21-008 and GP21-009) were sampled by stem drilling. Samples from four asymptomatic coconut trees (GP21-001 to GP21-004) were collected in the locality of Deshaies. DNA was extracted from the nine sawdust samples following a cetyltrimethylammonium bromide (CTAB) modified protocol (Doyle and Doyle, 1990). A quantitative polymerase chain reaction (PCR), following the protocol described by Christensen et al. (2004), was performed on DNA to diagnose the presence of phytoplasmas. An exponential amplification was observed for all DNA extracts from symptomatic palm samples (threshold number of PCR cycles (Ct) ranged from 18.50 to 23.58). DNA from asymptomatic samples yielded negative results (undetermined Ct). To identify the phytoplasma associated with LY, DNA samples were subjected to PCR, based on the 16SrRNA gene, plus internal transcribed spacers (ITS) using P1-1T (Pilet et al., 2021)/P7 (Schneider et al., 1995) primers, and secA gene using the primer pair secAFor1/secARev1 (Hodgetts et al. 2008). Amplicons of 1.8 kb covering the 16S ribosomal operon and 830 bp for the secA gene were produced using DNA from symptomatic trees. All amplicons were double strand sequenced (Genewiz, UK). The corresponding sequences were deposited in GenBank and subjected to BLASTn on NCBI. Sequences of the ribosomal operon gene (accession no. ON521114 to ON521118) were identical for the five positive samples. Sequencing revealed two distinct ribosomal operons with heterozygous peaks on the DNA chromatogram. The first aMino ambiguity (M = Adenine or Cytosine) was observed in the 16Sr RNA gene. The second was observed in the first intergenic transcript spacer. The 16S rDNA sequence (M = Cytosine) presented 100% identity with accession no. HQ613874 and 99.93% with accession no. U18747, the reference sequence for 'Candidatus Phytoplasma palmae'. The virtual RFLP pattern derived from the 16S rDNA F2nR2 fragment and identified using iPhyclassifier (Zhao et al. 2009) was identical to the reference pattern for the 16SrIV-A subgroup. A unique sequence was obtained for the partial secA gene (OP136139 to OP136143), sharing 100% identity with EU267187 for the palm LY phytoplasma preprotein translocase subunit (secA) gene. This is the first report of 'Ca. Phytoplasma palmae' (subgroup 16SrIV-A) associated with palm LY disease on Cocos nucifera and Pritchardia sp. in Guadeloupe. Measures to eradicate LY were implemented as soon as its presence was confirmed in Guadeloupe. LY phytoplasmas continue to spread in the Caribbean and are approaching South America, where the known vector, Haplaxius crudus, has already been reported (Silva et al., 2019). This poses a major threat to the coconut economy and the diversity of palm trees.

Pathotyping Citrus Ornamental Relatives with Xanthomonas citri pv. citri and X. citri pv. aurantifolii Refines Our Understanding of Their Susceptibility to These Pathogens.

Xanthomonas citri pv. citri (Xcc) and X. citri pv. aurantifolii (Xca) are causal agents of Citrus Bacterial Canker (CBC), a devastating disease that severely affects citrus plants. They are harmful organisms not reported in Europe or the Mediterranean Basin. Host plants are in the Rutaceae family, including the genera Citrus, Poncirus, and Fortunella, and their hybrids. In addition, other genera of ornamental interest are reported as susceptible, but results are not uniform and sometimes incongruent. We evaluated the susceptibility of 32 ornamental accessions of the Rutaceae family belonging to the genera Citrus, Fortunella, Atalantia, Clausena, Eremocitrus, Glycosmis, Microcitrus, Murraya, Casimiroa, Calodendrum, and Aegle, and three hybrids to seven strains of Xcc and Xca. Pathotyping evaluation was assessed by scoring the symptomatic reactions on detached leaves. High variability in symptoms and bacterial population was shown among the different strains in the different hosts, indicative of complex host-pathogen interactions. The results are mostly consistent with past findings, with the few discrepancies probably due to our more complete experimental approach using multiple strains of the pathogen and multiple hosts. Our work supports the need to regulate non-citrus Rutaceae plant introductions into areas, like the EU and Mediterranean, that are currently free of this economically important pathogen.

Diversity and Geographical Structure of Xanthomonas citri pv. citri on Citrus in the South West Indian Ocean Region.

A thorough knowledge of genotypic and phenotypic variations (e.g., virulence, resistance to antimicrobial compounds) in bacteria causing plant disease outbreaks is key for optimizing disease surveillance and management. Using a comprehensive strain collection, tandem repeat-based genotyping techniques and pathogenicity assays, we characterized the diversity of X. citri pv. citri from the South West Indian Ocean (SWIO) region. Most strains belonged to the prevalent lineage 1 pathotype A that has a wide host range among rutaceous species. We report the first occurrence of genetically unrelated, nonepidemic lineage 4 pathotype A* (strains with a host range restricted to Mexican lime and related species) in Mauritius, Moheli and Réunion. Microsatellite data revealed that strains from the Seychelles were diverse, grouped in three different clusters not detected in the Comoros and the Mascarenes. Pathogenicity data suggested a higher aggressiveness of strains of one of these clusters on citron (Citrus medica). With the noticeable exception of the Comoros, there was no sign of recent interisland movement of the pathogen. Consistent with this finding, the copL gene, a marker for the plasmid-borne copLAB copper resistance that was recently identified in Réunion, was not detected in 568 strains from any islands in the SWIO region apart from Réunion.

High genomic diversity of novel phages infecting the plant pathogen Ralstonia solanacearum, isolated in Mauritius and Reunion islands.

Bacterial wilt caused by the Ralstonia solanacearum species complex (RSSC) is among the most important plant diseases worldwide, severely affecting a high number of crops and ornamental plants in tropical regions. Only a limited number of phages infecting R. solanacearum have been isolated over the years, despite the importance of this bacterium and the associated plant disease. The antibacterial effect or morphological traits of these R. solanacearum viruses have been well studied, but not their genomic features, which need deeper consideration. This study reports the full genome of 23 new phages infecting RSSC isolated from agricultural samples collected in Mauritius and Reunion islands, particularly affected by this plant bacterial pathogen and considered biodiversity hotspots in the Southwest Indian Ocean. The complete genomic information and phylogenetic classification is provided, revealing high genetic diversity between them and weak similarities with previous related phages. The results support our proposal of 13 new species and seven new genera of R. solanacearum phages. Our findings highlight the wide prevalence of phages of RSSC in infected agricultural settings and the underlying genetic diversity. Discoveries of this kind lead more insight into the diversity of phages in general and to optimizing their use as biocontrol agents of bacterial diseases of plants in agriculture.

Draft Genome Sequences of 284 Xanthomonas citri pv. citri Strains Causing Asiatic Citrus Canker.

High-quality Illumina assemblies were produced from 284 Xanthomonas citri pv. citri pathotype A strains mostly originating from the Southwest Indian Ocean region, a subset of which was also sequenced using MinION technology. Some strains hosted chromosomally encoded transcription activator-like effector (TALE) genes, an atypical feature for this bacterium.

Time-calibrated genomic evolution of a monomorphic bacterium during its establishment as an endemic crop pathogen.

Horizontal gene transfer is of major evolutionary importance as it allows for the redistribution of phenotypically important genes among lineages. Such genes with essential functions include those involved in resistance to antimicrobial compounds and virulence factors in pathogenic bacteria. Understanding gene turnover at microevolutionary scales is critical to assess the pace of this evolutionary process. Here, we characterized and quantified gene turnover for the epidemic lineage of a bacterial plant pathogen of major agricultural importance worldwide. Relying on a dense geographic sampling spanning 39 years of evolution, we estimated both the dynamics of single nucleotide polymorphism accumulation and gene content turnover. We identified extensive gene content variation among lineages even at the smallest phylogenetic and geographic scales. Gene turnover rate exceeded nucleotide substitution rate by three orders of magnitude. Accessory genes were found preferentially located on plasmids, but we identified a highly plastic chromosomal region hosting ecologically important genes such as transcription activator-like effectors. Whereas most changes in the gene content are probably transient, the rapid spread of a mobile element conferring resistance to copper compounds widely used for the management of plant bacterial pathogens illustrates how some accessory genes can become ubiquitous within a population over short timeframes.

A duplex quantitative real-time PCR assay for the detection and quantification of Xanthomonas phaseoli pv. dieffenbachiae from diseased and latently infected anthurium tissue.

Anthurium bacterial blight caused by Xanthomonas phaseoli pv. dieffenbachiae (formerly Xanthomonas axonopodis pv. dieffenbachiae) is the major phytosanitary threat in many anthurium growing areas worldwide. Reliable and sensitive diagnostic tools are required for surveillance and certification programs. A duplex real-time quantitative PCR assay was developed for the detection and quantification of X. phaseoli pv. dieffenbachiae from anthurium tissue. This PCR assay targeted a X. phaseoli pv. dieffenbachiae-specific gene encoding an ABC transporter and an internal control encoding for chalcone synthase in Anthurium andreanum. A cycle threshold (Ct), using a receiver-operating characteristic approach (ROC), was implemented to ensure that the declaration of a positive sample was reliable. The duplex real-time assay displayed very high performance with regards to analytical specificity (100% inclusivity, 98.9% exclusivity), analytical sensitivity (LOD95% = 894 bacteria/ml corresponding to 18 bacteria per reaction) and repeatability. We demonstrated the pertinence of this real-time quantitative PCR assay for detecting X. phaseoli pv. dieffenbachiae from diseased leaf tissue (collected from outbreaks on anthurium) and from asymptomatic, latently infected anthurium plants. This assay could be useful for surveillance, as well as for indexing propagative plant material for the presence of X. phaseoli pv. dieffenbachiae.

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

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.

Xanthomonas citri pv. citri Pathotypes: LPS Structure and Function as Microbe-Associated Molecular Patterns.

Xanthomonas citri pv. citri is the pathogen responsible for Asiatic citrus canker, one of the most serious citrus diseases worldwide. The lipopolysaccharide (LPS) molecule has been demonstrated to be involved in X. citri pv. citri virulence. Despite enormous progress in investigations of the molecular mechanisms for bacterial pathogenicity, determination of the detailed LPS structure-activity relationship is limited, as the current knowledge is mainly based on structural determination of one X. citri pv. citri strain. As X. citri pv. citri strains are distinguished into three main pathogenicity groups, we characterized the full structure of the LPS from two pathotypes that differ in their host-range specificity. This revealed an intriguing difference in LPS O-chain structure. We also tested the LPSs and isolated lipid A moieties for their ability to act as microbe-associated molecular patterns in Arabidopsis thaliana. Both LPS/lipid As induced ROS accumulation, but no difference was observed between the two pathotypes.

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.

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.

Sequevar Diversity and Virulence of Ralstonia solanacearum Phylotype I on Mayotte Island (Indian Ocean).

The genetic and phenotypic diversity of the Ralstonia solanacearum species complex, which causes bacterial wilt to Solanacae, was assessed in 140 strains sampled from the main vegetable production areas of the Mayotte island. Only phylotype I strains were identified in the five surveyed areas. The strains were distributed into the following 4 sequevars: I-31 (85.7%), I-18 (5.0%), I-15 (5.7%), and I-46 (3.6%). The central area of Mayotte was the most diverse region, harboring 4 sequevars representing 47.1% of the collected strains. Virulence tests were performed under field and controlled conditions on a set of 10 tomato breeding line accessions and two commercial hybrid tomato cultivars. The strains belonging to sequevar I-31 showed the highest virulence on the tomatoes (pathotypes T-2 and T-3), whereas sequevars I-18, I-15, and I-46 were grouped into the weakly T-1 pathotype. When the tomato accessions were challenged in the field and growth chambers, the highest level of resistance were observed from the genetically related accessions Hawaii 7996, R3034, TML46, and CLN1463. These accessions were considered moderately to highly resistant to representative strains of the most virulent and prevalent sequevar (I-31). Interestingly, the Platinum F1 cultivar, which was recently commercialized in Mayotte for bacterial wilt resistance, was highly or moderately resistant to all strains. This study represents the first step in the rationalization of resistance deployment strategies against bacterial wilt-causing strains in Mayotte.

Complete Genome Sequence of a Copper-Resistant Bacterium from the Citrus Phyllosphere, Stenotrophomonas sp. Strain LM091, Obtained Using Long-Read Technology.

The Stenotrophomonas genus shows great adaptive potential including resistance to multiple antimicrobials, opportunistic pathogenicity, and production of numerous secondary metabolites. Using long-read technology, we report the sequence of a plant-associated Stenotrophomonas strain originating from the citrus phyllosphere that displays a copper resistance phenotype.

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.

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.

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.

Populations of Xanthomonas citri pv. mangiferaeindicae from asymptomatic mango leaves are primarily endophytic.

Epiphytic survival of several Xanthomonas pathovars has been reported, but most studies failed to determine whether such populations were resident epiphytes, resulting from latent infections, or casual epiphytes. This study aimed at understanding the nature of Xanthomonas citri pv. mangiferaeindicae populations associated with asymptomatic leaves. When spray-inoculated on mango leaves cv. Maison Rouge, the pathogen multiplied markedly in association with juvenile leaves, but was most often detected as low population sizes (<1 x 10(3) cfu g(-1)) in association with mature leaves. Our results suggest a very low biological significance of biofilm-associated populations of X. citri pv. mangiferaeindicae, while saprophytic microbiota associated with mango leaves survived frequently as biofilms. A chloroform vapor-based disinfestation assay which kills cells specifically located on the leaf surface and not those located within the leaf mesophyll was developed. When applied to spray-inoculated leaves maintained under controlled environmental conditions, 155 out of the 168 analyzed datasets collected over three assessment dates for seven bacterial strains representative of the genetic diversity of the pathogen failed to demonstrate a significant X. citri pv. mangiferaeindicae population decrease on chloroform treated leaves up to 13 days after inoculation. We conclude that an efficient survival of X. citri pv. mangiferaeindicae present on mango leaf surfaces following a limited dissemination event is largely dependent on the availability of juvenile plant tissues. The bacterium gains access to protected sites (e.g., mesophyll) through stomata where it becomes endophytic and eventually causes disease. Chloroform vapor-based disinfestation assays should be useful for further studies aiming at evaluating survival sites of bacteria associated with the phyllosphere.