Oak declines - New definitions and new episodes in Britain (PDF-842K)
An update on the latest (2009) developments and clarifying some of the confusion surrounding this worrying problem.
By Sandra Denman and Joan Webber.
Acute Oak Decline in Britain
Sandra Denman , Nathan Brown, Susan Kirk, Gavin Hunter, Carrie Brady, James McDonald, Mike Jeger, Joan Webber - 2013
Dieback and decline on pedunculate (Quercus robur) and sessile (Q. petraea) oak is recognised as a complex syndrome present in Europe at least since the early 1900s. However, recently a closer and discerning evaluation of the disorder in Britain revealed a discreet and distinctive condition called Acute Oak Decline (AOD), within the broader concept of oak decline. It is identified by weeping patches emanating from fissures between bark plates on trunks of established trees (>50 yrs-old). Beneath the outer bark extensive necrosis of the inner bark occurs frequently resulting in the formation of cavities. Using a draw knife to pare through three symptomatic bark panels (W*L=47*62cm2 each), consecutively removed from one side of a felled tree and tracing the necrotic tissue using digital image analysis software, it was shown that 20% of the surface area in these panels was necrotic. Damage occurred throughout the phloem but decreased in the sapwood. The distribution of the necrosis occurred over the entire tangential arc of the panels causing severe disruption and damage to the vascular tissue with consequent dysfunction that would inflict considerable carbon starvation of roots and fluid stress on the tree. Larval galleries of the oak Buprestid, Agrilus biguttatus, were also present on the cambial-phloem interface. Similar disorders have previously been described in Europe but the causes remain unresolved or attributed to A. biguttatus. In AOD we hypothesised that tissue necrosis was caused by biotic factors. A study using conventional isolation techniques to determine putative biotic causal agents was carried out on 21 symptomatic and 9 healthy trees from 17 sites across England. A range of selective media was used to plate pieces of surface disinfested tissue. Various fungal species were isolated but at low incidence and frequency. By contrast, bacteria were isolated with high incidence. Most bacterial taxa proved to be novel species and in some cases novel genera were created to accommodate them. The bacterial species composition of healthy trees was significantly different to symptomatic ones (P=0.001). Healthy trees were characterised by mostly Gram-positive bacteria which were fewer in symptomatic trees but high levels of Gram-negative bacteria particularly members of the Pseudomonadaceae and Enterobacteriaceae, were present in symptomatic oak. Two Enterobacterial species Brenneria goodwinii and Gibbsiella quercinecans, were consistently isolated from symptomatic tissue but not healthy trees suggesting a possible causal role in lesion formation; whereas multiple Pseudomonas spp. were present in both healthy and symptomatic trees negating that probability. The pathogenicity of B. goodwinii and G. quercinecans is currently under investigation. Inoculation studies demonstrated the necrogenic ability of both organisms although there was interaction with the host wound response in some cases. In vitro polysaccharide and tobacco tests also gave supportive evidence of necrogenic capability. Although further work on necrosis still needs to be carried out we can conclude provisionally that B. goodwinii and G. quercinecans play a role in causing the tissue necrosis that characterises AOD.
Development of a real-time PCR assay for the identification of Brenneria goodwinii, associated with Acute Oak Decline (AOD) of Quercus spp. in the United Kingdom
Gavin C. Hunter, Sarah Plummer, Sandra Denman - 2013
Acute Oak Decline (AOD) is a complex disorder of native Oak species, Quercus robur and Q. petrea in England. Recent studies have identified two Enterobacterial species, Gibbsiella quercinecans and Brenneria goodwinii and a buprestid, Agrilus biguttatus associated with AOD. Over the past 5 years, reports submitted to Forest Research Advisory Service revealed that the prevalence and distribution of AOD in England has steadily increased. Techniques used to identify the bacteria associated with AOD rely on culturing techniques and DNA sequencing of informative housekeeping genes. However, these techniques are labour intensive and expensive. The aim of this study was therefore to develop a rapid, specific and reliable molecular diagnostic assay for the identification of B. goodwinii. In order to achieve this, a large and comprehensive DNA sequence dataset based on gyraseB gene sequences was established for species of Brenneria and related genera within the Enterobacteriaceae. This DNA sequence dataset was used to identify potential species-specific DNA sequence targets for B. goodwinii. Using a commercial facility (PrimerDesign) B. goodwinii specific oligonucleotide primers and fluorescently labeled hydrolysis probes were developed. To validate their specificity for B. goodwinii a series of specificity tests were conducted on the LightCycler® real-time PCR platform using DNA from representative species of Brenneria including B. alni, B. nigrifluens, B. rubrifaciens, B. salicis, and Brenneria sp. nov. for comparison. Additionally, the limit of detection (LOD) and quantification framework for the assay was developed by preparing standard curves based on B. goodwinii samples of known DNA concentrations. Results from these studies indicate that the primers and hydrolysis probe have good specificity toward B. goodwinii and that the assay is able to detect very low amounts of B. goodwinii DNA. It is envisaged that the assay will be put into routine diagnostic use within the laboratory of Forest Research (FR) and will be added to the existing molecular diagnostic pipeline employed by FR staff in order to identify forestry pathogens.
Epidemiology of acute oak decline in England
Nathan Brown,Sandra Denman, Xiangming Xu and Mike Jeger - 2013
Oak has long been affected by a chronic decline in which tree vigour is being reduced by many interacting causes. The decline has typically been observed as a gradual process occurring over decades. In recent years a rapid form of decline has been observed in southern and central England, affecting both native species Quercus petraea and Q. robur. This syndrome has been termed Acute Oak Decline (AOD) and in all investigations thus far has involved a key bacterial component. Typical symptoms include sticky dark exudates (“bleeding cankers”) from lesions on the tree trunk often (>90%) associated with larval galleries in the inner bark and distinctive exit holes of the beetle Agrilus biguttatus. Whether a causal relationship is implied by this association is currently under investigation. A four-year study on the spatial and temporal dynamics of AOD and occurrence of A. biguttatus at eight geographically separated sites in England was completed in 2013. The main aims of the study included (1) to examine the correlation of stem symptoms with tree health and mortality, (2) to examine the within-site spread of AOD, and (3) to assess possible links between stem bleeds and beetle exit holes. At each site a complete mapping was made in which the status of all oak trees (115-260 trees per site) was assessed in terms of: (i) presence and absence of AOD symptoms (stem bleed); (ii) presence and absence of A. biguttatus exit holes; (iii) co-occurrence (bleeds and exit holes); and (iv) overall tree condition, including mortality. A modified version of the Ripley’s k-function was used to characterise the dynamic within-year and between-year spatial patterns of trees with AOD symptoms at each site. In addition, the spatial association of AOD symptoms with A. biguttatus exit holes was analysed. The spatial and temporal dynamics of AOD varied across the sites, with a range of epidemic stages from establishment, through to exponential growth, and to the late plateau stage occurring at different sites. Where exponential growth of the epidemic was in progress the spread of the disorder from tree to tree was documented adding support to the idea of a causal role for biotic agents.
Taxonomy and phylogeny of bacteria associated with Acute Oak Decline (AOD)
Carrie Brady, Gavin Hunter,Susan Kirk, Dawn Arnold and Sandra Denman - 2013
An episode of Acute Oak Decline (AOD) has recently been identified in Britain on native oak (Q. robur and Q. petraea) and has a rapid effect on tree health. Affected trees are characterized by extensive bleeding of dark, sticky exudates from vertical cracks between the bark plates. Tissues underlying the stem-bleed are stained and necrotic. A similar condition has been noted on Mediterranean oak (Q. pyrenacia and Q. ilex) in Spain, on Q. robur in Belgium, France, Germany, Poland and the Czech Republic and on black oak (Q. kelloggii) in the USA. Since 2008, numerous bacterial strains, representing several novel species, have been isolated from necrotic lesions and fluid exudates of symptomatic trees in Britain. The most frequently isolated species belong to Gibbsiella quercinecans and Brenneria goodwinii, both identified as novel species and in the case of Gibbsiella, a novel genus. The majority of bacterial strains isolated from symptomatic oak are Gram-negative and belong to the family Enterobacteriaceae. Species belonging to this family are typically difficult to identify and classify, owing to a high degree of both phenotypic and phylogenetic similarity. Partial sequencing of the 16S rRNA gene can possibly assign strains to the correct genus, but this gene lacks resolving power at the species level. In contrast, MLSA (multilocus sequence analysis), based on partial sequencing of 3-5 protein-encoding genes, is a robust and reliable method for identification, classification and phylogenetic studies of the Enterobacteriaceae. Symptomatic oak samples are currently screened by sequencing a single gene, gyrB, which is commonly included in MLSA schemes.. Phylogenetic analyses of the gyrB sequences have indicated that the bacteria recently isolated from symptomatic oak may belong to several additional novel species within the Enterobacteriaceae. The aim of this study was to characterize the strains recently isolated from symptomatic oak in the UK and USA using a polyphasic approach based on MLSA of partial gyrB, rpoB, infB and atpD sequences, DNA relatedness studies and phenotypic assays. Phylogenetic trees generated from the concatenated MLSA data revealed that the strains form seven discrete clusters, five within the Rahnella clade and one each in the Gibbsiella and Brenneria clades, suggesting that these strains constitute seven novel species. Initial DNA relatedness data confirm the phylogenetic results and support the proposal of novel species. Additionally, the potential novel species from each genus can be differentiated from their closest phylogenetic neighbours on the basis of several phenotypic characteristics. Following further DNA relatedness tests, seven novel species will be formally described and classified in three genera in the family Enterobacteriaceae. The role of these novel species in the current episode of AOD has yet to be determined.
Evaluation of a commercial semi-selective culture medium for detection and isolation of putative necrogenic Enterobacteriaceae associated with Acute Oak Decline
Jonathan Partridge,Susan Kirk,Sandra Denman - 2013
First described in 2009, Acute Oak Decline (AOD) is a disorder of native Oak (Quercus robur and Q. petraea) in the UK. To determine possible causes, comparative investigations to elucidate putative fungal and bacterial causal agents were carried out on healthy and symptomatic trees. Pieces of symptomatic and non-symptomatic tissue were plated onto Proteose Peptone Yeast Glucose agar (PYGA) to isolate bacterial flora. PCR amplification of the Gyrase B gene region followed by DNA purification and sequencing enabled the identification of strains using BLAST®. Many bacterial taxa, both Gram-negative and Gram-positive, were present, but the frequent and consistent occurrence of Gibbsiella quercinecans and regular presence of Brenneria goodwinii (both Enterobacteriaceae) in only symptomatic oak tissue identifies them as having a possible causal role in the condition. It is therefore of key importance that detection and identification of these bacteria is completed rapidly, even when dealing with large numbers of isolations. Since conventional isolation techniques are used in the first step towards identification and in research work, a less labour intensive method that facilitates this process and makes it more cost effective would be useful. One way of accelerating identification is through the use of a selective medium. A search for commercially available products highlighted Gassner agar (GA) as a suitable candidate for testing. GA is used for the detection and isolation of pathogenic Enterobacteriaceae from organic samples. It contains metachrome yellow (1.25 g L-1) which inhibits Gram-positive microbial flora. Many bacteria identified in oak tissue are Gram-positive, including Enterococcus spp. and Paenibacillus spp. This medium may remove strains of little interest, with respect to AOD, that are normally isolated. The effectiveness of GA to permit the selective growth of bacteria relevant to AOD (Enterobacteriaceae) was investigated. Fifty strains representing 21 species were used to test the selective medium including members of the Enterobacteriaceae: Brenneria, Gibbsiella, Lonsdalea and Rahnella. There was a highly significant reduction in the growth of all the Gram-positive bacteria compared to the members of the Enterobacteriaceae (P<0.001), with only some of the Brenneria strains showing slightly reduced growth. Interestingly each species produced different colour changes in the selective medium: After 24 h G. quercinecans produced an orange colour but Rahnella spp. produced a very dark blue colour while B. goodwinii produced the dark blue only after 48 h. Although this medium is not specific for the isolation of G. quercinecans, it is useful to suppress species of no interest thereby reducing time and effort in the identification of relevant species. Further tests are required, but it may be a starting place for the production of a species specific selective medium.
Metagenomic analysis of microbial communities associated with acute oak decline in the UK
Melanie Sapp, John Elphinstone, Richard Thwaites, Neil Parkinson, Susan Kirk, Gavin Hunter and Sandra Denman - 2013
A novel metagenomic approach was used to supplement investigations into the causes of an episode of acute oak decline (AOD) which has spread rapidly through the Midlands and South East England since 2006. DNA extraction and next generation (Roche 454) amplicon sequencing procedures were optimised to identify potential plant pathogens and insect pests exclusive to symptomatic oak tissues. Symptoms included lesions in the inner bark which also often extend to surrounding galleries left by boring larvae of the buprestid beetle Agrilus biguttatus. In this study primers targetted bacteria (16S rRNA gene; Gyrase B (GyrB) and DNA-J genes), fungi (ITS) and insects (Co-A). In the initial investigation, two healthy and symptomatic trees each, were identified at a site in Oxfordshire and Berkshire, central England. After tree felling, equivalent sets of symptomatic and asymptomatic tissue pieces were dissected. One set was processed by conventional isolation on a large number of selective and general culture media plating pieces from the inner bark, outer bark, sapwood, heartwood and the larval galleries, which were thought to be created by A. biguttatus. The second set of tissues was used for DNA extraction and next generation (Roche 454) amplicon sequencing. Work presented here reports bacterial results only. The bacterial community structure was assessed based on sequence diversity in the V1-V3 region of the 16S rRNA gene. Clustering and BLAST analyses were used to compare sequences with those in the curated Ribosomal Database Project (RDP) and gyrB sequences was used as an alternative taxonomic marker. 16S rRNA gene sequencing resulted in 119,757 sequences compared with 73,566 gyrB sequences. Clustering and BLASTn analyses with those in the curated Ribosomal Database Project (RDP) were carried out. The results indicated that bacterial diversity was higher in symptomatic than in healthy tissue. The families Sphingobacteriaceae, Pseudomonadaceae, Enterobacteriaceae and Rhodobacteraceae were represented. Where similarities to known genera could be established, it appeared that most bacteria were of likely environmental origin, many associated with soil, water or woodland plants. Of most interest to date are the potential oak pathogens Gibbsiella quercinecans and Brenneria goodwinii, (Enterobacteriaceae), found only in symptomatic tissues at different sites, and which were also isolated from the AOD-affected tissues. Baseline data on bacterial communities present in healthy oak tissue are also emerging, of which there is little published information available to date. Our results showed the presence of Acetobacteraceae in particular. Similar approaches are being developed to assess fungal community diversity in diseased and healthy oak tissues according to Internal Transcribed Spacer (ITS) sequence diversity.
Description of Gibbsiella quercinecans gen. nov., sp. nov., associated with Acute Oak Decline
Carrie Brady, Sandra Denman, Susan Kirk, Stephanus Venter, Pablo Rodríguez-Palenzuela, Teresa Coutinho. (2010).
Systematic and Applied Microbiology: Volume 33, Issue 8, Pages 427-478.
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 97T=LMG 25500T=NCPPB 4470T) is proposed.
Brenneria goodwinii sp. nov., a novel species associated with Acute Oak Decline in Britain
Sandra Denman, Carrie Brady, Susan Kirk, Ilse Cleenwerck, Stephanus Venter, Teresa Coutinho and Paul de Vos.
Int J Syst Evol Microbiol ijs.0.037879-0; published ahead of print November 25, 2011, doi:10.1099/ijs.0.037879-0
A group of nine Gram-negative staining, facultatively anaerobic bacterial strains isolated from native oak trees displaying symptoms of Acute Oak Decline (AOD) in Britain 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). MLSA 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. The name Brenneria goodwinii sp. nov. (type strain = FRB 141T = LMG 26270T = NCPPB 4484T) is proposed.
Proposal to reclassify Brenneria quercina (Hildebrand & Schroth 1967) Hauben et al. 1999 into a novel 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 Dickeya dadantii subsp. dieffenbachiae comb. nov., and emendation of the description of Dickeya dadantii
Carrie Brady, Ilse Cleenwerck, Sandra Denman, Stephanus Venter, Pablo Rodríguez-Palenzuela, Teresa A Coutinho and Paul de Vos.
Int J Syst Evol Microbiol ijs.0.035055-0; published ahead of print September 2, 2011, doi:10.1099/ijs.0.035055-0
Bacterial isolates from oak 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, revealed that the isolates were separated into two genetic groups according to their origin. Their closest phylogenetic relative was B. quercina, the causal agent of drippy nut disease of oak, which clustered distant to the remaining Brenneria species. MLSA data of Brenneria, Pectobacterium, Dickeya, Erwinia, Pantoea and Samsonia species confirmed the polyphyletic nature of the genus Brenneria, and indicated synonymy of Dickeya dadantii and Dickeya dieffenbachiae. DNA-DNA hybridizations confirmed this synonymy, and also revealed values of 58-73 % between the oak isolates and B. quercina. Phenotypic and/or chemotaxonomic methods allowed discrimination of B. quercina and the two genetic groups of oak isolates from the remaining Brenneria species, and from the closely related genera Dickeya, Pectobacterium and Samsonia. Based on the data obtained, the following taxonomic proposals are made: (1) reclassification of B. quercina into a novel genus, Lonsdalea gen. nov., as Lonsdalea quercina comb. nov., (2) classification of the oak isolates as Lonsdalea quercina subsp. iberica subsp. nov. (LMG 26264T =NCPPB 4490T) and Lonsdalea quercina subsp. britannica subsp. nov. (LMG 26267T =NCPPB 4481T), (3) emendation of the description of the genus Brenneria, (4) and reclassification of Dickeya dieffenbachiae as Dickeya dadantii subsp. dieffenbachiae comb. nov.
The utility of the intergenic transcribed spacer region 1 as a molecular marker for the identification and discrimination of the Enterobacteriaceae associated with Acute Oak Decline
James Doonan,Sandra Denman, Justin Pachebat, Peter Golyshin and James McDonald,School of Biological Sciences, Bangor University; Centre for Forestry and Climate Change, Forest Research; IBERS, Aberystwyth University.
The molecular identification of environmental Enterobacteriaceae strains from trees affected by Acute Oak Decline (AOD) is a laborious and costly process, particularly when a large number of isolates are obtained. Currently, strain identification relies on PCR amplification, DNA sequencing and phylogenetic analysis of marker genes such as 16S rRNA or DNA gyrase B. Here, we describe the use of the intergenic spacer region 1 (ITS1) for the rapid, inexpensive and accurate typing of bacterial strains belonging to the Enterobacteriaceae. The method was validated using of seven cultured Enterobacteriaceae strains isolated from oak trees. The ITS1 and gyrase B genes of five sub-isolates of each strain (n=35) were amplified using PCR followed by sequencing of ITS1 and gyrB amplicons. The number and size of ITS1 amplicons for each sub-isolate was subsequently determined using both 3% agarose gel electrophoresis and polyacrylamide gel electrophoresis. Each bacterial species was found to have a unique ITS1 profile (between 3 and 7 amplicons). The validated technique was subsequently used to screen a further 270 sub-isolates derived from 54 Enterobacteriaceae strains, providing accurate species specific profiles of ITS1 amplicons. The method was found to have equivalent sensitivity to PCR amplification and sequence analysis of the DNA gyrase B sequencing gene, but with significantly reduced processing time and cost.