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Improved Understanding of the Causes, Distribution and Scale of Acute Oak Decline in the UK - TH0108

Description
Acute Oak Decline (AOD) is a process caused by multiple factors and results in rapid loss of health of oak trees. AOD in the UK is identified by dark weeping patches on stems of established trees, (>50 years-old). Close inspection of bark plates on symptomatic trees may also reveal ¡®D-shaped¡¯ exit holes of the oak jewel beetle (Agrilus biguttatus). Internal tree tissues underneath the weeping patches are under attack, and lesions and sinuous beetle-larval tracks (galleries) can be seen in the inner bark. Two agents are consistently associatedwith stem symptoms of AOD viz. larvae of Agrilus and bacteria, but many microbes escape detection if they are not culturable. One aspect of this project aims to improve understanding the causes of AOD. As a first step to ascertaining the organisms that might be involved, the microbial communities of healthy and symptomatic oak will be compared. Using ¡®Next Generation Sequencing¡¯ (NGS) total bacterial, fungal and insect DNA will be analysed and by employing shotgun pyrosequencing of reverse-transcribed Small Sub-unit (SSU) ribosomal RNA (rRNA) both phylogenetic and quantitative data will be obtained, giving a comparative overview of the diversity of microbial communities on healthy and symptomatic trees. Concurrently conventional laboratory culture methods will secure a collection of culturable organisms. Because Agrilus galleries are usually present in symptomatic trees and European studies suggest beetle attack precedes lesion development questions arise about what attracts the beetles to oak trees and at what point a tree becomes susceptible to attack. Volatile chemical compounds given off by trees attacked by the beetles will be analysed, defined and tested to ascertain whether or not they attract beetles and these volatiles will be linked to tree health status. Similarly compounds given off by bacteria associated with AOD, to the beetle, will also be assessed as attractants. This work will tie in
with studies on the combined effects of the beetles and bacteria on lesion development, and transmission of bacteria by the adult and larval stages of Agrilus. Looking to the future in a changing climate with warmer temperatures the present range of the beetle could expand and its generation time shorten which may cause populations to increase, enhancing the risk of AOD intensifying and spreading. The beetle will be reared in captivity and effects of temperature on its life cycle determined to inform predictive risk assessment. The extent and scale of the problem will be estimated using a scientifically derived matrix to optimise sampling points and collect data that will be applied to a model developed to estimate the incidence and severity of the condition.
Drawing on epidemiological information already to hand and incorporating results of the studies outlined above, the rates of increase of AOD at eight monitored sites in the UK will be estimated. This key parameter will ultimately be used to predict the risk of spread of AOD. All the above and other existing studies on AOD underpin and inform management strategies. This work clearly addresses all three questions posed by the Defra Call and will significantly improve the understanding of risk and potential impact from AOD, as required by the Defra-FC Tree Health and Plant Biosecurity Action Plan (see http://www.defra.gov.uk/publications/files/pb13657-tree-health-action plan.pdf). It will address questions of with clear relevance to risk based management of AOD through developing our understanding of this disorder.
Objective
Overall Aims of the Project

This project will improve our understanding of the causes of Acute Oak Decline (AOD) through estimating its current distribution, incidence and severity; predicting future distribution, incidence and severity, and thus will assist policy development and to inform the development of possible management approaches. The project is divided into three work packages (WP1, WP2 and WP3) that address scientific issues while overall project management and knowledge and information transfer will be undertaken by Dr Sandra Denman with the full support of FR. In terms of scientific content WP1 and WP2 focus on improving knowledge of understanding the causes of AOD by concentrating on the biotic component of AOD. In WP1 microbial aspects are investigated and detection and diversity of the insect component on a molecular level particularly in the early stages of the condition is included, while in WP2 behavioural aspects pertaining to the involvement of Agrilus are studied. Distribution, incidence and severity aspects are addressed through survey and modelling in WP3.

Workpackage 1: Causes of AOD - identification and quantification of the biotic component of AOD

General Aims WP1 - Causes: The overall aim of WP1 is to improve our understanding of the causes of AOD.

We hypothesise that (i) certain members of microbial communities and insect activity are consistently associated with AOD; (ii) bacteria have a role in causing necrosis and degradation of tissue beneath the stem bleeds (iii) interaction between Agrilus and bacteria will lead to the development of typical AOD symptoms viz. stem bleeding, lesion formation and galleries in inner bark and cambial tissues.

Specific Evidence Objectives WP1 - Causes: In order to test hypothesis (i) stated above ¨C i.e. that members of microbial communities and insects are associated with AOD our specific objectives are:
i. To refine techniques that will enable the extraction and purification of DNA from symptomatic and healthy trees
ii. Separation of host DNA from microbial DNA
iii. Extraction of rRNA from symptomatic and healthy oak tissues and analysis of microbial Small Sub-unit rRNA
iv. To acquire an overview of the microbial and insect communities associated with AOD
v. Determine the structure and function of microbial populations associated with AOD via Small Sub-Unit rRNA abundance and functional shotgun metagenomics
vi. To acquire cultures of putative causal organisms associated with AOD
vii. To derive quantitative data on the relative proportion of the different agents associated with AOD
viii. To carry out bioinformatics and comparative analyses on the different techniques applied to the problem.
ix. Establish microbial communities directly associated with any Agrilus populations collected during WP2 from sites with and without AOD, determine the diversity of Agrilus and estimate potential for Agrilus to vector microorganisms of key taxa.
x. To compile a sequence base to inform development of rapid molecular diagnostics for subsequent deployment in a more statistically comprehensive investigation of the association of key taxa with AOD symptoms of varying severity and across more locations.

Note (1): Hypotheses (ii) and (iii) mentioned above are being addressed by FR with the support of the Forestry Commission (FC) and so are not discussed further here. A number of results of WP1 will however, add to supporting or rejecting hypothesis (ii).
Note (2): All outputs from the work carried out will be communicated as described later and so only scientific objectives are listed in this section.

Measurement and duration of objectives in WP1 - Causes. Please note details in the approaches and methodology section as well as the milestones. The numbering below corresponds with the objective listed immediately above here.
i. Extraction and purification of DNA and refinement of the 454 pyrosequencing methodology will take 9 months (expected completion date end September 2013). The measurement of fulfilment of this objective is the development of the technology and compiling a standard operating procedure (SOP) document. It will be necessary to complete objective (i) before the other objectives can be achieved in full. Preliminary collaborative research between the partners (Elphinstone et al., 2012) has already developed NGS methods for analysis of bacterial communities but further development will be needed in year 1 to improve the resolution of these methods and allow their application to investigate fungal and insect communities (see 2.3). Co-operative cross-working between FR who will carry out site visits, sampling and conventional culture and Fera who will apply NGS methodology and others to the process of obtaining pure DNA for use in assessing the microbial and insect communities associated with AOD; this part of the work is ongoing over two years and will dovetail with the sampling to be carried out at some of the sites in the modelling exercise. Acquisition of sequence data is the measurement of fulfilling the objective (Fera).
ii. It will take 9 months to develop the methodology to separation of host DNA from microbial DNA and this work will be carried out at Bangor University (BU). Bangor will cross-link with FR in obtaining samples. The measurement of fulfilment of this objective is the development of the technology and compiling a standard operating procedure (SOP) document.
iii. It will take 9 months to develop the methodology to extract rRNA from symptomatic and healthy oak tissues and analyse the microbial Small Sub-unit rRNA from oak tissue and this work will be carried out at Bangor University (BU). The measurement of fulfilment of this objective is the development of the technology and compiling a standard operating procedure (SOP) document.
iv. An overview of the microbial and insect communities associated with AOD is achieved once sequence data are analysed. This will be ongoing throughout the three year period (FR, Fera and BU).
v. The quantification of microbial DNA of the bacterial groups associated with AOD using analysis of microbial Small Sub-unit rRNA from oak tissue (abundance and activity) will take 9 months and will be measurable by the production of a report/publication of results. This work is to be led by BU.
vi. Acquisition of cultures of putative causal organisms associated with AOD as well as obtaining isolation data for comparative analyses is ongoing throughout the project period and is led by FR.
vii. Bioinformatics and comparative analyses between symptomatic and healthy tissues using the different sequence-based techniques applied to the problem will be ongoing for the duration of the project, led by Fera but with input from FR and BU.
viii. Integrate results from all work stands above FR, BU and Fera.
ix. Establish microbial communities directly associated with any Agrilus populations collected during WP2 from sites with and without AOD and estimate potential for Agrilus to vector microorganisms of key taxa FR and Fera ongoing over 24 months.
x. To compile a sequence base to inform development of rapid molecular diagnostics for subsequent deployment in a more statistically comprehensive investigation of the association of key taxa with AOD symptoms of varying severity and across more locations FR, BU and Fera ongoing over 36 months, the
measurable item in fulfilment of this objective is the database itself.

Linkages WP1 - Causes: Linkage between WP1, WP2 and WP3 will ensure that data on the distribution of Agrilus at different sites (with and without AOD) is correlated with consistent evidence of its presence in symptomatic but not healthy tissues and incorporated in the models (WP3). For example sampling from the same trees will be used in WP1, objective1; WP2 objectives iii and v and information from the research results will be fed into the models in WP3 (parameterization). The overall result will be to build a holistic picture of factors and interactions present at a limited number of sites, because these same sites will also be used in abiotic studies which will be carried out under a separate funding stream. Additionally analysis in WP1 of insects collected in WP2 will allow assessment of the potential of Agrilus to vector those microbes associated with AOD. Surveys conducted in WP3 will allow selection of representative symptomatic and healthy trees from key sites for detailed analyses in WP3.

Workpackage 2: Causes of AOD ¨C Agrilus behaviour and role in AOD and future predictions in changing climates

General Aims WP2 - Agrilus: In order to compliment other work carried out on the causes of AOD in WP1 and to develop our understanding of the nature and behaviour of Agrilus, WP2 will focus on the diversity and life history of the beetle and drivers determining/affecting host selection, so that knowledge of the underlying mechanisms affecting Agrilus population dynamics will be elucidated, but also, to clarify the context of the proposed causal role A. biguttatus plays in causing AOD and the distribution/range of the buprestid.

The general aims of WP2 are thus (i) to develop knowledge of the life cycle of Agrilus and acquire empirical data on factors that affect it; (ii) to develop knowledge on mechanisms, such as chemical ecology, which drive certain behaviours such as host selection, which will ultimately affect population dynamics and give insights into the distribution of AOD. By revealing the chemical ecology of A. biguttatus, a platform for more applied approaches,
e.g. trapping for future surveillance and management, will be created. Semiochemicals identified in this project will create the basis of the development of effective detection and monitoring systems for the control of A. biguttatus. Based on the result on the closely related A. planipennis, we hypothesise that A. biguttatus exploit (i) volatiles from oak foliage to find mating sites and (ii) oak bark volatiles to locate suitable oviposition sites.

A third important step in developing our understanding of Agrilus biguttatus populations is to understand its life cycle and voltinism. This will allow predictions of future change to be grounded in good science. We anticipate being able to locate pre-pupal larvae in their overwintering stage by using thermal imaging to detect these larvae in the bark of oak trees. We plan on capturing emerging adults from bark samples and establishing a breeding colony in an insectory and then testing the effect of temperature on voltinism. We hypothesise that temperature will be the driving factor limiting generation time.

The information obtained in WP2 will add context to A. biguttatus's proposed causal role in AOD, and inform modelling (WP3) but data relevant for future Climex mapping on the predicted distribution of Agrilus, which is beyond the scope of this project, will be also obtained for future studies in this regard.
Specific Evidence Objectives WP2 - Agrilus: In order to test the hypotheses listed above our specific objectives are:
i. Study the chemical basis of the attraction of unmated adult females and males to oak foliage. We will compare the chemistry of foliage volatiles from symptomatic and non-symptomatic oak trees, and assess the attractiveness of these to A. biguttatus.
ii. Characterise volatiles from oak tree bark as putative olfactory cues for ovipositing females. We will compare the chemistry of bark volatiles from symptomatic and non-symptomatic oak trees, and assess the attractiveness of the volatiles to gravid A. biguttatus females.
iii. Collect live A. biguttatus and establish a breeding colony. We will trial a thermal imaging technique to detect Agrilus activity and locate individuals. In addition emergence traps and field collection should ensure a supply of live beetles for use in breeding experiments.
iv. Investigate the role of temperature in Agrilus development, and predict the likely effects of climate change upon its voltinism and distribution.

Measurement and duration of objectives in WP2 - Agrilus Please note details in the approaches and methodology section as well as the milestones. Numbering below corresponds with objective listed immediately above here.
i. Attractive volatiles from oak tree foliage are identified. Duration: 8 months.
ii. Attractive volatiles from oak tree bark are identified. Duration: 8 months.
iii. Thermal trial duration 3 months; extend if successful. Emergence traps 5 months (repeat year 2). Insectory rearing ongoing after successful capture and mating of adults.

Linkages WP2 - Agrilus:
Cross-linkages will take place between WP1 and WP3 as already indicated above. As beetles or larvae are obtained some will be used by FR and Fera for detection of bacteria associated with these life stages. Information generated in WP2 will ultimately be used by WP3 so that the developmental life history components that affect epidemiology can be included in the models.

Workpackage 3: Distribution, incidence and severity of AOD - Modelling component
(to be considered and revised as necessary at first steeering groyup meeting).

General Aims WP3 - Modelling

The main aims of the sampling, modelling and mapping component of the proposal are to estimate the boundaries and extent of the condition, estimate severity/incidence for certain sites, and to predict the risk and spatial spread of AOD in Britain.

Specific Evidence Objectives WP3 - Modelling: The objectives of the sampling and mapping component of the project are:
i. Develop an optimal sampling plan to map the geographical distribution of AOD, carry out training, arrange site visits, carry out field surveys, estimate AOD incidence /severity; convert field data into electronic format
ii. Develop and parameterise a flexible model for the spread of AOD
iii. Create maps showing the estimated geographical distribution and boundaries of AOD
iv. Estimate the severity/incidence of the condition at specific locations

Note: In section 2.3 (Approach and methodology) we provide a more detailed description of the modelling approaches and methods used to address these objectives.

Measurement and duration of objectives in WP3 ¨C Modelling: Please refer to section 2.3 for more details
i. An optimal sampling plan to map the geographical distribution of AOD will be provided to FR and this will take 6 months (RRes). Based on the plan the first round of surveys will be carried out (at an estimated 150-200 sites) and this will take 4 months (FR). The data will be collated and transformed into electronic format which will take 3 months (FR). The electronic data will then be handed to RRes (December 2013) and initial models developed and the sampling plan revised for the next samping round - duration 6 months (RRes). Revised sampling plan to be given to FR in June 2014. Second surveys and data collation as above - duration 7 months (FR). Final data to be handed to RRes December 2014. Final modelling and mapping - duration 12 months (RRes) expected completion date December 2015.
ii. Develop and parameterise a flexible model for the spread of AOD - ongoing over 36 months completed by December 2015.
iii. Create maps showing the estimated geographical distribution and boundaries of AOD - ongoing over 36 months with the production of maps being the measurement of fulfilment of the objective completed by December 2015.
iv. Estimate the severity/incidence of the condition at specific locations - ongoing over 36 months using existing (FR) data at first and incorporating new data as it becomes available. Fulfilment of objectives is measured by production of incidence and severity maps and refined predicitve models.

Linkages WP3 - Modelling: There will be a high degree of cross-linkage with all the other WPs in this section of the project and possibly with projects beyond this Defra project. For example FR is developing partnerships with other organisations to investigate abiotic factors, such as soil types and properties associated with AOD. Should significant information be uncovered this will be supplied to the modellers. Within the current project however, there is strong linkage between the modellers and FR, in terms of supplying information already owned by FR but also in feeding new information about causes and epidemiology into models.
Time-Scale and Cost
From: 2013

To: 2016

Cost: £1,109,764
Contractor / Funded Organisations
Harper Adams University College, F E R A (FERA), Forest Research Agency, University of Cambridge, Bangor University, Rothamsted Research (BBSRC)
Keywords
Forestry              
Plant health