Phylodynamic Insights into Global Emergence and Diversification of the Tomato Pathogen Xanthomonas hortorum pv. gardneri.

The emergence of plant pathogens is often associated with waves of unique evolutionary and epidemiological events. Xanthomonas hortorum pv. gardneri is one of the major pathogens causing bacterial spot disease of tomatoes. After its first report in the 1950s, there were no formal reports on this pathogen until the 1990s, despite active global research on the pathogens that cause tomato and pepper bacterial spot disease. Given the recently documented global distribution of X. hortorum pv. gardneri, our objective was to examine genomic diversification associated with its emergence. We sequenced the genomes of X. hortorum pv. gardneri strains collected in eight countries to examine global population structure and pathways of emergence using phylodynamic analysis. We found that strains isolated post-1990 group by region of collection and show minimal impact of recombination on genetic variation. A period of rapid geographic expansion in X. hortorum pv. gardneri is associated with acquisition of a large plasmid conferring copper tolerance by horizontal transfer and coincides with the burgeoning hybrid tomato seed industry through the 1980s. The ancestry of X. hortorum pv. gardneri is consistent with introduction to hybrid tomato seed production and dissemination during the rapid increase in trade of hybrid seeds.

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 historical genome of a crop bacterial pathogen from herbarium specimen: Insights into citrus canker emergence.

Over the past decade, ancient genomics has been used in the study of various pathogens. In this context, herbarium specimens provide a precious source of dated and preserved DNA material, enabling a better understanding of plant disease emergences and pathogen evolutionary history. We report here the first historical genome of a crop bacterial pathogen, Xanthomonas citri pv. citri (Xci), obtained from an infected herbarium specimen dating back to 1937. Comparing the 1937 genome within a large set of modern genomes, we reconstructed their phylogenetic relationships and estimated evolutionary parameters using Bayesian tip-calibration inferences. The arrival of Xci in the South West Indian Ocean islands was dated to the 19th century, probably linked to human migrations following slavery abolishment. We also assessed the metagenomic community of the herbarium specimen, showed its authenticity using DNA damage patterns, and investigated its genomic features including functional SNPs and gene content, with a focus on virulence factors.

A network approach to decipher the dynamics of Lysobacteraceae plasmid gene sharing.

Plasmids provide an efficient vehicle for gene sharing among bacterial populations, playing a key role in bacterial evolution. Network approaches are particularly suitable to represent multipartite relationships and are useful tools to characterize plasmid-mediated gene sharing events. The bacterial family Lysobacteraceae includes plant commensal, plant pathogenic and opportunistic human pathogens for which plasmid-mediated adaptation has been reported. We searched for homologues of plasmid gene sequences from this family in the entire diversity of available bacterial genome sequences and built a network of plasmid gene sharing from the results. While plasmid genes are openly shared between the bacteria of the family Lysobacteraceae, taxonomy strongly defined the boundaries of these exchanges, which only barely reached other families. Most inferred plasmid gene sharing events involved a few genes only, and evidence of full plasmid transfers were restricted to taxonomically closely related taxa. We detected multiple plasmid-chromosome gene transfers, including the known sharing of a heavy metal resistance transposon. In the network, bacterial lifestyles shaped substructures of isolates colonizing specific ecological niches and harbouring specific types of resistance genes. Genes associated with pathogenicity or antibiotic and metal resistance were among those that most importantly structured the network, highlighting the imprints of human-mediated selective pressure on pathogenic populations. A massive sequencing effort on environmental Lysobacteraceae is therefore required to refine our understanding of how this reservoir fuels the emergence and the spread of genes among this family and its potential impact on plant, animal and human health.

Molecular Epidemiology of Ralstonia pseudosolanacearum Phylotype I Strains in the Southwest Indian Ocean Region and Their Relatedness to African Strains.

The increasing requirement for developing tools enabling fine strain traceability responsible for epidemics is tightly linked with the need to understand factors shaping pathogen populations and their environmental interactions. Bacterial wilt caused by the Ralstonia solanacearum species complex (RSSC) is one of the most important plant diseases in tropical and subtropical regions. Sadly, little, outdated, or no information on its epidemiology is reported in the literature, although alarming outbreaks are regularly reported as disasters. A large set of phylotype I isolates (n = 2,608) was retrieved from diseased plants in fields across the Southwest Indian Ocean (SWIO) and Africa. This collection enabled further assessment of the epidemiological discriminating power of the previously published RS1-MLVA14 scheme. Thirteen markers were validated and characterized as not equally informative. Most had little infra-sequevar polymorphism, and their performance depended on the sequevar. Strong correlation was found with a previous multilocus sequence typing scheme. However, 2 to 3% of sequevars were not correctly assigned through endoglucanase gene sequence. Discriminant analysis of principal components (DAPC) revealed four groups with strong phylogenetic relatedness to sequevars 31, 33, and 18. Phylotype I-31 isolates were highly prevalent in the SWIO and Africa, but their dissemination pathways remain unclear. Tanzania and Mauritius showed the greatest diversity of RSSC strains, as the four DAPC groups were retrieved. Mauritius was the sole territory harboring a vast phylogenetic diversity and all DAPC groups. More research is still needed to understand the high prevalence of phylotype I-31 at such a large geographic scale.

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.

Development and comparative validation of genomic-driven PCR-based assays to detect Xanthomonas citri pv. citri in citrus plants.

BACKGROUND: Asiatic Citrus Canker, caused by Xanthomonas citri pv. citri, severely impacts citrus production worldwide and hampers international trade. Considerable regulatory procedures have been implemented to prevent the introduction and establishment of X. citri pv. citri into areas where it is not present. The effectiveness of this surveillance largely relies on the availability of specific and sensitive detection protocols. Although several PCR- or real-time PCR-based methods are available, most of them showed analytical specificity issues. Therefore, we developed new conventional and real-time quantitative PCR assays, which target a region identified by comparative genomic analyses, and compared them to existing protocols. RESULTS: Our assays target the X. citri pv. citri XAC1051 gene that encodes for a putative transmembrane protein. The real-time PCR assay includes an internal plant control (5.8S rDNA) for validating the assay in the absence of target amplification. A receiver-operating characteristic approach was used in order to determine a reliable cycle cut-off for providing accurate qualitative results. Repeatability, reproducibility and transferability between real-time devices were demonstrated for this duplex qPCR assay (XAC1051-2qPCR). When challenged with an extensive collection of target and non-target strains, both assays displayed a high analytical sensitivity and specificity performance: LOD95% = 754 CFU ml- 1 (15 cells per reaction), 100% inclusivity, 97.2% exclusivity for XAC1051-2qPCR; LOD95% = 5234 CFU ml- 1 (105 cells per reaction), 100% exclusivity and inclusivity for the conventional PCR. Both assays can detect the target from naturally infected citrus fruit. Interestingly, XAC1051-2qPCR detected X. citri pv. citri from herbarium citrus samples. The new PCR-based assays displayed enhanced analytical sensitivity and specificity when compared with previously published PCR and real-time qPCR assays. CONCLUSIONS: We developed new valuable detection assays useful for routine diagnostics and surveillance of X. citri pv. citri in citrus material. Their reliability was evidenced through numerous trials on a wide range of bacterial strains and plant samples. Successful detection of the pathogen was achieved from both artificially and naturally infected plants, as well as from citrus herbarium samples, suggesting that these assays will have positive impact both for future applied and academic research on this bacterium.

CRISPR elements provide a new framework for the genealogy of the citrus canker pathogen Xanthomonas citri pv. citri.

BACKGROUND: Xanthomonads are an important clade of Gram-negative bacteria infecting a plethora of economically important host plants, including citrus. Knowledge about the pathogen's diversity and population structure are prerequisite for epidemiological surveillance and efficient disease management. Rapidly evolving genetic loci, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), are of special interest to develop new molecular typing tools. RESULTS: We analyzed CRISPR loci of 56 Xanthomonas citri pv. citri strains of world-wide origin, a regulated pathogen causing Asiatic citrus canker in several regions of the world. With one exception, 23 unique sequences built up the repertoire of spacers, suggesting that this set of strains originated from a common ancestor that already harbored these 23 spacers. One isolate originating from Pakistan contained a string of 14 additional, probably more recently acquired spacers indicating that this genetic lineage has or had until recently the capacity to acquire new spacers. Comparison of CRISPR arrays with previously obtained molecular typing data, such as amplified fragment length polymorphisms (AFLP), variable-number of tandem-repeats (VNTR) and genome-wide single-nucleotide polymorphisms (SNP), demonstrated that these methods reveal similar evolutionary trajectories. Notably, genome analyses allowed to generate a model for CRISPR array evolution in X. citri pv. citri, which provides a new framework for the genealogy of the citrus canker pathogen. CONCLUSIONS: CRISPR-based typing will further improve the accuracy of the genetic identification of X. citri pv. citri outbreak strains in molecular epidemiology analyses, especially when used concomitantly with another genotyping method.

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.

Comparative genomics of 43 strains of Xanthomonas citri pv. citri reveals the evolutionary events giving rise to pathotypes with different host ranges.

BACKGROUND: The identification of factors involved in the host range definition and evolution is a pivotal challenge in the goal to predict and prevent the emergence of plant bacterial disease. To trace the evolution and find molecular differences between three pathotypes of Xanthomonas citri pv. citri that may explain their distinctive host ranges, 42 strains of X. citri pv. citri and one outgroup strain, Xanthomonas citri pv. bilvae were sequenced and compared. RESULTS: The strains from each pathotype form monophyletic clades, with a short branch shared by the A(w) and A pathotypes. Pathotype-specific recombination was detected in seven regions of the alignment. Using Ancestral Character Estimation, 426 SNPs were mapped to the four branches at the base of the A, A*, A(w) and A/A(w) clades. Several genes containing pathotype-specific nonsynonymous mutations have functions related to pathogenicity. The A pathotype is enriched for SNP-containing genes involved in defense mechanisms, while A* is significantly depleted for genes that are involved in transcription. The pathotypes differ by four gene islands that largely coincide with regions of recombination and include genes with a role in virulence. Both A* and A(w) are missing genes involved in defense mechanisms. In contrast to a recent study, we find that there are an extremely small number of pathotype-specific gene presences and absences. CONCLUSIONS: The three pathotypes of X. citri pv. citri that differ in their host ranges largely show genomic differences related to recombination, horizontal gene transfer and single nucleotide polymorphism. We detail the phylogenetic relationship of the pathotypes and provide a set of candidate genes involved in pathotype-specific evolutionary events that could explain to the differences in host range and pathogenicity between them.

Genomics and transcriptomics of Xanthomonas campestris species challenge the concept of core type III effectome.

BACKGROUND: The bacterial species Xanthomonas campestris infects a wide range of Brassicaceae. Specific pathovars of this species cause black rot (pv. campestris), bacterial blight of stock (pv. incanae) or bacterial leaf spot (pv. raphani). RESULTS: In this study, we extended the genomic coverage of the species by sequencing and annotating the genomes of strains from pathovar incanae (CFBP 1606R and CFBP 2527R), pathovar raphani (CFBP 5828R) and a pathovar formerly named barbareae (CFBP 5825R). While comparative analyses identified a large core ORFeome at the species level, the core type III effectome was limited to only three putative type III effectors (XopP, XopF1 and XopAL1). In Xanthomonas, these effector proteins are injected inside the plant cells by the type III secretion system and contribute collectively to virulence. A deep and strand-specific RNA sequencing strategy was adopted in order to experimentally refine genome annotation for strain CFBP 5828R. This approach also allowed the experimental definition of novel ORFs and non-coding RNA transcripts. Using a constitutively active allele of hrpG, a master regulator of the type III secretion system, a HrpG-dependent regulon of 141 genes co-regulated with the type III secretion system was identified. Importantly, all these genes but seven are positively regulated by HrpG and 56 of those encode components of the Hrp type III secretion system and putative effector proteins. CONCLUSIONS: This dataset is an important resource to mine for novel type III effector proteins as well as for bacterial genes which could contribute to pathogenicity of X. campestris.

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.

Highly polymorphic markers reveal the establishment of an invasive lineage of the citrus bacterial pathogen Xanthomonas citri pv. citri in its area of origin.

Investigating the population biology of plant pathogens in their native areas is essential to understand the factors that shape their population structure and favour their spread. Monomorphic pathogens dispatch extremely low genetic diversity in invaded areas, and native areas constitute a major reservoir for future emerging strains. One of these, the gammaproteobacterium Xanthomonas citri pv. citri, causes Asiatic canker and is a considerable threat to citrus worldwide. We studied its population genetic structure by genotyping 555 strains from 12 Vietnam provinces at 14 tandem repeat loci and insertion sequences. Discriminant analysis of principal components identified six clusters. Five of them were composed of endemic strains distributed heterogeneously across sampled provinces. A sixth cluster, VN6, displayed a much lower diversity and a clonal expansion structure, suggesting recent epidemic spread. No differences in aggressiveness on citrus or resistance to bactericides were detected between VN6 and other strains. VN6 likely represents a case of bioinvasion following introduction in a native area likely through contaminated plant propagative material. Highly polymorphic markers are useful for revealing migration patterns of recently introduced populations of a monomorphic bacterial plant pathogen.

Genome sequence of Xanthomonas fuscans subsp. fuscans strain 4834-R reveals that flagellar motility is not a general feature of xanthomonads.

BACKGROUND: Xanthomonads are plant-associated bacteria responsible for diseases on economically important crops. Xanthomonas fuscans subsp. fuscans (Xff) is one of the causal agents of common bacterial blight of bean. In this study, the complete genome sequence of strain Xff 4834-R was determined and compared to other Xanthomonas genome sequences. RESULTS: Comparative genomics analyses revealed core characteristics shared between Xff 4834-R and other xanthomonads including chemotaxis elements, two-component systems, TonB-dependent transporters, secretion systems (from T1SS to T6SS) and multiple effectors. For instance a repertoire of 29 Type 3 Effectors (T3Es) with two Transcription Activator-Like Effectors was predicted. Mobile elements were associated with major modifications in the genome structure and gene content in comparison to other Xanthomonas genomes. Notably, a deletion of 33 kbp affects flagellum biosynthesis in Xff 4834-R. The presence of a complete flagellar cluster was assessed in a collection of more than 300 strains representing different species and pathovars of Xanthomonas. Five percent of the tested strains presented a deletion in the flagellar cluster and were non-motile. Moreover, half of the Xff strains isolated from the same epidemic than 4834-R was non-motile and this ratio was conserved in the strains colonizing the next bean seed generations. CONCLUSIONS: This work describes the first genome of a Xanthomonas strain pathogenic on bean and reports the existence of non-motile xanthomonads belonging to different species and pathovars. Isolation of such Xff variants from a natural epidemic may suggest that flagellar motility is not a key function for in planta fitness.

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.

Herbarium specimen sequencing allows precise dating of Xanthomonas citri pv. citri diversification history.

Herbarium collections are an important source of dated, identified and preserved DNA, whose use in comparative genomics and phylogeography can shed light on the emergence and evolutionary history of plant pathogens. Here, we reconstruct 13 historical genomes of the bacterial crop pathogen Xanthomonas citri pv. citri (Xci) from infected Citrus herbarium specimens. Following authentication based on ancient DNA damage patterns, we compare them with a large set of modern genomes to estimate their phylogenetic relationships, pathogenicity-associated gene content and several evolutionary parameters. Our results indicate that Xci originated in Southern Asia ~11,500 years ago (perhaps in relation to Neolithic climate change and the development of agriculture) and diversified during the beginning of the 13th century, after Citrus diversification and before spreading to the rest of the world (probably via human-driven expansion of citriculture through early East-West trade and colonization).

A minisatellite-based MLVA for deciphering the global epidemiology of the bacterial cassava pathogen Xanthomonas phaseoli pv. manihotis.

Cassava Bacterial Blight (CBB) is a destructive disease widely distributed in the different areas where this crop is grown. Populations studies have been performed at local and national scales revealing a geographical genetic structure with temporal variations. A global epidemiology analysis of its causal agent Xanthomonas phaseoli pv. manihotis (Xpm) is needed to better understand the expansion of the disease for improving the monitoring of CBB. We targeted new tandem repeat (TR) loci with large repeat units, i.e. minisatellites, that we multiplexed in a scheme of Multi-Locus Variable number of TR Analysis (MLVA-8). This genotyping scheme separated 31 multilocus haplotypes in three clusters of single-locus variants and a singleton within a worldwide collection of 93 Xpm strains isolated over a period of fifty years. The major MLVA-8 cluster 1 grouped strains originating from all countries, except the unique Chinese strain. On the contrary, all the Xpm strains genotyped using the previously developed MLVA-14 microsatellite scheme were separated as unique haplotypes. We further propose an MLVA-12 scheme which takes advantage of combining TR loci with different mutation rates: the eight minisatellites and four faster evolving microsatellite markers, for global epidemiological surveillance. This MLVA-12 scheme identified 78 haplotypes and separated most of the strains in groups of double-locus variants (DLV) supporting some phylogenetic relationships. DLV groups were subdivided into closely related clusters of strains most often sharing the same geographical origin and isolated over a short period, supporting epidemiological relationships. The main MLVA-12 DLV group#1 was composed by strains from South America and all the African strains. The MLVA-12 scheme combining both minisatellite and microsatellite loci with different discriminatory power is expected to increase the accuracy of the phylogenetic signal and to minimize the homoplasy effects. Further investigation of the global epidemiology of Xpm will be helpful for a better control of CBB worldwide.

Suspicions of two bridgehead invasions of Xylella fastidiosa subsp. multiplex in France.

Of American origin, a wide diversity of Xylella fastidiosa strains belonging to different subspecies have been reported in Europe since 2013 and its discovery in Italian olive groves. Strains from the subspecies multiplex (ST6 and ST7) were first identified in France in 2015 in urban and natural areas. To trace back the most probable scenario of introduction in France, the molecular evolution rate of this subspecies was estimated at 3.2165 × 10-7 substitutions per site per year, based on heterochronous genome sequences collected worldwide. This rate allowed the dating of the divergence between French and American strains in 1987 for ST6 and in 1971 for ST7. The development of a new VNTR-13 scheme allowed tracing the spread of the bacterium in France, hypothesizing an American origin. Our results suggest that both sequence types were initially introduced and spread in Provence-Alpes-Côte d'Azur (PACA); then they were introduced in Corsica in two waves from the PACA bridgehead populations.

Molecular epidemiology of the citrus bacterial pathogen Xanthomonas citri pv. citri from the Arabian Peninsula reveals a complex structure of specialist and generalist strains.

Molecular epidemiology studies are essential to refine our understanding of migrations of phytopathogenic bacteria, the major determining factor in their emergence, and to understand the factors that shape their population structure. Microsatellite and minisatellite typing are useful techniques for deciphering the population structure of Xanthomonas citri pv. citri, the causal agent of Asiatic citrus canker. This paper presents a molecular epidemiology study, which has improved our understanding of the history of the pathogen's introductions into the Arabian Peninsula, since it was first reported in the 1980s. An unexpectedly high genetic diversity of the pathogen was revealed. The four distinct genetic lineages within X. citri pv. citri, which have been reported throughout the world, were identified in the Arabian Peninsula, most likely as the result of multiple introductions. No copper-resistant X. citri pv. citri strains were identified. The pathogen's population structure on Mexican lime (their shared host species) was closely examined in two countries, Saudi Arabia and Yemen. We highlighted the marked prevalence of specialist pathotype A* strains in both countries, which suggests that specialist strains of X. citri pv. citri may perform better than generalist strains when they occur concomitantly in this environment. Subclade 4.2 was the prevailing lineage identified. Several analyses (genetic structure deciphered by discriminant analysis of principal components, RST-based genetic differentiation, geographic structure) congruently suggested the role of human activities in the pathogen's spread. We discuss the implications of these results on the management of Asiatic citrus canker in the region.

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.