Description of a novel species of Leclercia, Leclercia tamurae sp. nov. and proposal of a novel genus Silvania gen. nov. containing two novel species Silvania hatchlandensis sp. nov. and Silvania confinis sp. nov. isolated from the rhizosphere of oak.

BACKGROUND: Acute Oak Decline (AOD) is a decline disease first reported on native oaks in the UK, but in recent years reports from further afield such as Europe and the Middle East, indicate that the distribution and host range is increasing at an alarming rate. The stem weeping symptoms of the disease partially develop due to polymicrobial-host interaction, caused by several members of the order Enterobacterales. While investigating the rhizosphere soil of AOD-unaffected trees, termed 'healthy' trees, and diseased oaks suffering from Acute Oak Decline (AOD), an enrichment method designed for enhanced recovery of Enterobacterales led to the recovery of several isolates that could not be classified as any existing species. These isolates showed a close relationship to the genus Leclercia, of which both species are of clinical importance, but the type species Leclercia adecarboxylata also displays plant growth-promoting properties in the rhizosphere. RESULTS: Partial sequencing of four housekeeping genes revealed similarity to the genus Leclercia with varying degrees of relatedness. As such a complete polyphasic approach was used to determine the true taxonomic position of these isolates. This involved whole genome sequencing, phylogenomic analysis, phylogenetic analysis of both the 16S rRNA and four housekeeping gene sequences, combined with phenotypic testing and fatty acid analysis. Both the phylogenomic and phylogenetic analyses separated the isolates into four clusters, two of which were contained in the Leclercia clade. The remaining two clusters formed a separate lineage far removed from any currently defined species. Further investigation into the role of the isolates as plant growth-promoting bacteria as well as plant pathogens was investigated computationally, revealing a number of plant growth-promoting traits as well as virulence genes related to motility, adhesion and immune modulation. CONCLUSION: Based on the genotypic and phenotypic data presented here, these isolates could be differentiated from each other and their closest neighbours. As such we propose the description of Leclercia tamurae sp. nov. (type strain H6S3T = LMG 32609T = CCUG 76176T), Silvania gen. nov., Silvania hatchlandensis sp. nov. (type strain H19S6T = LMG 32608T = CCUG 76185T) and Silvania confinis sp. nov. (type strain H4N4T = LMG 32607T = CCUG 76175T). Due to their interesting protein annotations and alignments, these species warrant further investigation for their role in relation to plant health.

Brenneria tiliae sp. nov., isolated from symptomatic Tilia × moltkei and Tilia × europaea trees in the UK.

Several strains of a previously undescribed bacterial species were isolated from mature Tilia hybrid trees suffering from bleeding cankers at various geographic locations in the UK. The strains were Gram-negative, facultatively anaerobic, and partial sequencing of the gyrB gene revealed that the strains belong to the genus Brenneria with the closest phylogenetic neighbours being Brenneria corticis and Brenneria nigrifluens. Further investigation using a polyphasic approach was undertaken to determine the taxonomic position of the novel species. Phylogenies based on the 16S rRNA gene and multilocus sequence analysis of partial housekeeping gene sequences of gyrB, rpoB, infB and atpD revealed that the strains formed an independent cluster within the genus Brenneria. The phenotypic and chemotaxonomic assays demonstrated that the strains could be differentiated from the closest relatives. Genome analysis of representative strains revealed in silico DNA-DNA hybridization values below the threshold for species delimitation, although the average nucleotide identity values obtained when compared to B. corticis (95.9-96%) were slightly higher than the suggested cut-off value of 95%. However, as all other data suggests that the strains belong to a novel taxon that can be differentiated from the closest relatives, we propose that the strains represent a novel species in the genus Brenneria, Brenneria tiliae sp. nov. (type strain WC1b.1T=LMG 32575T=NCPPB 4697T).

Rahnella perminowiae sp. nov., Rahnella bonaserana sp. nov., Rahnella rivi sp. nov. and Rahnella ecdela sp. nov., isolated from diverse environmental sources, and emended description of the genus Rahnella.

Bacteria isolated from onion bulbs suffering from bacterial decay in the United States and Norway were previously shown to belong to the genus Rahnella based on partial housekeeping gene sequences and/or fatty acid analysis. However, many strains could not be assigned to any existing Rahnella species. Additionally, strains isolated from creek water and oak as well as a strain with bioremediation properties were assigned to Rahnella based on partial housekeeping gene sequences. The taxonomic status of these 21 strains was investigated using multilocus sequence analysis, whole genome analyses, phenotypic assays and fatty acid analysis. Phylogenetic and phylogenomic analyses separated the strains into five clusters, one of which corresponded to Rahnella aceris. The remaining four clusters could be differentiated both genotypically and phenotypically from each other and existing Rahnella species. Based on these results, we propose the description of four novel species: Rahnella perminowiae sp. nov. (type strain SL6T=LMG 32257T=DSM 112609T), Rahnella bonaserana sp. nov. (H11bT=LMG 32256T=DSM 112610T), Rahnella rivi sp. nov. (FC061912-KT=LMG 32259T=DSM 112611T) and Rahnella ecdela sp. nov. (FRB 231T=LMG 32255T=DSM 112612T).

Pseudomonas kirkiae sp. nov., a novel species isolated from oak in the United Kingdom, and phylogenetic considerations of the genera Pseudomonas, Azotobacter and Azomonas.

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.

Supercoiling of an excised genomic island represses effector gene expression to prevent activation of host resistance.

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.

Pseudomonas syringae: enterprising epiphyte and stealthy parasite.

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.

Pseudomonas daroniae sp. nov. and Pseudomonas dryadis sp. nov., isolated from pedunculate oak affected by acute oak decline in the UK.

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).

Taxonomy and identification of bacteria associated with acute oak decline.

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.

A low frequency persistent reservoir of a genomic island in a pathogen population ensures island survival and improves pathogen fitness in a susceptible 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.

Pseudomonas syringae Differentiates into Phenotypically Distinct Subpopulations During Colonization of a Plant Host.

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.

Early changes in apoplast composition associated with defence and disease in interactions between Phaseolus vulgaris and the halo blight pathogen Pseudomonas syringae Pv. phaseolicola.

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, ß-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.

Gibbsiella papilionis Kim et al. 2013 is a later heterotypic synonym of Gibbsiella dentisursi Saito et al. 2013.

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).

Bacterial pathogen evolution: breaking news.

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 rulB gene of plasmid pWW0 is a hotspot for the site-specific insertion of integron-like elements found in the chromosomes of environmental Pseudomonas fluorescens group bacteria.

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 (ILE(FH1) and ILE(FH5)) and the NR-II virulence region of genomic island PAGI-5 (ILE(FH4)). 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.

Two novel Raoultella species associated with bleeding cankers of broadleaf hosts, Raoultella scottia sp. nov. and Raoultella lignicola sp. nov.

Seventeen Gram-negative, facultatively anaerobic bacterial strains were isolated from bleeding cankers of various broadleaf hosts and oak rhizosphere soil in Great Britain. The strains were tentatively identified as belonging to the genus Raoultella based on 16S rRNA gene sequencing. Multilocus sequence analysis (MLSA), based on four protein-encoding genes (fusA, leuS, pyrG, and rpoB), separated the strains into three clusters within the Raoultella genus clade. The majority of strains clustered with the type strain of Raoultella terrigena, with the remaining strains divided into two clusters with no known type strain. Whole genome sequencing comparisons confirmed these two clusters of strains as belonging to two novel Raoultella species which can be differentiated phenotypically from their current closest phylogenetic relatives. Therefore, two novel species are proposed: Raoultella scottia sp. nov. (type strain = BAC 10a-01-01T = LMG 33072T = CCUG 77096T) and Raoultella lignicola sp. nov. (type strain = TW_WC1a.1T = LMG 33073T = CCUG 77094T).

The stealth episome: suppression of gene expression on the excised genomic island PPHGI-1 from Pseudomonas syringae pv. phaseolicola.

Pseudomonas syringae pv. phaseolicola is the causative agent of halo blight in the common bean, Phaseolus vulgaris. P. syringae pv. phaseolicola race 4 strain 1302A contains the avirulence gene avrPphB (syn. hopAR1), which resides on PPHGI-1, a 106 kb genomic island. Loss of PPHGI-1 from P. syringae pv. phaseolicola 1302A following exposure to the hypersensitive resistance response (HR) leads to the evolution of strains with altered virulence. Here we have used fluorescent protein reporter systems to gain insight into the mobility of PPHGI-1. Confocal imaging of dual-labelled P. syringae pv. phaseolicola 1302A strain, F532 (dsRFP in chromosome and eGFP in PPHGI-1), revealed loss of PPHGI-1::eGFP encoded fluorescence during plant infection and when grown in vitro on extracted leaf apoplastic fluids. Fluorescence-activated cell sorting (FACS) of fluorescent and non-fluorescent PPHGI-1::eGFP F532 populations showed that cells lost fluorescence not only when the GI was deleted, but also when it had excised and was present as a circular episome. In addition to reduced expression of eGFP, quantitative PCR on sub-populations separated by FACS showed that transcription of other genes on PPHGI-1 (avrPphB and xerC) was also greatly reduced in F532 cells harbouring the excised PPHGI-1::eGFP episome. Our results show how virulence determinants located on mobile pathogenicity islands may be hidden from detection by host surveillance systems through the suppression of gene expression in the episomal state.

In planta induced changes in the native plasmid profile of Pseudomonas syringae pathover phaseolicola strain 1302A.

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.

A Pseudomonas syringae diversity survey reveals a differentiated phylotype of the pathovar syringae associated with the mango host and mangotoxin production.

Pseudomonas syringae pv. syringae, the causal agent of bacterial apical necrosis (BAN) in mango crops, has been isolated in different mango-producing areas worldwide. An extensive collection of 87 P. syringae pv. syringae strains isolated from mango trees affected by BAN from different countries, but mainly from Southern Spain, were initially examined by repetitive sequence-based polymerase chain reaction (rep-PCR) to analyze the genetic diversity with an epidemiological aim. rep-PCR was powerful in assessing intrapathovar distribution and also allowing clustering of the P. syringae pv. syringae strains isolated from mango, depending on the isolation area. A clear pattern of clustering was observed for all the P. syringae pv. syringae strains isolated from mango distinct from strains from other hosts, including strains for the same geographical regions as the mango isolates. For this reason, a representative group of 51 P. syringae pv. syringae strains isolated from mango and other hosts, as well as some P. syringae strains from other pathovars, were further characterized to determine their possible genetic, phenotypic, and phylogenetic relationships. Similar to the rep-PCR results, the randomly amplified polymorphic DNA PCR (RAPD-PCR) and catabolic diversity analysis using the Biolog GN2 profile grouped 90% of the mango isolates together in a unique cluster. Interestingly, the majority of P. syringae pv. syringae strains isolated from mango produced mangotoxin. The analysis of the phylogenetic distribution using the multilocus sequence typing analysis strongly supports the existence of a differentiated phylotype of the pathovar syringae mainly associated with the mango host and characterized by the mangotoxin production.

Bacteria Associated with Acute Oak Decline: Where Did They Come From? We Know Where They Go.

Acute oak decline is a high-impact disease causing necrotic lesions on the trunk, crown thinning and the eventual death of oak. Four bacterial species are associated with the lesions-Brenneria goodwinii, Gibbsiella quercinecans, Rahnella victoriana and Lonsdalea Britannica-although an epi-/endophytic lifestyle has also been suggested for these bacteria. However, little is known about their environmental reservoirs or their pathway to endophytic colonisation. This work aimed to investigate the ability of the four AOD-associated bacterial species to survive for prolonged periods within rhizosphere soil, leaves and acorns in vitro, and to design an appropriate method for their recovery. This method was trialled on field samples related to healthy and symptomatic oaks. The in vitro study showed that the majority of these species could survive for at least six weeks within each sample type. Results from the field samples demonstrated that R. victoriana and G. quercinecans appear environmentally widespread, indicating multiple routes of endophytic colonisation might be plausible. B. goodwinii and L. britannica were only identified from acorns from healthy and symptomatic trees, indicating they may be inherited members of the endophytic seed microbiome and, despite their ability to survive outside of the host, their environmental occurrence is limited. Future research should focus on preventative measures targeting the abiotic factors of AOD, how endophytic bacteria shift to a pathogenic cycle and the identification of resilient seed stock that is less susceptible to AOD.