Acute Oak Decline Research Articles

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.

Multilocus sequence typing provides insights into the population structure and evolutionary potential of Brenneria goodwinii, associated with acute oak decline.

Brenneria goodwinii is one of the most frequently isolated Gram-negative bacteria from native oak species, Quercus robur and Q. petraea, affected by acute oak decline (AOD) in the UK. We investigated the population biology of this bacterial species using a multilocus sequence analysis to determine the population structure and evolutionary potential. Seven partial housekeeping genes were used in the analyses. Amongst 44 bacterial strains from seven different locations, we identified 22 unique sequence types [STs]; only one ST was found at two separate locations. Phylogenetic and cluster-based analyses suggested that B. goodwinii STs form two main distinct groups; however, no geographical pattern of their distribution could be observed. Clonality and recombination tests demonstrated that the studied population is primarily clonal, however both mutation and recombination processes play a role in shaping the genetic structure and evolution of the population. Our study suggests that the B. goodwinii population on oak in the UK has an endemic form, with background recombination appearing to generate new alleles more frequently than mutation, despite the introduction of nucleotide substitutions being approximately twice less likely than mutation. The newly emerged STs subsequently undergo clonal expansion to become dominant genotypes within their specific geographical locations and even within the individual host oak trees.

Acute Oak Decline and Agrilus biguttatus: The Co-Occurrence of Stem Bleeding and D-Shaped Emergence Holes in Great Britain

Acute Oak Decline (AOD) is a new condition affecting both species of native oak, Quercus robur and Quercus petraea, in Great Britain. The decline is characterised by a distinctive set of externally visible stem symptoms; bark cracks that “weep” dark exudate are found above necrotic lesions in the inner bark. Emergence holes of the buprestid beetle, Agrilus biguttatus are often also seen on the stems of oak within affected woodlands. This investigation assesses the extent to which the external symptoms of these two agents co-occur and reveals the spatial and temporal patterns present in affected woodland. Annual monitoring in eight affected woodlands showed that stem bleeding and emergence holes frequently occur on the same trees, with new emergence holes significantly more likely to occur when trees already have stem bleeds. Trials with coloured prism traps confirm A. biguttatus was present at all experimental sites. Beetle emergence is linked primarily to a few heavily declining trees, indicating that susceptibility may vary between hosts and that those with reduced health may be predisposed to AOD. Stem bleeds occur on trees in close proximity to the locations of trees with exit holes.

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.

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

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. 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 effects of host age and spatial location on bacterial community composition in the English Oak tree (Quercus robur).

Drivers of bacterial community assemblages associated with plants are diverse and include biotic factors, such as competitors and host traits, and abiotic factors, including environmental conditions and dispersal mechanisms. We examine the roles of spatial distribution and host size, as an approximation for age, in shaping the microbiome associated with Quercus robur woody tissue using culture-independent 16S rRNA gene amplicon sequencing. In addition to providing a baseline survey of the Q. robur microbiome, we screened for the pathogen of acute oak decline. Our results suggest that age is a predictor of bacterial community composition, demonstrating a surprising negative correlation between tree age and alpha diversity. We find no signature of dispersal limitation within the Wytham Woods plot sampled. Together, these results provide evidence for niche-based hypotheses of community assembly and the importance of tree age in bacterial community structure, as well as highlighting that caution must be applied when diagnosing dysbiosis in a long-lived plant host.

Metabarcoding of Bacteria Associated with the Acute Oak Decline Syndrome in England

Outbreaks of acute oak decline (AOD) have been documented in England from 2006. Both species of native oaks (Quercus robur and Quercus petraea) are affected. To complement isolation efforts for identification of putative causative biotic agents and increase our understanding of bacteria associated with oak tissue, five sites in England were chosen for this study. Samples of outer bark, inner bark, sapwood and heartwood were taken from healthy oak and trees with symptoms at varying stages of the syndrome. Furthermore, larval galleries attributed to infestation with Agrilus biguttatus were included. After DNA extraction and amplification of the V3–V5 fragment of the bacterial 16S rRNA genes by pyrosequencing, the dataset was analyzed to identify patterns in bacterial communities in oak tissue samples with and without AOD symptoms at each site. The composition of bacterial communities differed greatly according to the site from which the samples were obtained. Within each site, the composition of the bacteria associated with symptomatic tissue varied between advanced stages of the syndrome and healthy tissue. Key players in healthy and symptomatic tissue were identified and included members of the Gammaproteobacteria related to Pseudomonas sp. or Brenneria goodwinii and members of the Firmicutes.

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.RESULTSBiologically 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.CONCLUSIONSThese 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. © 2015 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Spatial and temporal patterns in symptom expression within eight woodlands affected by Acute Oak Decline

Acute Oak Decline (AOD) is a newly defined condition in the UK that forms a distinct component within the wider oak decline complex. It can be identified by distinctive stem symptoms, dark liquid seeping out between the bark plates from areas of necrosis in the cambial tissue, which are found in conjunction with signs of the two spotted oak buprestid Agrilus biguttatus. Novel bacterial species have been isolated from the inner bark necrosis, but little is known regarding the spatial and temporal patterns of AOD symptoms expression. This study presents the results from eight plots across southern and central England monitored for three to four years between 2009 and 2012. These data give a first description of the epidemiology of AOD. Findings suggest affected trees occur in localised clusters rather than at random through the plots, pointing to biotic rather than wider scale environmental effects such as drought. In addition, contagion (spread of symptoms between neighbouring trees) was demonstrated. Previously, mortality was described as occurring soon after infection; current data suggests this is longer than two years. 23.5% of oak with both stem bleeds and exit holes in 2009 died during the monitoring period. Exit holes were observed on hosts for the first time in the final years before death linking them to the later stages of decline. Finally, a large proportion of lightly infected individuals have been shown to form callus over the previous year’s stem symptoms and enter remission. This finding suggests host resistance may provide some scope for management of AOD.

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

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. 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. 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. 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.

A review of Agrilus biguttatus in UK forests and its relationship with acute oak decline

Agrilus biguttatus Fab. (Coleoptera: Buprestidae) is a European bark-boring beetle whose larvae feed in the vascular tissue of oak trees. Until recently, it was considered rare in Britain, but sightings have become more frequent and it is often found on weakened trees suffering from Acute Oak Decline (AOD). This rapidly acting syndrome is characterized by patches of dark sticky fluid exuding from cracks on the trunk, with areas of necrotic tissue beneath, probably caused by a pathogenic bacterial component. However, the frequent association of AOD with the larval galleries and distinctive adult exit holes of A. biguttatus has raised concerns that the beetle may be contributing to the AOD syndrome or hastening the mortality of affected trees. This review evaluates the potential role of A. biguttatus in the AOD complex. Information on the beetle's life cycle and ecology is assessed along with the apparent increase in its abundance and distribution in the UK, and likely mechanisms of host selection. Oak tree defences against the beetle are discussed, as well as risk factors influencing susceptibility. Research on related Agrilus species is reviewed so that insights into the relationship between the beetle, the bacteria and the host tree can be made through comparisons with more extensively studied species. Possible management options in an AOD context are considered, and priority areas for future research are identified.

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 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).

A description of the symptoms of Acute Oak Decline in Britain and a comparative review on causes of similar disorders on oak in Europe

Acute Oak Decline (AOD) is a relatively new decline-disease affecting both native oak species (Quercus robur and Q. petraea) in Britain. The key aim of this study was to describe the symptoms, and signs of AOD, to set a baseline. The second aim was to compare and review the European literature on what appear to be similar disorders on oak. AOD is characterized by four key features: weeping patches more-or-less vertically aligned on oak tree trunks; cracks between bark plates from which dark fluid seeps; inner bark necrosis and the presence (in >90 per cent of cases) of larval galleries of the oak buprestid, Agrilus biguttatus, on the phloem–sapwood interface. In this study, it was noted that although larval galleries were present in the inner bark in 19 of 21 trees, the ‘D-shaped’ exit holes of the adult beetles were seen less frequently on bark plates of affected trees (33 per cent of cases). Similar disorders reported in Europe are compared with AOD in Britain and potential causes of the condition discussed. Based on the unmistakable symptoms, it is hypothesized that AOD is a distinctive, identifiable condition within the broader oak decline syndrome.

Proposal to reclassify Brenneria quercina (Hildebrand and Schroth 1967) Hauben et al. 1999 into a new genus, Lonsdalea gen. nov., as Lonsdalea quercina comb. nov., descriptions of Lonsdalea quercina subsp. quercina comb. nov., Lonsdalea quercina subsp. iberica subsp. nov. and Lonsdalea quercina subsp. britannica subsp. nov., emendation of the description of the genus Brenneria , reclassification of Dickeya dieffenbachiae as

Bacterial isolates from oak trees in Spain and Britain, showing symptoms of bark canker and Acute Oak Decline (AOD), respectively, were examined by a polyphasic approach. Both 16S rRNA gene sequencing and multilocus sequence analysis (MLSA), based on partial sequences of gyrB, rpoB, infB and atpD genes, revealed that the isolates were separated into two genetic groups according to their origin. Their closest phylogenetic relative was Brenneria quercina, the causal agent of drippy nut disease of oak, which clustered distant to the other species of the genus Brenneria. MLSA data for species of the genera Brenneria, Pectobacterium, Dickeya, Erwinia, Pantoea and Samsonia confirmed the polyphyletic nature of the genus Brenneria and indicated synonymy of Dickeya dadantii and Dickeya dieffenbachiae. DNA-DNA hybridization experiments confirmed this synonymy and also revealed DNA-DNA relatedness values of 58-73% between the new oak isolates and B. quercina. Phenotypic and/or chemotaxonomic methods allowed B. quercina and the two genetic groups of new oak isolates to be discriminated from other recognized species of the genus Brenneria and from members of the closely related genera Dickeya, Pectobacterium and Samsonia. Based on the data obtained, the following taxonomic proposals are made: (1) reclassification of B. quercina as the type species of a novel genus, Lonsdalea gen. nov., as Lonsdalea quercina comb. nov. (type strain LMG 2724(T)=ATCC 29281(T)=CCUG 48867(T)=CFBP 3617(T)=CIP 105201(T)=DSM 4561(T)=ICMP 1845(T)), (2) classification of the oak isolates as Lonsdalea quercina subsp. iberica subsp. nov. (type strain LMG26264(T)=NCPPB 4490(T)) and Lonsdalea quercina subsp. britannica subsp. nov. (type strain LMG 26267(T)=NCPPB 4481(T)) and leading to the automatic creation of Lonsdalea quercina subsp. quercina subsp. nov. (type strain LMG 2724(T)=ATCC 29281(T)), (3) emendation of the description of the genus Brenneria, and (4) reclassification of Dickeya dieffenbachiae as Dickeya dadantii subsp. dieffenbachiae comb. nov. (type strain LMG 25992(T)=CFBP 2051(T)), with the automatic creation of Dickeya dadantii subsp. dadantii subsp. nov. (type strain LMG 25991(T)=CFBP 1269(T)).

Brenneria goodwinii sp. nov., associated with acute oak decline in the UK

A group of nine Gram-negative staining, facultatively anaerobic bacterial strains isolated from native oak trees displaying symptoms of acute oak decline (AOD) in the UK were investigated using a polyphasic approach. 16S rRNA gene sequencing and phylogenetic analysis revealed that these isolates form a distinct lineage within the genus Brenneria, family Enterobacteriaceae, and are most closely related to Brenneria rubrifaciens (97.6 % sequence similarity to the type strain). Multilocus sequence analysis based on four housekeeping genes (gyrB, rpoB, infB and atpD) confirmed their position within the genus Brenneria, while DNA-DNA hybridization indicated that the isolates belong to a single taxon. The isolates can be differentiated phenotypically from their closest phylogenetic neighbours. The phylogenetic and phenotypic data demonstrate that these isolates from oak with symptoms of AOD represent a novel species in the genus Brenneria, for which the name Brenneria goodwinii sp. nov. (type strain FRB 141(T) = R-43656(T) = BCC 845(T) = LMG 26270(T) = NCPPB 4484(T)) is proposed.

Description of Gibbsiella quercinecans gen. nov., sp. nov., associated with Acute Oak Decline

Gram-negative, facultatively anaerobic bacterial strains were consistently isolated from oak trees displaying symptoms of extensive stem bleeding. In Britain, this disorder is called Acute Oak Decline (AOD). A similar condition has been noted on species of Mediterranean oak in Spain. The identity of bacterial isolates from symptomatic trees in both countries was investigated using molecular techniques and phenotypic assays. 16S rRNA gene sequencing indicated that the strains were most closely related to the genera Serratia, Kluyvera, Klebsiella and Raoultella (all > 97%). Phylogenetic analysis revealed that the strains formed a distinct lineage within the family Enterobacteriaceae, which was confirmed by both gyrB- and rpoB-gene sequencing. DNA–DNA hybridization confirmed that the strains belonged to a single taxon which could also be differentiated phenotypically from its closest phylogenetic neighbours. The phylogenetic and phenotypic data both demonstrated that the strains isolated from oak represented a novel genus and species within the family Enterobacteriaceae for which the name Gibbsiella quercinecans gen. nov., sp. nov. (type strain=FRB 97 T=LMG 25500 T=NCPPB 4470 T) is proposed.