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.

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).

Host-microbiota-insect interactions drive emergent virulence in a complex tree disease.

Forest declines caused by climate disturbance, insect pests and microbial pathogens threaten the global landscape, and tree diseases are increasingly attributed to the emergent properties of complex ecological interactions between the host, microbiota and insects. To address this hypothesis, we combined reductionist approaches (single and polyspecies bacterial cultures) with emergentist approaches (bacterial inoculations in an oak infection model with the addition of insect larvae) to unravel the gene expression landscape and symptom severity of host-microbiota-insect interactions in the acute oak decline (AOD) pathosystem. AOD is a complex decline disease characterized by predisposing abiotic factors, inner bark lesions driven by a bacterial pathobiome, and larval galleries of the bark-boring beetle Agrilus biguttatus. We identified expression of key pathogenicity genes in Brenneria goodwinii, the dominant member of the AOD pathobiome, tissue-specific gene expression profiles, cooperation with other bacterial pathobiome members in sugar catabolism, and demonstrated amplification of pathogenic gene expression in the presence of Agrilus larvae. This study highlights the emergent properties of complex host-pathobiota-insect interactions that underlie the pathology of diseases that threaten global forest biomes.

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.

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).

Integrating regulatory surveys and citizen science to map outbreaks of forest diseases: acute oak decline in England and Wales.

The number of emerging tree diseases has increased rapidly in recent times, with severe environmental and economic consequences. Systematic regulatory surveys to detect and establish the distribution of pests are crucial for successful management efforts, but resource-intensive and costly. Volunteers who identify potential invasive species can form an important early warning network in tree health; however, what these data can tell us and how they can be best used to inform and direct official survey effort is not clear. Here, we use an extensive dataset on acute oak decline (AOD) as an opportunity to ask how verified data received from the public can be used. Information on the distribution of AOD was available as (i) systematic regulatory surveys conducted throughout England and Wales, and (ii) ad hoc sightings reported by landowners, land managers and members of the public (i.e. 'self-reported' cases). By using the available self-reported cases at the design stage, the systematic survey could focus on defining the boundaries of the affected area. This maximized the use of available resources and highlights the benefits to be gained by developing strategies to enhance volunteer efforts in future programmes.

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.

Rapid identification of bacteria associated with Acute Oak Decline by high-resolution melt analysis.

UNLABELLED: 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.

The intergenic transcribed spacer region 1 as a molecular marker for identification and discrimination of Enterobacteriaceae associated with acute oak decline.

AIMS: We assessed the veracity of intergenic spacer region 1 (ITS1) ribotyping for the rapid, inexpensive and accurate identification of Brenneria goodwinii and Gibbsiella quercinecans that are associated with acute oak decline (AOD) in the UK. METHODS AND RESULTS: Agarose gel electrophoresis and polyacrylamide gel electrophoresis (PAGE) were applied for the typing of ITS1 PCR amplicons from strains of B. goodwinii, G. quercinecans and related species (n = 34). The number and length of ITS1 amplicons varied significantly between strains. ITS1 profiles generated via PAGE were used to differentiate species using a neighbour-joining phylogram. The ITS1 phylogram was compared against DNA gyrase B (gyrB) gene sequences from the same strains, demonstrating that ITS1 ribotyping is as effective as gyrB at resolving G. quercinecans and B. goodwinii to the species level. CONCLUSIONS: The ITS1 gene has been successfully employed as a novel marker to resolve newly described AOD-associated Enterobacteriaceae, B. goodwinii and G. quercinecans, to species level. SIGNIFICANCE AND IMPACT OF THE STUDY: ITS1 ribotyping of B. goodwinii and G. quercinecans provides equivalent sensitivity to the current standard method for strain identification (sequence analysis of the gyrB gene), but with reduced processing time and cost. Furthermore, the ITS1 gene is widely applicable as a rapid and inexpensive typing system for Enterobacteriaceae.

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.

Survival of Brenneria goodwinii and Gibbsiella quercinecans, associated with acute oak decline, in rainwater and forest soil.

Acute oak decline (AOD) affects native UK oak species causing rapid decline and mortality in as little as five years. A major symptom of AOD is black weeping stem lesions associated with bacterial phytopathogens, Brenneria goodwinii and Gibbsiella quercinecans. However, there is limited knowledge on the ecological and environmental reservoirs of these phytopathogens. Rainwater and soils are common reservoirs of plant pathogens in a forest environment; therefore, the aim of this study was to investigate the survival of B. goodwinii and G. quercinecans in vitro when inoculated into rainwater and forest soil using a combination of agar-based colony counts and gyrB gene-targeted quantitative PCR (qPCR). Brenneria goodwinii lost viability on inoculation into soil and rainwater, but was detectable at low abundance in soil for 28 days using qPCR, suggesting a limited ability to persist outside of the host, potentially in a viable but non-culturable (VBNC) state. Conversely, Gibbsiella quercinecans, was re-isolated from rainwater for the entire duration of the experiment (84 days) and was re-isolated from forest soil after 28 days, with qPCR analysis corroborating these trends. These data demonstrate that B. goodwinii is unable to survive in forest soils and rainwater, suggesting that it may be an endosymbiont of oak trees, whereas G. quercinecans remains viable in soil and rainwater biomes, suggesting a broad ecological distribution. These data advance understanding of the potential epidemiology of AOD-associated bacteria and their ecological reservoirs, thus increasing the overall knowledge of the pathology of AOD, which assists the development of future management strategies.

Genomic analysis of bacteria in the Acute Oak Decline pathobiome.

The UK's native oak is under serious threat from Acute Oak Decline (AOD). Stem tissue necrosis is a primary symptom of AOD and several bacteria are associated with necrotic lesions. Two members of the lesion pathobiome, Brenneria goodwinii and Gibbsiella quercinecans, have been identified as causative agents of tissue necrosis. However, additional bacteria including Lonsdalea britannica and Rahnella species have been detected in the lesion microbiome, but their role in tissue degradation is unclear. Consequently, information on potential genome-encoded mechanisms for tissue necrosis is critical to understand the role and mechanisms used by bacterial members of the lesion pathobiome in the aetiology of AOD. Here, the whole genomes of bacteria isolated from AOD-affected trees were sequenced, annotated and compared against canonical bacterial phytopathogens and non-pathogenic symbionts. Using orthologous gene inference methods, shared virulence genes that retain the same function were identified. Furthermore, functional annotation of phytopathogenic virulence genes demonstrated that all studied members of the AOD lesion microbiota possessed genes associated with phytopathogens. However, the genome of B. goodwinii was the most characteristic of a necrogenic phytopathogen, corroborating previous pathological and metatranscriptomic studies that implicate it as the key causal agent of AOD lesions. Furthermore, we investigated the genome sequences of other AOD lesion microbiota to understand the potential ability of microbes to cause disease or contribute to pathogenic potential of organisms isolated from this complex pathobiome. The role of these members remains uncertain but some such as G. quercinecans may contribute to tissue necrosis through the release of necrotizing enzymes and may help more dangerous pathogens activate and realize their pathogenic potential or they may contribute as secondary/opportunistic pathogens with the potential to act as accessory species for B. goodwinii. We demonstrate that in combination with ecological data, whole genome sequencing provides key insights into the pathogenic potential of bacterial species whether they be phytopathogens, part-contributors or stimulators of the pathobiome.

Responses of the two-spotted oak buprestid, Agrilus biguttatus (Coleoptera: Buprestidae), to host tree volatiles.

BACKGROUND: Agrilus bigutattus (Fabricius) is a forest pest of increasing importance in the United Kingdom. The larvae damage weakened native oaks and are thought to contribute to premature tree death. Suspected links with acute oak decline (AOD) are not yet confirmed, but AOD-predisposed trees appear to become more susceptible to A. biguttatus attack. Thus, management may be necessary for control of this insect. To explore the possibility of monitoring beetle populations by baited traps, the host tree volatiles regulating A. biguttatus-oak interactions were studied. RESULTS: Biologically active volatile organic compounds in dynamic headspace extracts of oak foliage and bark were identified initially by coupled gas chromatography-electroantennography (GC-EAG) and GC-mass spectrometry (GC-MS), and the structures were confirmed by GC coinjection with authentic compounds. Of two synthetic blends of these compounds comprising the active leaf volatiles, the simpler one containing three components evoked strongly positive behavioural responses in four-arm olfactometer tests with virgin females and males, although fresh leaf material was more efficient than the blend. The other blend, comprising a five-component mixture made up of bark volatiles, proved to be as behaviourally active for gravid females as bark tissue. CONCLUSIONS: These initial results on A. biguttatus chemical ecology reveal aspects of the role of attractive tree volatiles in the host-finding of beetles and underpin the development of semiochemically based surveillance strategies for this forest insect.

Isolation studies reveal a shift in the cultivable microbiome of oak affected with Acute Oak Decline.

Acute Oak Decline is a syndrome within the Oak Decline complex in Britain. Profuse stem bleeding and larval galleries of the native buprestid, Agrilus biguttatus characterize the disease. A systematic study comparing healthy with diseased trees was undertaken. This work reports the result of isolations from healthy trees, diseased and non-symptomatic tissue within AOD affected trees, at five sites in England. Bacteria and fungi were identified using the DNA gyrase B gene, or ITS 1 sequencing. A significantly higher proportion of diseased tissues (82%) yielded more bacteria than either healthy (18%) or non-symptomatic tissue in diseased trees (33%). Overall bacterial community compositions varied at each site, but significant similarities were evident in diseased tissues at all sites. Enterobacteriaceae dominated in diseased trees whereas Pseudomonadaceae dominated healthy trees. Significant associations between diseased tissues and certain bacterial species occurred, implying that the cause of tissue necrosis was not due to random microbiota. Brenneria goodwinii and Gibbsiella quercinecans were key species consistently isolated from diseased tissue; Rahnella victoriana and an un-named Pseudomonas taxon were also frequently isolated from both healthy and diseased trees. Most fungi isolated were from the outer bark and had no significant association with tree health status. It was concluded that there was a shift in the cultivatable bacterial microbiome of diseased trees, with Enterobacteriaceae strongly represented in symptomatic but not healthy tissues. No single species dominated the isolations from diseased tissues and the tissue degradation in AOD is therefore likely to have a polymicrobial cause.

Description of Brenneria roseae sp. nov. and two subspecies, Brenneria roseae subspecies roseae ssp. nov and Brenneria roseae subspecies americana ssp. nov. isolated from symptomatic oak.

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.

Rahnella victoriana sp. nov., Rahnella bruchi sp. nov., Rahnella woolbedingensis sp. nov., classification of Rahnella genomospecies 2 and 3 as Rahnella variigena sp. nov. and Rahnella inusitata sp. nov., respectively and emended description of the genus Rahnella.

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 225(T)=LMG 27717(T)=DSM 27397(T)), Rahnella variigena sp. nov. (previously Rahnella genomosp. 2, type strain CIP 105588(T)=LMG 27711(T)), Rahnella inusitata sp. nov. (previously Rahnella genomosp. 3, type strain DSM 30078(T)=LMG 2640(T)), Rahnella bruchi sp. nov. (type strain FRB 226(T)=LMG 27718(T)=DSM 27398(T)) and Rahnella woolbedingensis sp. nov. (type strain FRB 227(T)=LMG 27719(T)=DSM 27399(T)).

Gibbsiella greigii sp. nov., a novel species associated with oak decline in the USA.

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.