A Multilocus Sequence Typing Scheme for Rapid Identification of Xanthomonas citri Based on Whole-Genome Sequencing Data.

Xanthomonas citri is a plant-pathogenic bacterium associated with a diverse range of host plant species. It has undergone substantial reclassification and currently consists of 14 different subspecies or pathovars that are responsible for a wide range of plant diseases. Whole-genome sequencing (WGS) provides a cutting-edge advantage over other diagnostic techniques in epidemiological and evolutionary studies of X. citri because it has a higher discriminatory power and is replicable across laboratories. WGS also allows for the improvement of multilocus sequence typing (MLST) schemes. In this study, we used genome sequences of Xanthomonas isolates from the NCBI RefSeq database to develop a seven-gene MLST scheme that yielded 19 sequence types (STs) that correlated with phylogenetic clades of X. citri subspecies or pathovars. Using this MLST scheme, we examined 2,911 Xanthomonas species assemblies from NCBI GenBank and identified 15 novel STs from 37 isolates that were misclassified in NCBI. In total, we identified 545 X. citri assemblies from GenBank with 95% average nucleotide identity to the X. citri type strain, and all were classified as one of the 34 STs. All MLST classifications correlated with a phylogenetic position inferred from alignments using 92 conserved genes. We observed several instances where strains from different pathovars formed closely related monophyletic clades and shared the same ST, indicating that further investigation of the validity of these pathovars is required. Our MLST scheme described here is a robust tool for rapid classification of X. citri pathovars using WGS and a powerful method for further comprehensive taxonomic revision of X. citri pathovars.

Development of a Long-Amplicon Propidium Monoazide-Quantitative PCR Assay for Detection of Viable Xanthomonas arboricola pv. pruni Cells in Peach Trees.

Bacterial spot is one of the most serious diseases of peach caused by the pathogen Xanthomonas arboricola pv. pruni (XAP), leading to early defoliation and unmarketable fruit. The pathogen can overwinter in peach twigs and form spring cankers, which are considered the primary inoculum source for early season leaf and fruitlet infection. The amount of overwintering bacterial inoculum plays a critical role for the bacterial spot development, but no reliable quantification method is available. Thus, we developed a long-amplicon propidium monoazide (PMA)-quantitative PCR (qPCR) assay for specific detection of viable XAP cells. The optimized PMA-qPCR assay used 20 µM of PMAxx for pure bacterial suspensions and 100 µM for peach twig tissues. The Qiagen Plant Pro Kit with an additional lysozyme digestion step was the DNA extraction protocol that yielded the best detection sensitivity with the bacteria-spiked peach twig extracts. The PMA-qPCR assay was tested with different mixtures of viable and heat-killed XAP cells in pure bacterial suspensions and bacteria-spiked peach twig tissues. The results showed that this assay enabled sensitive, specific, and accurate quantification of viable XAP cells as low as 103 CFU/ml with the presence of up to 107 CFU/ml of dead XAP cells, while suppressing the amplification of DNA from dead cells. For mixtures of viable and dead cells, the PMA-qPCR results were linearly correlated with the predicted concentrations of viable XAP (R2 > 0.98). Thus, the PMA-qPCR assay will be a suitable tool for quantifying overwintering XAP population on peach trees.

TaqMan Real-Time PCR Assay for Specific Detection and Differentiation of Xanthomonas translucens pv. undulosa from Other Pathovars Targeting a Recombination Mediator Gene, recF.

Bacterial leaf streak and black chaff diseases of wheat caused by Xanthomonas translucens pv. undulosa is becoming a major constraint to growers and trade since it is seedborne. Molecular tools for specific detection/differentiation of pv. undulosa are lacking. We report the development of a TaqMan real-time PCR for specific detection/identification of pv. undulosa targeting the recombination mediator gene (recF). Analysis of the complete recF (1,117 bp) sequences identified the gene as a reliable phylogenetic marker for identification of pv. undulosa, differentiating it from the other pathovars; recF-based sequence homology values among the 11 pathovars correlated well with genome-based DNA-DNA hybridization values. The discriminatory power of recF to differentiate pv. undulosa from the other pathovars is due to nucleotide polymorphic positions. We used these nucleotide polymorphisms to develop a TaqMan PCR for specific detection of pv. undulosa. The specificity of the assay was validated using 67 bacterial and fungal/oomycete strains. The selected primers and the double-quenched FAM-labeled TaqMan probe were specific for the detection of 11 pv. undulosa/secalis strains. The 56 strains of other X. translucens pathovars (n = 39) and non-Xanthomonas spp. (n = 17) did not exhibit any detectable fluorescence. Also, greenhouse-inoculated and naturally infected wheat leaf samples showed positive reactions for the presence of pv. undulosa DNA but not healthy control plants. The TaqMan assay reliably detected as low as 1-pg DNA amount and 10 colony forming units of the target pathogen per reaction. This TaqMan assay could be useful to regulatory agencies with economic benefits to wheat growers.

Ppe.XapF: High throughput KASP assays to identify fruit response to Xanthomonas arboricola pv. pruni (Xap) in peach.

Bacterial spot, caused by Xanthomonas arboricola pv. pruni (Xap), is a serious peach disease with symptoms that traverse severe defoliation and black surface pitting, cracking or blemishes on peach fruit with global economic impacts. A management option for control and meeting consumer demand for chemical-free, environmentally friendly fruit production is the development of resistant or tolerant cultivars. We developed simple, accurate, and efficient DNA assays (Ppe.XapF) based on SNP genotyping with KASP technology to quickly test for bacterial spot resistance alleles in peach fruit that allows breeders to cull seedlings at the greenhouse stage. The objective of this research was to validate newly developed DNA tests that target the two major QTLs for fruit resistance in peach with diagnostic utility in predicting fruit response to bacterial spot infection. Our study confirms that with only two Ppe.XapF DNA tests, Ppe.XapF1-1 and Ppe.XapF6-2, individuals carrying susceptible alleles can be identified. Use of these efficient and accurate Ppe.XapF KASP tests resulted in 44% reduction in seedling planting rate in the Clemson University peach breeding program.

Phenotypic and Molecular-Phylogenetic Analyses Reveal Distinct Features of Crown Gall-Associated Xanthomonas Strains.

In summer 2019, widespread occurrence of crown gall disease caused by Agrobacterium spp. was observed on commercially grown ornamental plants in southern Iran. Beside agrobacteria, pale yellow-pigmented Gram-negative strains resembling the members of Xanthomonas were also associated with crown gall tissues on weeping fig (Ficus benjamina) and Amaranthus sp. plants. The purpose of the present study was to characterize the crown gall-associated Xanthomonas strains using plant inoculation assays, molecular-phylogenetic analyses, and comparative genomics approaches. Pathogenicity tests showed that the Xanthomonas strains did not induce disease symptoms on their host of isolation. However, the strains induced hypersensitive reaction on tobacco, geranium, melon, squash, and tomato leaves via leaf infiltration. Multilocus sequence analysis suggested that the strains belong to clade IA of Xanthomonas, phylogenetically close to Xanthomonas translucens, X. theicola, and X. hyacinthi. Average nucleotide identity and digital DNA-DNA hybridization values between the whole-genome sequences of the strains isolated in this study and reference Xanthomonas strains are far below the accepted thresholds for the definition of prokaryotic species, signifying that these strains could be defined as two new species within clade IA of Xanthomonas. Comparative genomics showed that the strains isolated from crown gall tissues are genetically distinct from X. translucens, as almost all the type III secretion system genes and type III effectors are lacking in the former group. The data obtained in this study provide novel insight into the breadth of genetic diversity of crown gall-associated bacteria and pave the way for research on gall-associated Xanthomonas-plant interactions. IMPORTANCE Tumorigenic agrobacteria-members of the bacterial family Rhizobiaceae-cause crown gall and hairy root diseases on a broad range of plant species. These bacteria are responsible for economic losses in nurseries of important fruit trees and ornamental plants. The microclimate of crown gall and their accompanying microorganisms has rarely been studied for the microbial diversity and population dynamics of gall-associated bacteria. Here, we employed a series of biochemical tests, pathogenicity assays, and molecular-phylogenetic analyses, supplemented with comparative genomics, to elucidate the biological features, taxonomic position, and genomic repertories of five crown gall-associated Xanthomonas strains isolated from weeping fig and Amaranthus sp. plants in Iran. The strains investigated in this study induced hypersensitive reactions (HR) on geranium, melon, squash, tobacco, and tomato leaves, while they were nonpathogenic on their host of isolation. Phylogenetic analyses and whole-genome-sequence-based average nucleotide identity (ANI)/digital DNA-DNA hybridization (dDDH) calculations suggested that the Xanthomonas strains isolated from crown gall tissues belong to two taxonomically unique clades closely related to the clade IA species of the genus, i.e., X. translucens, X. hyacinthi, and X. theicola.

Xanthomonas hydrangeae sp. nov., a novel plant pathogen isolated from Hydrangea arborescens.

This paper describes a novel species isolated in 2011 and 2012 from nursery-grown Hydrangea arborescens cultivars in Flanders, Belgium. After 4 days at 28 °C, the strains yielded yellow, round, convex and mucoid colonies. Pathogenicity of the strains was confirmed on its isolation host, as well as on Hydrangea quercifolia. Analysis using MALDI-TOF MS identified the Hydrangea strains as belonging to the genus Xanthomonas but excluded them from the species Xanthomonas hortorum. A phylogenetic tree based on gyrB confirmed the close relation to X. hortorum. Three fatty acids were dominant in the Hydrangea isolates: anteiso-C15 : 0, iso-C15 : 0 and summed feature 3 (C16 : 1 ω7c/C16 : 1 ω6c). Unlike X. hortorum pathovars, the Hydrangea strains were unable to grow in the presence of lithium chloride and could only weakly utilize d-fructose-6-PO4 and glucuronamide. Phylogenetic characterization based on multilocus sequence analysis and phylogenomic characterization revealed that the strains are close to, yet distinct from, X. hortorum. The genome sequences of the strains had average nucleotide identity values ranging from 94.35-95.19 % and in silico DNA-DNA hybridization values ranging from 55.70 to 59.40 % to genomes of the X. hortorum pathovars. A genomics-based loop-mediated isothermal amplification assay was developed which was specific to the Hydrangea strains for its early detection. A novel species, Xanthomonas hydrangeae sp. nov., is proposed with strain LMG 31884T (=CCOS 1956T) as the type strain.

Novel Virulent Bacteriophages Infecting Mediterranean Isolates of the Plant Pest Xylella fastidiosa and Xanthomonas albilineans.

Xylella fastidiosa (Xf) is a plant pathogen causing significant losses in agriculture worldwide. Originating from America, this bacterium caused recent epidemics in southern Europe and is thus considered an emerging pathogen. As the European regulations do not authorize antibiotic treatment in plants, alternative treatments are urgently needed to control the spread of the pathogen and eventually to cure infected crops. One such alternative is the use of phage therapy, developed more than 100 years ago to cure human dysentery and nowadays adapted to agriculture. The first step towards phage therapy is the isolation of the appropriate bacteriophages. With this goal, we searched for phages able to infect Xf strains that are endemic in the Mediterranean area. However, as Xf is truly a fastidious organism, we chose the phylogenetically closest and relatively fast-growing organism X. albineans as a surrogate host for the isolation step. Our results showed the isolation from various sources and preliminary characterization of several phages active on different Xf strains, namely, from the fastidiosa (Xff), multiplex (Xfm), and pauca (Xfp) subspecies, as well as on X. albilineans. We sequenced their genomes, described their genomic features, and provided a phylogeny analysis that allowed us to propose new taxonomic elements. Among the 14 genomes sequenced, we could identify two new phage species, belonging to two new genera of the Caudoviricetes order, namely, Usmevirus (Podoviridae family) and Subavirus (Siphoviridae family). Interestingly, no specific phages could be isolated from infected plant samples, whereas one was isolated from vector insects captured in a contaminated area, and several from surface and sewage waters from the Marseille area.

Wastewater treatment alters microbial colonization of microplastics.

Microplastics are ubiquitous contaminants in aquatic habitats globally, and wastewater treatment plants (WWTPs) are point sources of microplastics. Within aquatic habitats microplastics are colonized by microbial biofilms, which can include pathogenic taxa and taxa associated with plastic breakdown. Microplastics enter WWTPs in sewage and exit in sludge or effluent, but the role that WWTPs play in establishing or modifying microplastic bacterial assemblages is unknown. We analyzed microplastics and associated biofilms in raw sewage, effluent water, and sludge from two WWTPs. Both plants retained >99% of influent microplastics in sludge, and sludge microplastics showed higher bacterial species richness and higher abundance of taxa associated with bioflocculation (e.g. Xanthomonas) than influent microplastics, suggesting that colonization of microplastics within the WWTP may play a role in retention. Microplastics in WWTP effluent included significantly lower abundances of some potentially pathogenic bacterial taxa (e.g. Campylobacteraceae) compared to influent microplastics; however, other potentially pathogenic taxa (e.g. Acinetobacter) remained abundant on effluent microplastics, and several taxa linked to plastic breakdown (e.g. Klebsiella, Pseudomonas, and Sphingomonas) were significantly more abundant on effluent compared to influent microplastics. These results indicate that diverse bacterial assemblages colonize microplastics within sewage and that WWTPs can play a significant role in modifying the microplastic-associated assemblages, which may affect the fate of microplastics within the WWTPs and the environment.

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.

Xanthomonas euroxanthea sp. nov., a new xanthomonad species including pathogenic and non-pathogenic strains of walnut.

We describe a novel species isolated from walnut (Juglans regia) which comprises non-pathogenic and pathogenic strains on walnut. The isolates, obtained from a single ornamental walnut tree showing disease symptoms, grew on yeast extract-dextrose-carbonate agar as mucoid yellow colonies characteristic of Xanthomonas species. Pathogenicity assays showed that while strain CPBF 424T causes disease in walnut, strain CPBF 367 was non-pathogenic on walnut leaves. Biolog GEN III metabolic profiles disclosed some differences between strains CPBF 367 and CPBF 424T and other xanthomonads. Multilocus sequence analysis with seven housekeeping genes (fyuA, gyrB, rpoD, atpD, dnaK, efp, glnA) grouped these strains in a distinct cluster from Xanthomonas arboricola pv. juglandis and closer to Xanthomonas prunicola and Xanthomonas arboricola pv. populi. Average nucleotide identity (ANI) analysis results displayed similarity values below 93 % to X. arboricola strains. Meanwhile ANI and digital DNA-DNA hybridization similarity values were below 89 and 50 % to non-arboricola Xanthomonas strains, respectively, revealing that they do not belong to any previously described Xanthomonas species. Furthermore, the two strains show over 98 % similarity to each other. Genomic analysis shows that strain CPBF 424T harbours a complete type III secretion system and several type III effector proteins, in contrast with strain CPBF 367, shown to be non-pathogenic in plant bioassays. Taking these data altogether, we propose that strains CPBF 367 and CPBF 424T belong to a new species herein named Xanthomonas euroxanthea sp. nov., with CPBF 424T (=LMG 31037T=CCOS 1891T=NCPPB 4675T) as the type strain.

Fluorescence-based immunoassay for the detection of Xanthomonas oryzae pv. oryzae in rice leaf.

The identification of rice bacterial leaf blight disease requires a simple, rapid, highly sensitive, and quantitative approach that can be applied as an early detection monitoring tool in rice health. This paper highlights the development of a turn-off fluorescence-based immunoassay for the early detection of Xanthomonas oryzae pv. oryzae (Xoo), a gram-negative bacterium that causes rice bacterial leaf blight disease. Antibodies against Xoo bacterial cells were produced as specific bio-recognition molecules and the conjugation of these antibodies with graphene quantum dots and gold nanoparticles was performed and characterized, respectively. The combination of both these bio-probes as a fluorescent donor and metal quencher led to changes in the fluorescence signal. The immunoreaction between AntiXoo-GQDs, Xoo cells, and AntiXoo-AuNPs in the immuno-aggregation complex led to the energy transfer in the turn-off fluorescence-based quenching system. The change in fluorescence intensity was proportional to the logarithm of Xoo cells in the range of 100-105 CFU mL-1. The limit of detection was achieved at 22 CFU mL-1 and the specificity test against other plant disease pathogens showed high specificity towards Xoo. The detection of Xoo in real plant samples was also performed in this study and demonstrated satisfactory results.

Clarifying the taxonomy of the causal agent of bacterial leaf spot of lettuce through a polyphasic approach reveals that Xanthomonas cynarae Trébaol et al. 2000 emend. Timilsina et al. 2019 is a later heterotypic synonym of Xanthomonas hortorum Vauterin et al. 1995.

Assessment of the taxonomy and diversity of Xanthomonas strains causing bacterial leaf spot of lettuce (BLSL), commonly referred to as Xanthomonas campestris pv. vitians, has been a long-lasting issue which held back the global efforts made to understand this pathogen. In order to provide a sound basis essential to its study, we conducted a polyphasic approach on strains obtained through sampling campaigns or acquired from collections. Results of a multilocus sequence analysis crossed with phenotypic assays revealed that the pathotype strain does not match the description of the nomenspecies provided by Brown in 1918. However, strain LMG 938=CFBP 8686 does fit this description. Therefore, we propose that it replaces LMG 937=CFBP 2538 as pathotype strain of X. campestris pv. vitians. Then, whole-genome based phylogenies and overall genome relatedness indices calculated on taxonomically relevant strains exhibited the intermediate position of X. campestris pv. vitians between closely related species Xanthomonas hortorum and Xanthomonas cynarae. Phenotypic profiles characterized using Biolog microplates did not reveal stable diagnostic traits legitimizing their distinction. Therefore, we propose that X. cynarae Trébaol et al. 2000 emend. Timilsina et al. 2019 is a later heterotypic synonym of X. hortorum, to reclassify X. campestris pv. vitians as X. hortorum pv. vitians comb. nov. and to transfer X. cynarae pathovars in X. hortorum as X. hortorum pv. cynarae comb. nov. and X. hortorum pv. gardneri comb. nov. An emended description of X. hortorum is provided, making this extended species a promising model for the study of Xanthomonas quick adaptation to different hosts.

Design of a new multiplex PCR assay for rice pathogenic bacteria detection and its application to infer disease incidence and detect co-infection in rice fields in Burkina Faso.

Crop diseases are responsible for considerable yield losses worldwide and particularly in sub-Saharan Africa. To implement efficient disease control measures, detection of the pathogens and understanding pathogen spatio-temporal dynamics is crucial and requires the use of molecular detection tools, especially to distinguish different pathogens causing more or less similar symptoms. We report here the design a new molecular diagnostic tool able to simultaneously detect five bacterial taxa causing important diseases on rice in Africa: (1) Pseudomonas fuscovaginae, (2) Xanthomonas oryzae, (3) Burkholderia glumae and Burkholderia gladioli, (4) Sphingomonas and (5) Pantoea species. This new detection tool consists of a multiplex PCR, which is cost effective and easily applicable. Validation of the method is presented through its application on a global collection of bacterial strains. Moreover, sensitivity assessment for the detection of all five bacteria is reported to be at 0.5 ng DNA by µl. As a proof of concept, we applied the new molecular detection method to a set of 256 rice leaves collected from 16 fields in two irrigated areas in western Burkina Faso. Our results show high levels of Sphingomonas spp. (up to 100% of tested samples in one field), with significant variation in the incidence between the two sampled sites. Xanthomonas oryzae incidence levels were mostly congruent with bacterial leaf streak (BLS) and bacterial leaf blight (BLB) symptom observations in the field. Low levels of Pantoea spp. were found while none of the 256 analysed samples was positive for Burkholderia or Pseudomonas fuscovaginae. Finally, many samples (up to 37.5% in one studied field) were positive for more than one bacterium (co-infection). Documenting co-infection levels are important because of their drastic consequences on epidemiology, evolution of pathogen populations and yield losses. The newly designed multiplex PCR for multiple bacterial pathogens of rice is a significant improvement for disease monitoring in the field, thus contributing to efficient disease control and food safety.

Multiplex real-time PCR for the detection of Clavibacter michiganensis subsp. michiganensis, Pseudomonas syringae pv. tomato and pathogenic Xanthomonas species on tomato plants.

A multiplex real-time PCR method based on fluorescent TaqMan® probes was developed for the simultaneous detection of the tomato pathogenic bacteria Clavibacter michiganensis subsp. michiganensis, Pseudomonas syringae pv. tomato and bacterial spot-causing xanthomonads. The specificity of the multiplex assay was validated on 44 bacterial strains, including 32 target pathogen strains as well as closely related species and nontarget tomato pathogenic bacteria. The designed multiplex real-time PCR showed high sensitivity when positive amplification was observed for one pg of bacterial DNA in the cases of Clavibacter michiganensis subsp. michiganensis and Pseudomonas syringae pv. tomato bacteria and 100 pg for bacterial spot-causing xanthomonads. The reliability of the developed multiplex real-time PCR assay for in planta detection was verified by recognition of the target pathogens in 18 tomato plants artificially inoculated by each of the target bacteria and tomato samples from production greenhouses.

Particle-size dependent bactericidal activity of magnesium oxide against Xanthomonas perforans and bacterial spot of tomato.

Bacterial spot, caused by Xanthomonas spp., is a highly destructive disease of tomatoes worldwide. Copper (Cu) bactericides are often ineffective due to the presence of Cu-tolerant strains. Magnesium oxide (MgO) is an effective alternative to Cu bactericides against Xanthomonas spp. However, the effects of particle size on bactericidal activity and fruit elemental levels are unknown. In this study, nano (20 nm) and micron (0.3 and 0.6 µm) size MgO particles were compared for efficacy. Nano MgO had significantly greater in vitro bactericidal activity against Cu-tolerant X. perforans than micron MgO at 25-50 µg/ml. In field experiments nano and micron MgO applied at 200 and 1,000 µg/ml were evaluated for disease control. Nano MgO at 200 µg/ml was the only treatment that consistently reduced disease severity compared to the untreated control. Inductively Coupled Plasma Optical Emission Spectroscopy revealed that nano MgO applications did not significantly alter Mg, Cu, Ca, K, Mn, P and S accumulation compared to fruits from the untreated plots. We demonstrated that although both nano MgO and micron MgO had bactericidal activity against Cu-tolerant strains in vitro, only nano MgO was effective in bacterial spot disease management under field conditions.

Independent Evolution with the Gene Flux Originating from Multiple Xanthomonas Species Explains Genomic Heterogeneity in Xanthomonas perforans.

Xanthomonas perforans is the predominant pathogen responsible for bacterial leaf spot of tomato and X. euvesicatoria for that of pepper in the southeast United States. Previous studies have indicated significant changes in the X. perforans population collected from Florida tomato fields over the span of 2 decades, including a shift in race and diversification into three phylogenetic groups driven by genome-wide homologous-recombination events derived from X. euvesicatoria In our sampling of Xanthomonas strains associated with bacterial spot disease in Alabama, we were readily able to isolate X. perforans from symptomatic pepper plants grown in several Alabama counties, indicating a recent shift in the host range of the pathogen. To investigate the diversity of these pepper-pathogenic strains and their relation to populations associated with tomatoes grown in the southeast United States, we sequenced the genomes of eight X. perforans strains isolated from tomatoes and peppers grown in Alabama and compared them with previously published genome data available from GenBank. Surprisingly, reconstruction of the X. perforans core genome revealed the presence of two novel genetic groups in Alabama that each harbored a different transcription activation-like effector (TALE). While one TALE, AvrHah1, was associated with an emergent lineage pathogenic to both tomato and pepper, the other was identified as a new class within the AvrBs3 family, here designated PthXp1, and was associated with enhanced symptom development on tomato. Examination of patterns of homologous recombination across the larger X. euvesicatoria species complex revealed a dynamic pattern of gene flow, with multiple donors of Xanthomonas spp. associated with diverse hosts of isolation.IMPORTANCE Bacterial leaf spot of tomato and pepper is an endemic plant disease with a global distribution. In this study, we investigated the evolutionary processes leading to the emergence of novel X. perforans lineages identified in Alabama. While one lineage was isolated from symptomatic tomato and pepper plants, confirming the host range expansion of X. perforans, the other lineage was isolated from tomato and acquired a novel transcription activation-like effector, here designated PthXp1. Functional analysis of PthXp1 indicated that it does not induce Bs4-mediated resistance in tomato and contributes to virulence, providing an adaptive advantage to strains on tomato. Our findings also show that different phylogenetic groups of the pathogen have experienced independent recombination events originating from multiple Xanthomonas species. This suggests a continuous gene flux between related xanthomonads associated with diverse plant hosts that results in the emergence of novel pathogen lineages and associated phenotypes, including host range.

Genome sequence of Xanthomonas fuscans subsp. fuscans strain Xff49: a new isolate obtained from common beans in Southern Brazil.

The genus Xanthomonas comprises Gram-negative bacteria, many of which are phytopathogens. Xanthomonas fuscans subsp. fuscans is one of the most devastating pathogens affecting the bean plant, resulting in the common bacterial blight of bean (CBB). The disease is mainly foliar and affects a wide variety of bean species, thus acting as the yield-limiting factor for the bean crop. Here, we report the whole-genome sequencing of a new strain of X. fuscans subsp. fuscans, named Xff49, isolated from the infected and symptomatic beans from Capão do Leão, Southern Brazil. The genetic analysis demonstrated the presence of single-nucleotide variants (SNVs) in this strain, potentially affecting the mobilome, cell mobility, and inorganic ion metabolism. In addition, the analysis resulted in the identification of a new plasmid similar to the pAX22 derived from Achromobacter denitrificans, which was named plX, along with plA and plC, previously reported in other strains of X. fuscans subsp. fuscans. Xff49 represents the first Brazilian genome of X. fuscans subsp. fuscans and might provide useful information applicable to the studies of phylogenetics, evolution, and pathogenomics, thereby allowing a better understanding of the genomic features present in the Brazilian strains.

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.

Role of the acquisition of a type 3 secretion system in the emergence of novel pathogenic strains of Xanthomonas.

Cases of emergence of novel plant-pathogenic strains are regularly reported that reduce the yields of crops and trees. However, the molecular mechanisms underlying such emergence are still poorly understood. The acquisition by environmental non-pathogenic strains of novel virulence genes by horizontal gene transfer has been suggested as a driver for the emergence of novel pathogenic strains. In this study, we tested such an hypothesis by transferring a plasmid encoding the type 3 secretion system (T3SS) and four associated type 3 secreted proteins (T3SPs) to the non-pathogenic strains of Xanthomonas CFBP 7698 and CFBP 7700, which lack genes encoding T3SS and any previously known T3SPs. The resulting strains were phenotyped on Nicotiana benthamiana using chlorophyll fluorescence imaging and image analysis. Wild-type, non-pathogenic strains induced a hypersensitive response (HR)-like necrosis, whereas strains complemented with T3SS and T3SPs suppressed this response. Such suppression depends on a functional T3SS. Amongst the T3SPs encoded on the plasmid, Hpa2, Hpa1 and, to a lesser extent, XopF1 collectively participate in suppression. Monitoring of the population sizes in planta showed that the sole acquisition of a functional T3SS by non-pathogenic strains impairs growth inside leaf tissues. These results provide functional evidence that the acquisition via horizontal gene transfer of a T3SS and four T3SPs by environmental non-pathogenic strains is not sufficient to make strains pathogenic. In the absence of a canonical effector, the sole acquisition of a T3SS seems to be counter-selective, and further acquisition of type 3 effectors is probably needed to allow the emergence of novel pathogenic strains.

Development of a genome-informed loop-mediated isothermal amplification assay for rapid and specific detection of Xanthomonas euvesicatoria.

Bacterial spot (BS), caused by Xanthomonas euvesicatoria, X. vesicatoria, X. gardneri and X. perforans, is an economically important bacterial disease of tomato and pepper. Symptoms produced by all four species are nearly indistinguishable. At present, no point-of-care diagnostics exist for BS. In this research, we examined genomes of X. euvesicatoria, X. vesicatoria, X. gardneri, X. perforans and other species of Xanthomonas; the unique gene recG was chosen to design primers to develop a loop-mediated isothermal amplification (LAMP) assay to rapidly and accurately identify and differentiate X. euvesicatoria from other BS causing Xanthomonas sp. using a field-deployable portable BioRangerTM instrument. Specificity of the developed assay was tested against 39 strains of X. euvesicatoria and 41 strains of other species in inclusivity and exclusivity panels, respectively. The assay detection limit was 100 fg (~18 genome copies) of genomic DNA and 1,000 fg in samples spiked with tomato DNA. The assay unambiguously detected X. euvesicatoria in infected tomato plant samples. Concordant results were obtained when multiple operators performed the test independently. No false positives and false negatives were detected. The developed LAMP assay has numerous applications in diagnostics, biosecurity and disease management.