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Fluorescent pseudomonads represent one of the largest groups of bacteria inhabiting the surfaces of plants, but their genetic composition in planta is poorly understood. Here, we examined the population structure and diversity of fluorescent pseudomonads isolated from sugar beet grown at two geographic locations (Oxford, United Kingdom and Auckland, New Zealand). To seek evidence for niche adaptation, bacteria were sampled from three types of leaves (immature, mature, and senescent) and then characterized using a combination of genotypic and phenotypic analysis. We first performed multilocus sequence analysis (MLSA) of three housekeeping genes (gapA, gltA, and acnB) in a total of 152 isolates (96 from Oxford, 56 from Auckland). The concatenated sequences were grouped into 81 sequence types and 22 distinct operational taxonomic units (OTUs). Significant levels of recombination were detected, particularly for the Oxford isolates (rate of recombination to mutation (r/m) = 5.23 for the whole population). Subsequent ancestral analysis performed in STRUCTURE found evidence of six ancestral populations, and their distributions significantly differed between Oxford and Auckland. Next, their ability to grow on 95 carbon sources was assessed using the Biolog™ GN2 microtiter plates. A distance matrix was generated from the raw growth data (A660) and subjected to multidimensional scaling (MDS) analysis. There was a significant correlation between substrate utilization profiles and MLSA genotypes. Both phenotypic and genotypic analyses indicated presence of a geographic structure for strains from Oxford and Auckland. Significant differences were also detected for MLSA genotypes between strains isolated from immature versus mature/senescent leaves. The fluorescent pseudomonads thus showed an ecotypic population structure, suggestive of adaptation to both geographic conditions and local plant niches.

As the current episode of Acute Oak Decline (AOD) continues to affect native British oak in the United Kingdom, ongoing isolations from symptomatic and healthy oak have yielded a large Pseudomonas species population. These strains could be divided into taxa representing three potential novel species. Recently, two of these taxa were described as novel Pseudomonas species in the Pseudomonas fluorescens lineage. Here, we demonstrate using a polyphasic approach that the third taxon represents another novel Pseudomonas species. The 16S rRNA gene sequencing assigned the strains to the Pseudomonas aeruginosa lineage, while multilocus sequence analysis (based on partial gyrB, rpoB and rpoD sequences) placed the 13 strains in a single cluster on the border of the Pseudomonas stutzeri group. Whole genome intra-species comparisons (based on average nucleotide identity and in silico DNA–DNA hybridization) confirmed that the strains belong to a single taxon, while the inter-species comparisons with closest phylogenetic relatives yielded similarity values below the accepted species threshold. Therefore, we propose these strains as a novel species, namely Pseudomonas kirkiae sp. nov., with the type strain FRB 229T (P4CT=LMG 31089T=NCPPB 4674T). The phylogenetic analyses performed in this study highlighted the difficulties in assigning novel species to the genus Pseudomonas due to its polyphyletic nature and close relationship to the genus Azotobacter . We further propose that a thorough taxonomic re-evaluation of the genus Pseudomonas is essential and should be performed in the near future.

Bacterial canker is a major disease of stone fruits and is a critical limiting factor to sweet cherry ( ) production worldwide. One important strategy for disease control is the development of resistant varieties. Partial varietal resistance in sweet cherry is discernible using shoot or whole tree inoculations; however, these quantitative differences in resistance are not evident in detached leaf assays. To identify novel sources of resistance to canker, we used a rapid leaf pathogenicity test to screen a range of wild cherry, ornamental species and sweet cherry × ornamental cherry hybrids with the canker pathogens, pvs , races 1 and 2, and . Several accessions exhibited limited symptom development following inoculation with each of the pathogens, and this resistance extended to 16 . strains pathogenic on sweet cherry and plum. Resistance was associated with reduced bacterial multiplication after inoculation, a phenotype similar to that of commercial sweet cherry towards nonhost strains of . . Progeny resulting from a cross of a resistant ornamental species with susceptible sweet cherry ( . ) exhibited resistance indicating it is an inherited trait. Identification of accessions with resistance to the major bacterial canker pathogens is the first step towards characterizing the underlying genetic mechanisms of resistance and introducing these traits into commercial germplasm. [Abstract copyright: © 2021 The Authors. Plant Pathology published by John Wiley & Sons Ltd on behalf of British Society for Plant Pathology.]

Bacterial canker is a major disease of stone fruits and is a critical limiting factor to sweet cherry (

Bacterial canker of Prunus, affecting economically important stone fruit crops including cherry, peach, apricot and plum, is caused by the plant pathogen Pseudomonas syringae (P.s.). Strains from two pathovars—P.s. pv. syringae (Pss) and P.s. pv. morsprunorum race 1 (PsmR1) and 2 (PsmR2)—in three phylogenetically distant clades have convergently evolved to infect Prunus. The bacteria enter woody tissues through wounds and leaf scars, causing black necrotic cankers. Symptoms are also produced on blossom, fruit and leaves. Little is known about the mechanisms P.s. uses to colonise tree hosts such as Prunus. Here, we created transposon (Tn) mutant libraries in one strain of P.s. from each of the three clades and screened the mutants on immature cherry fruit to look for changes in virulence. Mutants (242) with either reduced or enhanced virulence were detected and further characterised by in vitro screens for biofilm formation, swarming ability, and pathogenicity on leaves and cut shoots. In total, 18 genes affecting virulence were selected, and these were involved in diverse functions including motility, type III secretion, membrane transport, amino acid synthesis, DNA repair and primary metabolism. Interestingly, mutation of the effector gene, hopAU1, led to an increase in virulence of Psm R2.

International audience; The strict relationships between bacteria and plants represent one of the major facets of terrestrial ecology. Depending on the type of interaction and amount of metabolic advantage one organism can obtain from such relationships, these are classified as mutualistic, commensal or parasitic interactions. Within this context, Pseudomonas and Xanthomonas are bacterial genera with a worldwide spread, capable of establishing all of the above mentioned interactions with plants. Therefore, they represent good models for studying different lifestyles and, accordingly, deciphering distinct evolutionary trajectories followed by different lineages of a single genus to infect and/or to establish a mutualistic relationships with the plant. Some members of these two genera are regulated pests that are recognized as economically major threats for their host crop(s) both in temperate and tropical environments. Some Pseudomonas and Xanthomonas are key examples of different lifestyles (i.e., mesophyll or vessel-colonizing pathogens, epiphytic pathogens, plant growth-promoting rhizobacteria, non-pathogenic strains of recognized pathogenic species, etc). Refining our knowledge on the ecology and epidemiology of these bacterial groups, as well as deciphering their evolutionary dynamics are keys for understanding their contrasting lifestyles and consequently improving plant disease control. At the same time, insights on the activation of different plant defense mechanisms as challenged by the different repertoires of virulence factors displayed by pseudomonads and xanthomonads, would yield new achievements to reduce the threats they pose to cultivated and wild plant species. This Research Topic focuses on microbial and evolutionary ecology of plant associated Pseudomonas and Xanthomonas, as well as the genomic and molecular diversity of lineages and the virulence and fitness features involved in the interaction with the host-plant. Most of the literature available for this Research Topic has been performed for strains isolated in temperate zones. In line with the long-recognized high social and environmental impact of pests and pathogens in tropical countries, we have welcomed submissions of studies covering such situations for these areas. This Research Topic gathers high-quality contributions (Original Research, Methods, Protocols, Hypothesis & Theory, Reviews, Mini Reviews, Focused Reviews) and in order to promote complementary and original research approaches to improve our knowledge on pseudomonads and xanthomonads-host interactions and their control, it benefited from the scientific communities currently working on Pseudomonas and Xanthomonas such as the teams dealing with the Pseudomonas syringae species complex and the French Network on Xanthomonads (FNX).

In order to achieve saturating transposon mutagenesis of the genome of plant pathogenic strains of Pseudomonas syringae we needed to improve plasmid conjugation frequency. Manipulation of the growth stage of donor and recipient cells allowed the required increase in frequency and facilitated conjugation of otherwise recalcitrant strains.

Pseudomonas syringae is best known as a plant pathogenic bacterium that causes diseases in a multitude of hosts, and it has been used as a model organism to understand the biology of plant disease. Pathogenic and non-pathogenic isolates of P. syringae are also commonly found living as epiphytes and in the wider environment, including water sources such as rivers and precipitation. Ice-nucleating strains of P. syringae are associated with frost damage to crops. The genomes of numerous strains of P. syringae have been sequenced and molecular genetic studies have elucidated many aspects of this pathogen’s interaction with its host plants.

Hydrogen sulfide (H2S) is known to be an important signalling molecule in both animals and plants, despite its toxic nature. In plants it has been seen to control stomatal apertures, so altering the ability of bacteria to invade plant tissues. Bacteria are known to generate H2S as well as being exposed to plant-generated H2S. During their interaction with plants pathogenic bacteria are known to undergo alterations to their genomic complement. For example Pseudomonas syringae pv. phaseolicola (Pph) strain 1302A undergoes loss of a section of DNA known as a genomic island (PPHGI-1) when exposed to the plants resistance response. Loss of PPHGI-1 from Pph 1302A enables the pathogen to overcome the plants resistance response and cause disease. Here, with the use of H2S donor molecules, changes induced in Pph 1302A genome, as demonstrated by excision of PPHGI-1, were investigated. Pph 1302A cells were found to be resistant to low concentrations of H2S. However, at sub-lethal H2S concentrations an increase in the expression of the PPHGI-1 encoded integrase gene (xerC), which is responsible for island excision, and a subsequent increase in the presence of the circular form of PPHGI-1 were detected. This suggests that H2S is able to initiate excision of PPHGI-1 from the Pph genome. Therefore, H2S that may emanate from the plant has an effect on the genome structure of invading bacteria and their ability to cause disease in plants. Modulation of such plant signals may be a way to increase plant defence responses for crops in the future.

Recent advances in genomics and single-cell analysis have demonstrated the extraordinary complexity reached by microbial populations within their hosts. Communities range from complex multispecies groups to homogeneous populations differentiating into lineages through genetic or non-genetic mechanisms. Diversity within bacterial populations is recognized as a key driver of the evolution of animal pathogens. In plants, however, little is known about how interactions between different pathogenic and non-pathogenic variants within the host impact on defence responses, or how the presence within a mixture may affect the development or the fate of each variant. Using confocal fluorescence microscopy, we analysed the colonization of the plant apoplast by individual virulence variants of Pseudomonas syringae within mixed populations. We found that non-pathogenic variants can proliferate and even spread beyond the inoculated area to neighbouring tissues when in close proximity to pathogenic bacteria. The high bacterial concentrations reached at natural entry points promote such interactions during the infection process. We also found that a diversity of interactions take place at a cellular level between virulent and avirulent variants, ranging from dominant negative effects on proliferation of virulent bacteria to in trans suppression of defences triggered by avirulent bacteria. Our results illustrate the spatial dynamics and complexity of the interactions found within mixed infections, and their potential impact on pathogen evolution.

Twenty-two cream-coloured bacterial strains were isolated from oak trees affected by acute oak decline (AOD) in Southern England. Isolates were Gram-negative, motile, slightly curved rods, aerobic, non-spore-forming, catalase positive and oxidase positive. 16S rRNA gene sequence analysis placed the strains in two separate phylogenetic clusters in the Pseudomonas straminea group, with Pseudomonas flavescens as the closest phylogenetic relative. Multilocus sequence analyses of the gyrB, rpoD and rpoB genes supported the delineation of the strains into two separate taxa, which could be differentiated phenotypically and chemotaxonomically from each other, and their closest relatives. Average nucleotide identity and in silico DNA-DNA hybridization values revealed percentages of genome similarity below the species threshold (95 and 70 %, respectively) between the two taxa and the closest relatives, confirming their novel species status. Therefore, on the basis of this polyphasic approach we propose two novel Pseudomonas species, Pseudomonasdaroniae sp. nov. (type strain FRB 228T=LMG 31087T=NCPPB 4672T) and Pseudomonasdryadis sp. nov. (type strain FRB 230T=LMG 31087T=NCPPB 4673T).

Bacterial microcolonies with heterogeneous sizes are formed during colonization of Phaseolus vulgaris by Pseudomonas syringae. Heterogeneous expression of structural and regulatory components of the P. syringae type III secretion system (T3SS), essential for colonization of the host apoplast and disease development, is likewise detected within the plant apoplast. T3SS expression is bistable in the homogeneous environment of nutrient-limited T3SS-inducing medium, suggesting that subpopulation formation is not a response to different environmental cues. T3SS bistability is reversible, indicating a non-genetic origin, and the T3SSHIGH and T3SSLOW subpopulations show differences in virulence. T3SS bistability requires the transcriptional activator HrpL, the double negative regulatory loop established by HrpV and HrpG, and may be enhanced through a positive feedback loop involving HrpA, the main component of the T3SS pilus. To our knowledge, this is the first example of phenotypic heterogeneity in the expression of virulence determinants during colonization of a non-mammalian host.

The co-evolution of bacterial plant pathogens and their hosts is a complex and dynamic process. Host resistance imposes stress on invading pathogens that can lead to changes in the bacterial genome enabling the pathogen to escape host resistance. We have observed this phenomenon with the plant pathogen Pseudomonas syringae pv. phaseolicola where isolates that have lost the genomic island PPHGI-1 carrying the effector gene avrPphB from its chromosome are infective against previously resistant plant hosts. However, we have never observed island extinction from the pathogen population within a host suggesting the island is maintained. Here, we present a mathematical model which predicts different possible fates for the island in the population; one outcome indicated that PPHGI-1 would be maintained at low frequency in the population long term, if it confers a fitness benefit. We empirically tested this prediction and determined that PPHGI-1 frequency in the bacterial population drops to a low but consistently detectable level during host resistance. Once PPHGI-1-carrying cells encounter a susceptible host, they rapidly increase in the population in a negative frequency-dependent manner. Importantly, our data show that mobile genetic elements can persist within the bacterial population and increase in frequency under favourable conditions.

The apoplast is the arena in which endophytic pathogens such as Pseudomonas syringae grow and interact with plant cells. Using metabolomic and ion analysis techniques, this study shows how the composition of Phaseolus vulgaris leaf apoplastic fluid changes during the first six hours of compatible and incompatible interactions with two strains of P. syringae pv. phaseolicola (Pph) that differ in the presence of the genomic island PPHGI-1. Leaf inoculation with the avirulent island-carrying strain Pph 1302A elicited effector-triggered immunity (ETI) and resulted in specific changes in apoplast composition, including increases in conductivity, pH, citrate, γ-aminobutyrate (GABA) and K(+) , that are linked to the onset of plant defence responses. Other apoplastic changes, including increases in Ca(2+) , Fe(2/3+) Mg(2+) , sucrose, β-cyanoalanine and several amino acids, occurred to a relatively similar extent in interactions with both Pph 1302A and the virulent, island-less strain Pph RJ3. Metabolic footprinting experiments established that Pph preferentially metabolizes malate, glucose and glutamate, but excludes certain other abundant apoplastic metabolites, including citrate and GABA, until preferred metabolites are depleted. These results demonstrate that Pph is well-adapted to the leaf apoplast metabolic environment and that loss of PPHGI-1 enables Pph to avoid changes in apoplast composition linked to plant defences.

Synonymy of Gibbsiella dentisursi DSM 23818T ( = NUM 1720T) and Gibbsiella papilionis JCM 18389T ( = LEN33T) was suspected following multilocus sequence analysis (MLSA) of both type strains in a previous classification study, where they were found to share >99.6 % gene sequence similarity. The taxonomic relationship between these two strains was re-examined here using a polyphasic approach. A DNA–DNA hybridization value of 98 % confirmed that the two type strains belong to a single taxon, while the phenotypic profiles were found to be nearly identical. Therefore we propose Gibbsiella papilionis as a later heterotypic synonym of Gibbsiella dentisursi, with the type strain as NUM 1720T ( = DSM 23818T = JCM 17201T).

Pseudomonas syringae is best known as a plant pathogenic bacterium that causes diseases in a multitude of hosts, and it has been used as a model organism to understand the biology of plant disease. Pathogenic and non-pathogenic isolates of P. syringae are also commonly found living as epiphytes and in the wider environment, including water sources such as rivers and precipitation. Ice-nucleating strains of P. syringae are associated with frost damage to crops. The genomes of numerous strains of P. syringae have been sequenced and molecular genetic studies have elucidated many aspects of this pathogen's interaction with its host plants.

Summary The plant pathogen Pseudomonas syringae pv. phaseolicola, which causes halo blight disease of beans, contains a 106 kb genomic island PPHGI‐1. PPHGI‐1 carries a gene, avrPphB, which encodes an effector protein that triggers a resistance response in certain bean cultivars. Previous studies have shown that when PPHGI‐1 is excised from the bacterial chromosome, avrPphB is downregulated and therefore the pathogen avoids triggering the host’s defence mechanism. Here, we investigate whether the downregulation of avrPphB is caused by the supercoiling of PPHGI‐1. We also investigate the effect of a PPHGI‐1‐encoded type 1A topoisomerase, TopB3, on island stability and bacterial pathogenicity in the plant. Supercoiling inhibitors significantly increased the expression of avrPphB but did not affect the excision of PPHGI‐1. An insertional mutant of topB3 displayed an increase in avrPphB expression and an increase in PPHGI‐1 excision as well as reduced population growth in resistant and susceptible cultivars of bean. These results suggest an important role for topoisomerases in the maintenance and stability of a bacterial‐encoded genomic island and demonstrate that supercoiling is involved in the downregulation of an effector gene once the island has been excised, allowing the pathogen to prevent further activation of the host defence response.

Understanding the molecular mechanisms underpinning the ecological success of plant pathogens is critical to develop strategies for controlling diseases and protecting crops. Recent observations have shown that plant pathogenic bacteria, particularly Pseudomonas, exist in a range of natural environments away from their natural plant host e.g., water courses, soil, non-host plants. This exposes them to a variety of eukaryotic predators such as nematodes, insects and amoebae present in the environment. Nematodes and amoeba in particular are bacterial predators while insect herbivores may act as indirect predators, ingesting bacteria on plant tissue. We therefore postulated that bacteria are probably under selective pressure to avoid or survive predation and have therefore developed appropriate coping mechanisms. We tested the hypothesis that plant pathogenic Pseudomonas syringae are able to cope with predation pressure and found that three pathovars show weak, but significant resistance or toxicity. To identify the gene systems that contribute to resistance or toxicity we applied a heterologous screening technique, called Rapid Virulence Annotation (RVA), for anti-predation and toxicity mechanisms. Three cosmid libraries for P. syringae pv. aesculi, pv. tomato and pv. phaseolicola, of approximately 2000 cosmids each, were screened in the susceptible/non-toxic bacterium Escherichia coli against nematode, amoebae and an insect. A number of potential conserved and unique genes were identified which included genes encoding haemolysins, biofilm formation, motility and adhesion. These data provide the first multi-pathovar comparative insight to how plant pathogens cope with different predation pressures and infection of an insect gut and provide a foundation for further study into the function of selected genes and their role in ecological success. All work was funded by the University of Reading.

AmpC β-lactamase is an enzyme commonly produced by Escherichia coli that causes resistance to cephalosporins and penicillins. Enzyme production is controlled by the strength of the promoter encoded by the chromosomal ampC gene, with the level of production affected by the presence of certain mutations in this region. This study sets out to determine the prevalence of ampC promoter mutations present in a group of uropathogenic E. coli strains. A total of 50 clinical strains of E. coli were collected from urine samples between June 2011 and November 2011. Strains were investigated for the presence of mutations in the chromosomal ampC promoter region by amplification and sequencing of a 271 bp product. The presence of ampC-carrying plasmids derived from other species was also determined, to exclude these from further analysis. ampC-carrying plasmids were found in 10 of the 50 strains, all of which were of the CIT-type. Analysis of the chromosomal ampC promoter region in the 40 remaining strains showed mutations at 16 different positions, with 18 different genotype patterns detected overall. The most common ampC chromosomal mutation, present in 25 of 40 strains, was a T --> A transition at position -32. This mutation has been shown by others to increase enzyme production by up to 46-fold. Altogether, three separate mutations (-32, -42 and -13ins) were present in 90% of the 40 non-plasmid strains, indicating a strong association with the resistance observed. It appears, therefore, that the majority of AmpC-mediated resistance in E. coli can be accounted for by just three point mutations in the chromosome.

Acute oak decline (AOD) is a relatively newly described disorder affecting native oak species in Britain. Symptomatic trees are characterised by stem bleeds from vertical fissures, necrotic lesions in the live tissue beneath and larval galleries of the two spotted oak buprestid (Agrilus biguttatus). Several abiotic and biotic factors can be responsible for tree death, however the tissue necrosis and stem weeping is thought to be caused by a combination of bacterial species. Following investigations of the current episode of AOD which began in 2008, numerous strains belonging to several different bacteria in the family Enterobacteriaceae have been consistently isolated from symptomatic tissue. The majority of these enterobacteria were found to be novel species, subspecies and even genera, which have now been formally classified. The most frequently isolated species from symptomatic oak are Gibbsiella quercinecans, Brenneria goodwinii and Rahnella victoriana. Identification of these bacteria is difficult due to similarities in colony morphology, phenotypic profile and 16S rRNA gene sequences. Current identification relies heavily on gyrB gene amplification and sequencing, which is time consuming and laborious. However, newer techniques based on detection of single nucleotide polymorphisms show greater promise for rapid and reliable identification of the bacteria associated with AOD.

In 2010, cream-coloured, Gram-negative staining, facultatively anaerobic enterobacteria were isolated from a single black oak tree (Quercus kelloggii) exhibiting decline symptoms in southern California, USA. These 12 isolates were tentatively identified as Gibbsiella quercinecans based on partial gyrB sequencing. Closer examination of the strains using multilocus sequence analysis, based on partial sequences of gyrB, rpoB, infB and atpD genes, and almost complete 16S rRNA gene sequencing suggested that the isolates belong to a novel taxon within the genus Gibbsiella with G. quercinecans as their closest phylogenetic relative. DNA-DNA relatedness studies confirmed that the strains belong to a single taxon in Gibbsiella, which can be differentiated from other members of the genus by several phenotypic traits. Therefore, the name Gibbsiella greigii sp. nov. is proposed for this novel species isolated from symptomatic Q. kelloggii in the USA with FRB 224(T) (=LMG 27716(T)=NCPPB 4583(T)) as the type strain.

Gram-negative, facultatively anaerobic bacteria were isolated from symptomatic oak tissue in the UK and USA. Partial gyrB sequencing placed ten strains in the genus Brenneria, with B. goodwinii as the closest phylogenetic relative. The strains were investigated further using a polyphasic approach including MLSA (based on partial gyrB, rpoB, infB and atpD gene sequences), 16S rRNA gene sequencing, DNA-DNA relatedness studies and both phenotypic and chemotaxonomic assays. The MLSA and 16S rRNA gene analyses separated the strains into two groups based on origin, suggesting that they belong to Brenneria as two novel species. However, the DNA-DNA relatedness values revealed a closer relationship between the groups and indicated that they should belong to the same species. As the two groups of strains from the UK and USA can be differentiated from each other phenotypically and by ERIC PCR fingerprints, it is proposed to classify them as novel subspecies of a novel Brenneria species. The name Brenneria roseae sp. nov. (FRB 222(T)=LMG 27714(T)=NCPPB 4581(T)) is proposed, with Brenneria roseae subsp. roseae ssp. nov. (FRB 222(T)=LMG 27714(T)=NCPPB 4581(T)) for the strains from the UK and Brenneria roseae subsp. americana ssp. nov. (FRB 223(T)=LMG 27715(T)=NCPPB 4582(T)) for the strains from the USA.

The rulAB operon of Pseudomonas spp. confers fitness traits on the host and has been suggested to be a hotspot for insertion of mobile elements that carry avirulence genes. Here, for the first time, we show that rulB on plasmid pWW0 is a hotspot for the active site-specific integration of related integron-like elements (ILEs) found in six environmental pseudomonads (strains FH1–FH6). Integration into rulB on pWW0 occurred at position 6488 generating a 3 bp direct repeat. ILEs from FH1 and FH5 were 9403 bp in length and contained eight open reading frames (ORFs), while the ILE from FH4 was 16 233 bp in length and contained 16 ORFs. In all three ILEs, the first 5.1 kb (containing ORFs 1–4) were structurally conserved and contained three predicted site-specific recombinases/integrases and a tetR homologue. Downstream of these resided ORFs of the ‘variable side’ with structural and sequence similarity to those encoding survival traits on the fitness enhancing plasmid pGRT1 (ILEFH1 and ILEFH5) and the NR-II virulence region of genomic island PAGI-5 (ILEFH4). Collectively, these ILEs share features with the previously described type III protein secretion system effector ILEs and are considered important to host survival and transfer of fitness enhancing and (a)virulence genes between bacteria.

Recent advances in genomics and single‐cell analysis have demonstrated the extraordinary complexity reached by microbial populations within their hosts. Communities range from complex multispecies groups to homogeneous populations differentiating into lineages through genetic or non‐genetic mechanisms. Diversity within bacterial populations is recognized as a key driver of the evolution of animal pathogens. In plants, however, little is known about how interactions between different pathogenic and non‐pathogenic variants within the host impact on defence responses, or how the presence within a mixture may affect the development or the fate of each variant. Using confocal fluorescence microscopy, we analysed the colonization of the plant apoplast by individual virulence variants of Pseudomonas syringae within mixed populations. We found that non‐pathogenic variants can proliferate and even spread beyond the inoculated area to neighbouring tissues when in close proximity to pathogenic bacteria. The high bacterial concentrations reached at natural entry points promote such interactions during the infection process. We also found that a diversity of interactions take place at a cellular level between virulent and avirulent variants, ranging from dominant negative effects on proliferation of virulent bacteria to in trans suppression of defences triggered by avirulent bacteria. Our results illustrate the spatial dynamics and complexity of the interactions found within mixed infections, and their potential impact on pathogen evolution.

Pseudomonas syringae pv. phaseolicola ( Pph ) strain 1302A, a causative agent of halo blight in the common bean Phaseolus vulgaris , contains four native plasmids designated pAV505 (150 kb), pAV506 (50 kb), pAV507 (47 kb) and pAV508 (42 kb). Pph 1302A also contains a 106 kb genomic island PPHGI-1 which shares features with integrative and conjugative elements (ICElands) and carries the effector gene avrPphB ( hopAR1 ) which triggers a defensive response in bean cultivars carrying the matching R3 resistance gene. It has been shown that when Pph 1302A is sequentially inoculated (passaged) through resistant bean cultivar Tendergreen (TG) in which the hypersensitive response (HR) is generated, the three largest plasmids are lost and an extra ∼100 kb plasmid is gained, which tests confirmed to be the 106 kb circular form of PPHGI-1. The aim of the current study was to determine if upon further passaging though bean plants, the plasmid profile of Pph 1302A would alter again and if the missing plasmids had been integrated into the chromosome. Pph 1302A-P6, the strain with the altered plasmid profile was passaged twice through TG and of the four re-isolated strains examined all displayed the plasmid profile associated with wildtype Pph 1302A, that is, all four native plasmids had reappeared and the PPHGI-1 plasmid was absent. This demonstrated that the plasmid composition of Pph 1302A-P6 could indeed change on further exposure to the plant environment and also that the seemingly missing native plasmids were still present within the genome, lending evidence to the theory that they had integrated into the chromosome. Furthermore two of these re-isolated strains had lost PPHGI-1 entirely, meaning they no longer triggered a HR on TG and instead generated a disease response. This study clearly demonstrates the plasticity of the bacterial genome and the extent it can be influenced by the plant environment and conditions generated during the HR.

Bacterial microcolonies with heterogeneous sizes are formed during colonization of Phaseolus vulgaris by Pseudomonas syringae. Heterogeneous expression of structural and regulatory components of the P. syringae type III secretion system (T3SS), essential for colonization of the host apoplast and disease development, is likewise detected within the plant apoplast. T3SS expression is bistable in the homogeneous environment of nutrient-limited T3SS-inducing medium, suggesting that subpopulation formation is not a response to different environmental cues. T3SS bistability is reversible, indicating a non-genetic origin, and the T3SS

Multi-locus sequence typing was used to reveal a high degree of diversity amongst the E. coli isolates with AmpC plasmid genes, and a high prevalence of the -32 mutation present.

Summary The co‐evolution of bacterial plant pathogens and their hosts is a complex and dynamic process. Host resistance imposes stress on invading pathogens that can lead to changes in the bacterial genome enabling the pathogen to escape host resistance. We have observed this phenomenon with the plant pathogen Pseudomonas syringae pv. phaseolicola where isolates that have lost the genomic island PPHGI‐1 carrying the effector gene avrPphB from its chromosome are infective against previously resistant plant hosts. However, we have never observed island extinction from the pathogen population within a host suggesting the island is maintained. Here, we present a mathematical model which predicts different possible fates for the island in the population; one outcome indicated that PPHGI‐1 would be maintained at low frequency in the population long term, if it confers a fitness benefit. We empirically tested this prediction and determined that PPHGI‐1 frequency in the bacterial population drops to a low but consistently detectable level during host resistance. Once PPHGI‐1‐carrying cells encounter a susceptible host, they rapidly increase in the population in a negative frequency‐dependent manner. Importantly, our data show that mobile genetic elements can persist within the bacterial population and increase in frequency under favourable conditions.

© 2016 The Society for Applied Microbiology Two Gram-negative Enterobacteriaceae, Gibbsiella quercinecans and Brenneria goodwinii, are frequently isolated from oak suffering from Acute Oak Decline. These two species are difficult to identify based on colony morphology, carbohydrate utilization or 16S rRNA gene sequence, and identification using gyrB gene sequencing is time-consuming and laborious. A rapid identification technique, based on high-resolution melt analysis of the atpD gene, was designed to efficiently process numerous isolates from an increasing number of affected woodlands and parks. Principal component analysis of the resulting melt curves from strains of G.quercinecans, B.goodwinii and their close phylogenetic relatives allowed differentiation into distinct clusters based on species or subspecies identity. Significance and Impact of the Study: Acute Oak Decline is an increasing threat to Britain's native oak population. Two novel bacterial species both belonging to the family Enterobacteriaceae, Gibbsiella quercinecans and Brenneria goodwinii, are thought to play an important role in symptom development. Here, we describe a rapid identification technique using high-resolution melt analysis of the atpD gene able to assign isolates to either G.quercinecans or B.goodwinii in a single assay, greatly reducing the time taken to identify if either or both of these species are present in symptomatic oak.

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Microdochium nivale (Fr.) Samuels and Hallett (teleomorph Monographella nivalis (Schafnitt) is one of the most damaging pathogens of cool-season amenity turfgrasses. The search for new or improved ...

Pseudomonas syringae pv. phaseolicola causes halo blight of the common bean, Phaseolus vulgaris, worldwide and remains difficult to control. Races of the pathogen cause either disease symptoms or a resistant hypersensitive response on a series of differentially reacting bean cultivars. The molecular genetics of the interaction between P. syringae pv. phaseolicola and bean, and the evolution of bacterial virulence, have been investigated in depth and this research has led to important discoveries in the field of plant-microbe interactions. In this review, we discuss several of the areas of study that chart the rise of P. syringae pv. phaseolicola from a common pathogen of bean plants to a molecular plant-pathogen supermodel bacterium. Taxonomy: Bacteria; Proteobacteria, gamma subdivision; order Pseudomonadales; family Pseudomonadaceae; genus Pseudomonas; species Pseudomonas syringae; Genomospecies 2; pathogenic variety phaseolicola. Microbiological properties: Gram-negative, aerobic, motile, rod-shaped, 1.5 µm long, 0.7-1.2 µm in diameter, at least one polar flagellum, optimal temperatures for growth of 25-30 °C, oxidase negative, arginine dihydrolase negative, levan positive and elicits the hypersensitive response on tobacco. Host range: Major bacterial disease of common bean (Phaseolus vulgaris) in temperate regions and above medium altitudes in the tropics. Natural infections have been recorded on several other legume species, including all members of the tribe Phaseoleae with the exception of Desmodium spp. and Pisum sativum. Disease symptoms: Water-soaked lesions on leaves, pods, stems or petioles, that quickly develop greenish-yellow haloes on leaves at temperatures of less than 23 °C. Infected seeds may be symptomless, or have wrinkled or buttery-yellow patches on the seed coat. Seedling infection is recognized by general chlorosis, stunting and distortion of growth. Epidemiology: Seed borne and disseminated from exudation by water-splash and wind occurring during rainfall. Bacteria invade through wounds and natural openings (notably stomata). Weedy and cultivated alternative hosts may also harbour the bacterium. Disease control: Some measure of control is achieved with copper formulations and streptomycin. Pathogen-free seed and resistant cultivars are recommended. Useful websites: Pseudomonas-plant interaction http://www.pseudomonas-syringae.org/; PseudoDB http://xbase.bham.ac.uk/pseudodb/; Plant Associated and Environmental Microbes Database (PAMDB) http://genome.ppws.vt.edu/cgi-bin/MLST/home.pl; PseudoMLSA Database http://www.uib.es/microbiologiaBD/Welcome.html.

The immense social and economic impact of bacterial pathogens, from drug-resistant infections in hospitals to the devastation of agricultural resources, has resulted in major investment to understand the causes and consequences of pathogen evolution. Recent genome sequencing projects have provided insight into the evolution of bacterial genome structures; revealing the impact of mobile DNA on genome restructuring and pathogenicity. Sequencing of multiple genomes of related strains has enabled the delineation of pathogen evolution and facilitated the tracking of bacterial pathogens globally. Other recent theoretical and empirical studies have shown that pathogen evolution is significantly influenced by ecological factors, such as the distribution of hosts within the environment and the effects of co-infection. We suggest that the time is ripe for experimentalists to use genomics in conjunction with evolutionary ecology experiments to further understanding of how bacterial pathogens evolve.

The co-evolution of bacterial plant pathogens and their hosts is a complex and dynamic process. Plant resistance can impose stress on invading pathogens that can lead to, and select for, beneficial changes in the bacterial genome. The Pseudomonas syringae pv. phaseolicola (Pph) genomic island PPHGI-1 carries an effector gene, avrPphB (hopAR1), which triggers the hypersensitive reaction in bean plants carrying the R3 resistance gene. Interaction between avrPphB and R3 generates an antimicrobial environment within the plant, resulting in the excision of PPHGI-1 and its loss from the genome. The loss of PPHGI-1 leads to the generation of a Pph strain able to cause disease in the plant. In this study, we observed that lower bacterial densities inoculated into resistant bean (Phaseolus vulgaris) plants resulted in quicker PPHGI-1 loss from the population, and that loss of the island was strongly influenced by the type of plant resistance encountered by the bacteria. In addition, we found that a number of changes occurred in the bacterial genome during growth in the plant, whether or not PPHGI-1 was lost. We also present evidence that the circular PPHGI-1 episome is able to replicate autonomously when excised from the genome. These results shed more light onto the plasticity of the bacterial genome as it is influenced by in planta conditions.

Although genome sequencing of microbial pathogens has shed light on the evolution of virulence, the drivers of the gain and loss of genes and of pathogenicity islands (gene clusters), which contribute to the emergence of new disease outbreaks, are unclear. Recent experiments with the bean pathogen Pseudomonas syringae pv. phaseolicola illustrate how exposure to resistance mechanisms acts as the driving force for genome reorganization. Here we argue that the antimicrobial conditions generated by host defences can accelerate the generation of genome rearrangements that provide selective advantages to the invading microbe. Similar exposure to environmental stress outside the host could also drive the horizontal gene transfer that has led to the evolution of pathogenicity towards both animals and plants.

The ability to form biofilms is seen as an increasingly important colonization strategy among both pathogenic and environmental bacteria. A survey of 185 plant-associated, phytopathogenic, soil and river Pseudomonas isolates resulted in 76% producing biofilms at the air-liquid (A-L) interface after selection in static microcosms. Considerable variation in biofilm phenotype was observed, including waxy aggregations, viscous and floccular masses, and physically cohesive biofilms with continuously varying strengths over 1500-fold. Calcofluor epifluorescent microscopy identified cellulose as the matrix component in biofilms produced by Pseudomonas asplenii, Pseudomonas corrugata, Pseudomonas fluorescens, Pseudomonas marginalis, Pseudomonas putida, Pseudomonas savastanoi and Pseudomonas syringae isolates. Cellulose expression and biofilm formation could be induced by the constitutively active WspR19 mutant of the cyclic-di-GMP-associated, GGDEF domain-containing response regulator involved in the P. fluorescens SBW25 wrinkly spreader phenotype and cellular aggregation in Pseudomonas aeruginosa PA01. WspR19 could also induce P. putida KT2440, which otherwise did not produce a biofilm or express cellulose, as well as Escherichia coli K12 and Salmonella typhimurium LT2, both of which express cellulose yet lack WspR homologues. Statistical analysis of biofilm parameters suggest that biofilm development is a more complex process than that simply described by the production of attachment and matrix components and bacterial growth. This complexity was also seen in multivariate analysis as a species-ecological habitat effect, underscoring the fact that in vitro biofilms are abstractions of those surface and volume colonization processes used by bacteria in their natural environments.

The use of bioluminescence was evaluated as a tool to study Pseudomonas syringae population dynamics in susceptible and resistant plant environments. Plasmid pGLITE, containing the luxCDABE genes from Photorhabdus luminescens, was introduced into Pseudomonas syringae pv. phaseolicola race 7 strain 1449B, a Gram-negative pathogen of bean (Phaseolus vulgaris). Bacteria recovered from plant tissue over a five-day period were enumerated by counting numbers of colony forming units and by measurement of bioluminescence. Direct measurement of bioluminescence from leaf disc homogenates consistently reflected bacterial growth as determined by viable counting, but also detected subtle effects of the plant resistance response on bacterial viability. This bioluminescence procedure enables real time measurement of bacterial metabolism and population dynamics in planta, obviates the need to carry out labour intensive and time consuming traditional enumeration techniques and provides a sensitive assay for studying plant effects on bacterial cells.

SummaryBacterial pathogenicity to plants and animals has evolved through an arms race of attack and defense. Key players are bacterial effector proteins, which are delivered through the type III secretion system and suppress basal defenses [1]. In plants, varietal resistance to disease is based on recognition of effectors by the products of resistance (R) genes [2]. When recognized, the effector or in this scenario, avirulence (Avr) protein triggers the hypersensitive resistance reaction (HR), which generates antimicrobial conditions [3]. Unfortunately, such gene-for-gene-based resistance commonly fails because of the emergence of virulent strains of the pathogen that no longer trigger the HR [4]. We have followed the emergence of a new virulent pathotype of the halo-blight pathogen Pseudomonas syringae pv. phaseolicola within leaves of a resistant variety of bean. Exposure to the HR led to the selection of strains lacking the avirulence (effector) gene avrPphB (or hopAR1 [5]), which triggers defense in varieties with the matching R3 resistance gene. Loss of avrPphB was through deletion of a 106 kb genomic island (PPHGI-1) that shares features with integrative and conjugative elements (ICElands) and also pathogenicity islands (PAIs) in diverse bacteria [6, 7]. We provide a molecular explanation of how exposure to resistance mechanisms in plants drives the evolution of new virulent forms of pathogens.

SUMMARY Pathogenicity islands (PAIs) were first described in uropathogenic E. coli. They are now defined as regions of DNA that contain virulence genes and are present in the genome of pathogenic strains, but absent from or only rarely present in non-pathogenic variants of the same or related strains. Other features include a variable G+C content, distinct boundaries from the rest of the genome and the presence of genes related to mobile elements such as insertion sequences, integrases and transposases. Although PAIs have now been described in a wide range of both plant and animal pathogens it has become evident that the general features of PAIs are displayed by a number of regions of DNA with functions other than pathogenicity, such as symbiosis and antibiotic resistance, and the general term genomic islands has been adopted. This review will describe a range of genomic islands in plant pathogenic bacteria including those that carry effector genes, phytotoxins and the type III protein secretion cluster. The review will also consider some medically important bacteria in order to discuss the range, acquisition and stabilization of genomic islands.

Isolations from oak symptomatic of Acute Oak Decline, alder and walnut log tissue, and buprestid beetles in 2009–2012 yielded 32 Gram-negative bacterial strains showing highest gyrB sequence similarity to Rahnella aquatilis and Ewingella americana. Multilocus sequence analysis (using partial gyrB, rpoB, infB and atpD gene sequences) delineated the strains into six MLSA groups. Two MLSA groups contained reference strains of Rahnella genomospecies 2 and 3, three groups clustered within the Rahnella clade with no known type or reference strains and the last group contained the type strain of E. americana. DNA–DNA relatedness assays using both the microplate and fluorometric methods, confirmed that each of the five Rahnella MLSA groups formed separate taxa. Rahnella genomospecies 2 and 3 were previously not formally described due to a lack of distinguishing phenotypic characteristics. In the present study, all five Rahnella MLSA groups were phenotypically differentiated from each other and from R. aquatilis. Therefore we propose to classify the strains from symptomatic oak, alder and walnut and buprestid beetles as: Rahnella victoriana sp. nov. (type strain FRB 225T = LMG 27717T = DSM 27397T), Rahnella variigena sp. nov. (previously Rahnella genomosp. 2, type strain CIP 105588T = LMG 27711T), Rahnella inusitata sp. nov. (previously Rahnella genomosp. 3, type strain DSM 30078T = LMG 2640T), Rahnella bruchi sp. nov. (type strain FRB 226T = LMG 27718T = DSM 27398T) and Rahnella woolbedingensis sp. nov. (type strain FRB 227T = LMG 27719T = DSM 27399T).

DNA sequences flanking two avr genes (avrPpiA1 and avrPpiB1) from Pseudomonas syringae pv. pisi show a high degree of similarity. Specific primers designed from the conserved regions were used in PCR amplifications with all P. syringae pv. pisi races. As well as amplifying the expected avrPpiA- and avrPpiB-containing fragments, two additional fragments were amplified: one contained a single open reading frame (ORF1) and was found in races of genomic group II (2, 3A, 4A and 6); the second fragment contained two open reading frames (ORF2 and ORF3), separated by 658 nt, and was detected in all races. All three ORFs had G+C ratios (46.9-48 mol%) that were significantly less than that for P. syringae and each was preceded by a potential hrp box promoter. In P. syringae pv. phaseolicola, ORF1 and ORF2 each elicited a strong non-host hypersensitive reaction on bean leaves; ORF1 was designated avrPpiG, the product of which had strong similarity to AvrRxv, AvrBsT and YopP. ORF2 was identical to a gene, designated avrPpiC, previously isolated from P. syringae pv. pisi race 5. ORF3 was always found in association with avrPpiC and both were detected in a wide range of P. syringae pathovars. In contrast, avrPpiG was only detected in strains of P. syringae pv. pisi genomic group II and P. syringae pv. coronafaciens (ICMP 3113). In P. syringae pv. pisi, avrPpiG was plasmid-borne and avrPpiC and ORF3 were chromosomal. This conservation of flanking sequences has implications for the horizontal transfer of avirulence and virulence genes, suggesting that specific regions of the bacterial genome act as sites for their integration/excision.

The largest plasmid (pPT23A, 100 kb) from Pseudomonas syringae pv.tomato PT23, carries the gene cluster for the synthesis of the phytotoxin coronatine and most of its DNA sequences are repeated in a co-residing plasmid, pPT23B (83 kb). Some DNA fragments from pPT23A are also repeated elsewhere in the genome. To investigate the role in virulence of these plasmids, avoiding the effects of gene redundancy, we constructed strain UPN2, which was derived by curing the four native plasmids from PT23. The necrotic lesions induced in tomato by UPN2 were four-fold smaller than those produced by PT23. This phenotype was only partially restored by pPT23A, and the production of lesions of wild type size was dependent on the concomitant presence in UPN2 of pPT23A and pPT23B. Mutants defective in the production of coronatine, but which still produced coronafacic acid, induced lesions of an intermediate size between PT23 and UPN2. The virulence determinants present on pPT23B could be modifying the amount, structure or uptake rate by the plant of the coronafacic acid molecules produced by genes present on pPT23A. Therefore, the production of necrotic lesions on tomato plants by strain PT23 is due substantially to the action of compounds whose determinants are located on plasmids pPT23A and pPT23B, including, but not limited to coronatine. The involvement of the coronafacic acid gene cluster in virulence in the absence of the cluster for the synthesis of coronamic acid, may help explain its maintenance in the bacterial population before being incorporated into the coronatine cluster. Likewise, the physical separation of the virulence determinants in the two independent replicons pPT23A and pPT23B, may have driven the evolution of strain PT23 so that it maintains two potentially incompatible plasmids.

Phylogenetic analysis of the genus Pseudomonas was conducted by using the combined gyrB and rpoD nucleotide sequences of 31 validly described species of Pseudomonas (a total of 125 strains). Pseudomonas strains diverged into two major clusters designated intrageneric cluster I (IGC I) and intrageneric cluster II (IGC II). IGC I was further split into two subclusters, the ‘P. aeruginosa complex’, which included P. aeruginosa, P. alcaligenes, P. citronellolis, P. mendocina, P. oleovorans and P. pseudoalcaligenes, and the ‘P. stutzeri complex’, which included P. balearica and P. stutzeri. IGC II was further split into three subclusters that were designated the ‘P. putida complex’, the ‘P. syringae complex’ and the ‘P. fluorescens complex’. The ‘P. putida complex’ included P. putida and P. fulva. The ‘P. syringae complex’ was the cluster of phytopathogens including P. amygdali, P. caricapapayae, P. cichorii, P. ficuserectae, P. viridiflava and the pathovars of P. savastanoi and P. syringae. The ‘P. fluorescens complex’ was further divided into two subpopulations, the ‘P. fluorescens lineage’ and the ‘P. chlororaphis lineage’. The ‘P. fluorescens lineage’ contained P. fluorescens biotypes A, B and C, P. azotoformans, P. marginalis pathovars, P. mucidolens, P. synxantha and P. tolaasii, while the ‘P. chlororaphis lineage’ included P. chlororaphis, P. agarici, P. asplenii, P. corrugata, P. fluorescens biotypes B and G and P. putida biovar B. The strains of P. fluorescens biotypes formed a polyphyletic group within the ‘P. fluorescens complex’.