This is a continuation of the previous PHRAME project.
Previous studies have shown that the pine wood nematodes (PWN) is a major threat to European forests. Project background
In order to ensure effective containment and eventual eradication of PWN, demands a detailed understanding of the behaviour and dynamics of PWN in infested trees. Summary of project aims
The key objective for this project was to extend the capability of existing models to identify the risk posed by PWN to the rest of Europe and the possible impact of climate change on its spread. It brings together Europe’s leading experts on PWN, together with colleagues from around the world, to address the key gaps in current knowledge.
Pine wood nematode (PWN), Bursaphelenchus xylophilus, is correctly regarded as a major threat to European forests, particularly following its establishment in Portugal and the fact that, even with the application of stringent measures, the pest has continued to spread and kill pine trees in that country.
The basis for classifying PWN as a highly significant threat is well documented and its track record of killing pine trees in:
In all countries where wilt expression has been recorded (leading to the description of the syndrome pine wilt disease) there has been extensive tree mortality. For example, in Japan annual losses peaked at 2.5 million m3 in the early 1980s but appear to be fluctuating around an average of about 1.0 million m3 to the present time.
The recent outbreak in Portugal illustrates the high costs of attempting eradication of a newly discovered organism (approximately €80million from 1999 – 2009 – source EU DG SANCO), but the direct losses from tree mortality must also be added to this amount. The campaign to eradicate the isolated finding of PWN in Spain has also incurred high management costs, of nearly €3m in 2010 (EU DG SANCO).
Faced with this evidence of direct and indirect financial effects, when combined with the environmental and social impacts, there is an urgent need to develop the most effective pest management strategies to deal both with established outbreaks and, particularly, for early detection and eradication of incipient infestations.
Considerable progress has been made in understanding the relationships between PWN, its vector insects and the host tree / environmental factors that result in pine wilt in all affected countries but, especially, in Portugal.
Apart from research and management initiated within Portugal directly, the EU Framework Programme 5 project PHRAME, addressed a number of important issues on B. xylophilus, its vectors in the genus Monochamus and the factors leading to wilt expression and tree mortality.
In developing the concept for the current project, the knowledge gained in PHRAME and through other research efforts internationally has been brought together to address the key interactions between the four main elements that determine risks from PWN namely:
The figure below, distinguishes between the ways in which the vector beetles in the genus Monochamus transmit nematodes to either living trees (maturation feeding) or dying/dead trees (oviposition).
The interactions between Bursaphelenchus xylophilus, its vectors in the genus Monochamus and living or dead host trees, with the 6 key questions then arising from knowledge gaps identified as the proposed research programme science Work Packages.
As virtually all conifer hosts (except the genera Thuja and Taxus) are suitable for the saprophytic (delivered by oviposition) phase of the nematode cycle, the availability of suitable host trees in Europe could be significantly extended. The expression of pine wilt, leading to tree mortality, is a phenomenon that is only noted under particular combinations of susceptible tree species and eco-climatic conditions, principally high temperatures and low soil moisture contents. The precise conditions leading to pathological impacts from PWN are not fully understood, although some of the factors have been elucidated, so this project hopes to provide solutions in an easily understood format.
The main aim of the research program is the development of improved methods for the detection, control and eradication of pine wood nematode (PWN) in support of EU Plant Health Policy.
The process governing association of the nematode with the vector, the nematode leaving the vector, the movement of the nematode in the tree and the expression or latency of wilt symptoms are poorly understood.
Thus, the research programme will address the following objectives:
Although there is a great deal of information available on the PWN worldwide and from the PHRAME Project there are still gaps in knowledge and the Work Packages in this project have been agreed to fill up the evidence bases.
The gathering of global knowledge within this project is a fundamental building block underpinning the main composite output of producing a PTK which will synthesise data, both from the wider international research and development community and the data produced by the REPHRAME consortium.
It will provide an innovative and interactive portal for stakeholders to interrogate the evolving state-of-the-art from REPHRAME and will enable customised outputs to be produced, ranging from basic advice through to precise and statistically valid procedures for sampling and management of PWN and its vectors.
Fundamentally, it will also be iterative and set up to ‘learn’ from new information from a disparate range of sources so that the outputs and recommendations will remain at the cutting edge. It will, therefore, be a key resource for those needing definitive and reliable information and advice on this highly damaging and invasive pest of conifers.
As well as developing the PTK, the project will actively seek to secure its sustainability, including establishing appropriate IPR arrangements.
The approach adopted enables the consortium to address the key questions posed by the REPHRAME project and which are interpreted in relation to how they relate to pine wood nematodes (PWN)/vector/tree interactions as detailed in the figure below.
The gaps in current knowledge are clearly understood and will form the basis of the research undertaken in the project, and will look into the relation of the saprophytic and pathogenic components of the nematode life cycle and, critically, to the interaction (or not) with the vector Monochamus spp.
The main benefits of the inter-related research will be that the infection, population build-up, distribution and spread of PWN in individual pine trees will be investigated, in particular in trees with long-term latent infections, and statistically reliable sampling strategies will be developed.
An important consideration both in assessing impacts and in developing improved sampling regimes will be the question of intrinsic pathogenicity of different races or strains of B. xylophilus. Thus, a range of molecular techniques have been employed to assess the potential origin and amount of genetic variation in the Portuguese populations of PWN, some within the PHRAME programme and others arising from international research effort and collaboration. These techniques are being developed as components of rapid and specific diagnostic and identification tools for PWN to supplement traditional extraction and morphological examination of nematodes.
Analyses of the natural spread of PWN from trees with the emphasis on the potential for long-distance dispersal with European Monochamus species, is critical for developing management strategies such as precautionary clearcuts. Detailed studies of the flight capabilities of Monochamus spp, especially M. alternatus in Japan and China, indicate that most flight is very local (up to 100 m), but that longer distance flight (various estimates between 1.8 and 3.3 km) can also take place. The balance between local and long-distance natural spread is therefore an area which requires urgent consideration and provision of definitive advice, taking account of variation in beetle age/physiology and its local surroundings. The dispersal tendency and any flight distance variation in relation to forest structure and tree density is a particular item to be assessed and improved Vector traps for early detection will be employed for determining the dispersal potential.
The magnitude of the risk of infested wood packaging material, wood chips and isolated bark as a pathway for spread of PWN to healthy forests will be quantified experimentally. The key element of this research requirement is the assessment of risks from non-vector transmission of PWN in wood-based pathways that do not have Monochamus spp. associated with them. Consequently, the risk profiles of the different pathways must be assessed in relation to whether non-vector transfer of PWN to host trees is possible and, if so, what is the significance of the risk.
This is essential. When pine wilt reaches epidemic proportions, as has been the case in Japan, the capacity to continue growing susceptible species of Pinus is lost. Indeed, in some areas of Japan the local species of pine have been entirely replaced by other conifer genera, such as Cryptomeria and Chamaecyparis. The alternative is therefore to assess those Pinus species that have been shown to be resistant to PWN or which have some tolerance to the nematode and then to evaluate them as potential replacements for known susceptible hosts.
This will be undertaken by taking into account possible climate change and the entire sylvo-climatic diversity of Europe. The information on the biology and pathogenesis of PWN in relation to a range of conifer hosts following introduction of the nematode to the crowns of trees during maturation feeding by Monochamus spp. is critical and essential to understanding and predicting likelihood of wilt expression.
Field experimentation in Portugal will validate the modelling as well as any latent infections. The latency in expression of wilt in PWN infested countries outside Europe which have cool climatic conditions is a factor that requires further detailed study to both refine the process models and also to design survey regimes to take account of the phenomenon in both wilt and non-wilt zones. As the reasons for and scale of latency are not well understood therefore, both direct experimentation and observation combined with refinement of the process-based models are required to quantify the extent of latency and to design survey regimes accordingly.
The project will interact intensively with ongoing EU-funded research projects on plant health (such as those addressing pest risk analysis, identification methods and the development of early PWN detection methods) and will utilise and expand the results as much as possible of previous projects (such as PHRAME).
Bringing the wide ranging PWN and Monochamus knowledge base together is important in ensuring that rapid and relevant progress is made in addressing the important requirements of the project. To this end, the gathering of this global knowledge is a fundamental building block underpinning the main composite output of REPHRAME, namely the PWN Tool Kit (PTK). This will:
The key deliverable of the project is the synthesis and development of a simple web based interface PWN Tool Kit which will provide analysed data from the project as well as practical advice and new or enhanced methodologies available for use by the end-user stakeholder community.
The consortium will aim to achieve this by:
See the description of the work packages for further details.
The main objective of Project Management in REPHRAME is detailed in Work Package 1: that is to ensure the project is managed in the most efficient and effective manner possible and meets all its obligations in terms of budget, reporting, milestones and deliverables.
With a natural synergy between the Consortium Partners, each having different expertise to bring to the partnership from within their specialist areas, this integration helped to deliver the overall project objective.
The project was managed by the Coordinator who had the overall responsibility for ensuring the project meets the above objective, whilst heading a small project management team.
With all project management information feeding into the Coordinator, the considerable experience of Partner 1 in the management of large projects, including coordination of EU Framework projects, will be utilised to ensure the European Commission is in receipt of all required information on the due date and that rapid and appropriate dissemination of results is achieved.
The Coordinator will report to the Management Committee established at the beginning of the project and will involve the Principal Investigator from each of the Consortium Partners.
The Management Committee will be chaired by the lead organisation in the project, Forest Research in Wales (Partner 1), and it will meet every six months. Its duties as well as providing an oversight of the management of the project, will oversee issues relating to technology, intellectual property, security and relationships with the Science Advisory Panel and Stakeholder Observer Group, together with exploitation management.
The Management Committee will confirm the project reporting structure in terms of communication, decision-making, conflict resolution and reporting requirements.
The Science Advisory Panel (SAP) when established will include EU and International experts in order to give the research a level of critical analysis leading to science and dissemination approaches to the highest international standards as well as high level validation through association.
The SAP will receive an annual briefing on the progress of the project and will be consulted on plans for future work. Its comments and advice will be fed into the Management Committee. In particular the SAP will be tasked with ensuring that the project takes full account of research being conducted elsewhere in order both to avoid duplication and to ensure the timeliest outcome of the research.
The SAP will be led by an eminent international researcher, chosen by the SAP, who will have a right of attendance at meetings of the Management Committee.
SAP meetings will be arranged alongside other project events to facilitate face-to-face interaction but with provision for remote participation to ensure optimal attendance.
Consultation on membership is continuing to ensure that the SAP includes leading scientists from North America, Japan and China as well as from other EU centres of excellence in the field, in particular those engaged in other relevant FP7 projects.
With all project management information feeding into the Coordinator, the considerable experience of Partner 1 in the management of large projects, including coordination of EU Framework projects, will be utilised to ensure the European Commission is in receipt of all required information on the due date and that rapid and appropriate dissemination of results is achieved.
Each work package will be led by a Work Package Manager who will have the knowledge and expertise within the precise activities to enable the achievement of objectives set and their effective and timely delivery.
In addition to managing the work within their own Work Packages, the managers will report to the Management Committee and take part in co-ordination activities across the entire project. To achieve this, each Work Package is divided into a number of Tasks of research and technological development (RTD) which will enable activities to be coordinated, each with a designated leader, and the necessary networks to build on and further develop strong working relationships between Partners.
A mid-term assessment report on the progress of the research and methodological development, and the REPHRAME project’s plans for its future exploitation strategy will be produced before the end of the 18th month from the date of commencement.
The Management Committee will organise a mid-term assessment meeting with all consortium members, the Science Advisory Panel and a representative from the Commission. The purpose of this meeting will be to report on the progress to date and to refine the Work Plan for the remaining part of the contract. Procedures for managing future exploitation of results will be discussed and assessed.
During the life of the Project the Consortium will hold, at six-monthly intervals, 7 plenary meetings of all project Partners. The initial meeting will be held at the outset of the research programme to develop common protocols for experimental and analytical procedures, data exchange and modelling procedures.
Additionally, scientists involved in single work-packages, and under the supervision of the leader for the work-package, will hold periodic progress meetings throughout the carrying out of research associated with the work-package.
Meeting minutes, notes and presentations
Location: Funchal, Madeira.
Date: 1st to 2nd April 2011.
Location: NRA Orleans, France.
Date: 13th to 14th March 2012.
The names and contact details of the principal scientist from each partner organisation are listed below.
| Dr. H. F. Evans Coordinator |
E-mail: hugh.evans@forestresearch.gov.uk
|
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| Prof. David Hall | The Natural Resources Institute of the University of Greenwich Old Royal Naval College Park Row Greenwich London SE10 9LS UK |
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| Dr. Christian Tomiczek | Federal Research + Training Centre for Forests, Natural Hazards + Landscape Seckendorff-Gudent-Weg Vienna 1131 AustriaTel direct: +43 1 87838 1133 Tel:+43 1 87838 1218 E-mail: christian.tomiczek@bfw.gv.at https://www.bfw.gv.at/ |
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| Dr. Alain Roques | Institut National de la Recherché Agronomique (INRA) Rue De L’Universite PARIS Cedex 07, 75338 France Working at the Forest Zoology Unit, Forest, Grassland and Freshwater Ecology Division Orleans Research Centre Tel direct: Tel.: 01 42 75 91 69 E-mail: Alain.Roques@orleans.inra.fr https://www.inrae.fr/en |
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| Dr. Thomas Schröder | Julius Kühn Institute Federal Research Centre for Cultivated Plants (JKI) Erwin Baur Strasse Quedlinburg 06484 Germany Tel: +49 531 299-3381 E-mail: thomas.schroeder@jki.bund.de https://www.julius-kuehn.de/ |
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| Dr. Edmundo de Sousa | Formerly of the National Institute of Biological Resources (INRB, IP) Portugal (Institute no longer open) |
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| Prof. Dr. Manuel M. Mota | University of Evora Largo dos Colegiais Evora 7000-803 Portugal Tel:+351 266 740 800 Fax: +351 266 740 806 E-mail: mmota@uevora.pt https://www.uevora.pt/ |
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| Dr. Alfonso Navas | Agencia Estatal Consejo Superior de Investigaciones Cientificas Calle Serrano MADRID 28006 Spain Tel.: +34 91 568 14 00 Fax: + 34 91 411 30 77 https://www.csic.es/en |
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| Dr. Juan A. Pajares | University of Valladolid, Autonomous Community of Castile + Leon Plaza Santa Cruz 8 VALLADOLID 47002 Spain Tel: +34 983 423 000 Fax: +34 983 423 234 E-mail: jpajares@pvs.uva.es https://universityofvalladolid.uva.es/ |
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| Dr. Jiang-Hua Sun | Institute of Zoology, Chinese Academy of Sciences Beichen West Road 1 BEIJING 100101 China (People’s Republic of) Tel direct: +86-10-64807121 Tel:+86-10-64807098 Fax: +86-10-64807099 E-mail: sunjh@ioz.ac.cn |
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| Dr. Christer Magnusson | Formerly of the Norwegian Institute for Agricultural and Environmental Research – BIOFORSK Norway (Institute no longer open) |
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Note: P1 to P11 refer to the 11 project Partners.
The following will need to be implemented to ensure the smooth operation of the project:
The principal objective is the determination of factors governing association of the nematode with the vector.
In spring, B. xylophilus third stage dispersal juveniles (JIII) begin to aggregate near the larval galleries and pupal chambers of the vector and then moult into dispersal JIV larvae (dauer larvae). Subsequently, these dispersal stages associate with the M. galloprovincialis callow adults inside the pupal chamber, just a few days before the emergence from the wood. The stimuli promoting the attraction, moulting and entry of the nematodes into this specific development stage of the Monochamus vector insect will be studied in tests under controlled conditions in the laboratory.
A further objective will be the determination of factors governing the departure of PWN from the vector. The fourth-stage dispersal larva of PWN (also known as JIV or dauer larvae) is the developmental stage carried by the insect vector into a new tree host, either by maturation feeding or oviposition activity. The stimuli that mediate and regulate the exiting of the nematodes from the vector’s body into the new pine host are not known, and may partially explain why some pines are attacked by the nematode and may suffer from wilt disease while others do not. Investigation of movement of PWN inside the host tree will be studied under greenhouse conditions with 2-3 year-old trees and in the field in Portugal.
Recent observations by Chinese colleagues have indicated a possible role for bacteria associated with PWN and B. mucronatus in the pathogenic process of PWN. A national project investigating this issue will provide partial support for this task. This work will address key issues such as Isolation and characterisation of bacteria associated with the nematode in different stages of the B. xylophilus life-cycle, in vector pupae and within infested trees.
P10 in China has developed a sampling method involving attraction of the different stages of PWN in infested trees, symptomatic or asymptomatic, by specific blends of host volatiles for either juvenile or dispersive nematodes. They allow rapid detection of PWN within two hours. Their applicability to the European species of conifers susceptible to infestation by PWN will be assessed in Portugal by P6.
Several methods regarding detection of PWN DNA have already been developed, mainly by Japanese and Chinese colleagues. One method in particular, using the “nested-PCR” technique appears to be promising (Takeuchi et al., 2006) and is based on finely crushing a wood sample, utilizing a specific probe to detect the presence of the nematode DNA, amongst the wood material, with no need to isolate the nematodes from the wood. P7 will conduct testing of this method to samples obtained either from PROLUNP (annual survey) or using healthy wood artificially inoculated with PWN. Other similar DNA detecting methods will be tested in parallel by P4. Novel methods for detecting gene products, resulting from the pathogenic action of the nematode will also be investigated by P7.
A second method will also be tested in Portugal by P6. This new method was developed in Japan and is available as a kit. It is based on an enzymatic reaction that can extract nematode DNA from the wood tissue followed by Loop-mediated Isothermal Amplification (LAMP), targeting a specific DNA region of the PWN gene. It requires no expertise in microscopic/morphological techniques and is a time and labour-saving way to identify the nematode.
The principal objective will be the study of the vector flight phenology under different climate conditions in central and northern Portugal by keeping pine wood containing M. galloprovincialis in meshed boxes at ambient temperatures in both completely shaded and sun-exposed situations, recording the air temperatures and the solar radiation of the wood. P6 will coordinate.
The developmental thresholds and thermal requirements of M. galloprovincialis larvae have already been studied in Portugal, resulting in a simple linear method (the modified sine wave) driven by air temperatures used to predict the emergence in southern Portugal. The model can be a useful tool to predict insect emergence in other European regions after refinement. Thus, refinement and adjustment of the existing model with the emergence patterns from other locations of central and northern Portugal and Europe will be carried out. In addition, a validated map of annual M. galloprovincialis emergence patterns for the Iberian Peninsula based on degree-day accumulations in the different climatic regions will be developed. Similar studies will be made for other Monochamus species, as indicated above.
Flight capabilities and flight behaviour of Monochamus galloprovincialis will be tested using sophisticated flight mills adapted from those designed for insects of the same size and weight, and including electronic recording of both single and multiple, successive flights. The tested insects will be reared under controlled conditions. Populations originating from different geographic areas and host trees will be compared in order to assess possible variability in flight capabilities within and between populations.
There are several approaches to estimate dispersal of animals, including the mark and recapture of released individuals, which is frequently used for insect population studies. The efficiency of this method is dependent on the effectiveness of the recapture method, and systems based on effective traps and attractants are usually suitable. The kairomone-pheromone attractant recently developed for M. galloprovincialis and other potential attractants as developed in WP4, will represent convenient tools to assess flight distance for this species. P3, P4, P6 and P9 in collaboration.
Dispersal of Monochamus adults will be tested by mark-release-recapture experiments under different forest conditions within and between forests. Additionally, to investigate the dispersal of the wilt disease in the new nematode focus in the centre of Portugal.
Monitoring of flight behaviour with remote detection techniques (radio tracking) have proved useful for close tracking of animal movements for some species, Development of smaller transmitters is, however, rapidly increasing and using these new techniques will be investigated to understand better the flight behaviour of Monochamus in real conditions. P3, P4, P6 and P9 will agree and carry out components of this study on a range of Monochamus spp.
Development of molecular techniques to assess vector dispersal and dispersal routes Dispersal of the vector depends on both its population density and on host availability and the study will be developed in areas of 30 km radius in PWN-infested zones in the Iberian Peninsula, and non-infested zones in Portugal, Spain and southern France, using 2 areas per country.
The principal objective will be the testing and improvement of synthetic lures for M. galloprovincialis.
Attractants recently discovered for M. galloprovincialis by P9 and P2 are based on a blend of kairomone compounds from hosts and bark beetle competitors with a male-emitted aggregation pheromone that is strongly synergized by the kairomones. New variations on the kairomone-pheromone blends and dispensers will be tested against M. galloprovincialis in different forests in Austria, (P3), France (P4), Germany (P5), Portugal (P6), Spain (P9) and other countries as appropriate, deployed in multiple-funnel traps in a randomized block design. Overall coordination of this work will be by P9 and the development of age-specific attractants based on host pine volatiles will be investigated (P2, P3, P6 and P9). Candidate compounds will be tested in contact recognition and short range olfaction bioassays and then on their ability to improve catches in traps baited with the optimised kairomone/pheromone lure.
A further objective will be the development of traps for live trapping and for mass-trapping of Monochamus spp with different trap designs to maximise captures of M. galloprovincialis will be tested under different field conditions. P9 will work closely with the other Partners to carry out this work.
The potential for management of vector populations by mass-trapping will be studied under different stand and landscape characteristics (host tree density, stand management, landscape fragmentation and heterogeneity) with different beetle population densities (from very low to outbreak). Field testing will be carried out in Austria, (P3), France (P4), Germany (P5), Portugal (P6), Spain (P9), and other countries as appropriate.
The development of attractants for other European Monochamus species and their responses to potential kairomone compounds by M. sutor will be laboratory tested in olfaction and electrophysiological (GC-EAD) bioassays (P9 and P2) with input from P3, P4 and P5.
The principal objective will be the study of B. xylophilus distribution in wood and wood chips: survival and population dynamics depending on wood moisture content.
Studies concerning the long term survival of PWN in wood will be established immediately after project start with different variants using fresh wood. P6 to lead, and similar investigations will be done with wood chips by P5.
A further objective will be the transmission of B. xylophilus with wood, wood chips or bark to healthy trees. Laboratory scale investigations in Portugal will be carried out concerning potential transfer of B. xylophilus in wood chips to living pine trees. P5 will coordinate with inputs by other Partners and by collaboration with international contacts. Corresponding outdoor investigations are planned to be carried out in the native range of PWN (i.e. Canada and USA) in areas where pine wilt expression has not been recorded and which, therefore, would provide a proxy for conditions in northern Europe where it is not currently expected that pine wilt would be expressed.
The transmission of B. xylophilus or closely related species of the genus from sawn wood boards to healthy trees will be looked at by laboratory experiments which will be conducted using 4- to 5-year-old seedlings of the species P. sylvestris. Small pieces of wood infested with PWN will be attached to the stems either with wounding the tree (e.g. attaching the wood with needles or thumbnails) or by fitting the wood pieces by lashing. Outdoor experiments using trees of different ages and therefore different thickness of bark will be carried out with boards infested with the non quarantine species B. mucronatus. P5 will coordinate.
Studies will be carried out in field and laboratory to clarify the possibility of nematode infestation from infected trees to the soil or by root contact. Its persistence within this element and subsequent entrance to nearby healthy hosts will be carried out at the premises of P6, working with P11. A similar experiment but using Pinus sylvestris will be carried out by P5 under quarantine conditions in a climate chamber.
A study on the transmission of B. xylophilus from infested wood to non-infested wood in storage and in transit Laboratory scale experiments will be carried out with artificially infested pine wood. Horizontal and vertical distribution potential of PWN to neighbouring wood samples will be investigated. P5 to lead with links to other Partners.
Importance of infection bridges from forest soil to sawn wood. Nematode free blocks of Pinus sylvestris intended for pallet construction will be laid out on the forest floor in a randomized block design. The experiment will be carried out in a pine forest (P. sylvestris) in eastern Norway and conducted by P11 in collaboration with forest mycologists of the Forest and Landscape institute in Austria.
The validation of a set of PWN-specific microsatellite markers usable at the individual level and in order to do this it is proposed to use a set of microsatellite markers identified from the nematode genome to locally characterise PWN populations and their genetic relationships, resulting from direct field sampling (including the newly infested area in Madeira) but also in retrospective analysis of the variability in the existing distribution of PWN in Portugal. Recently developed computer simulation methods (Ciosi et al., 2008; Guillemaud et al., 2010) will be used to quantitatively compare the different introduction scenarios for the European populations.
The assessment of genetic diversity of European PWN populations in relation to invasions will also be an objective. Proteomics are extremely useful when looking for differential “functional” elements (parasitism, adaptability, ecological value) within a species’ common genetic pool, and is becoming increasingly valuable when studying the natural variations within a population of any species Adaptive processes are the key in parasite evolution so it is therefore proposed to describe the proteome of B. xylophilus by creating a database of the positions of the proteins in bidimensional gels isolated from B. xylophilus populations of European and Asian origin and identify the genetic changes which have occurred during the evolution and distribution/movement of B. xylophilus.
The principal objective will be a symptom development of resistance of Pinus to PWN and the task will be coordinated by P5 and use a standardised artificial inoculation procedure developed and used successfully in PHRAME.
In Portugal the same experiments will be done by P6 in the laboratory using pine shoots collected from different origins which will be tested using a hierarchical approach, i.e. pine species > provenances > families. B6 will use plant material from different Pinus pinaster and P. pinea provenance trials.
Confirmation of nematode breeding will be made in all trees tested, irrespective of symptom development. P6 and P7 will link to the other Partners in this work package to coordinate the biological and molecular elements of the work.
A further objective will be the comparison of the transcriptomes of Pinus pinaster and resistant genotypes of Pinus in response to Bursaphelenchus xylo. To identify candidate genes involved in disease resistance to PWN, the transcriptomes of infested and healthy tissues, collected from sensitive and resistant genotypes of Pinus sp. will be sequenced, using ultra high throughput pyrosequencing to identify, characterise and quantify the transcripts and putative candidate genes involved in the molecular mechanism of response to biotic stress related with PWN infestation.
The testing of resistance of Pinus spp. to PWN insect vectors and the evaluation of resistance level and identification of resistance markers during the maturation feeding phase by Monochamus spp. on cut shoots. Preliminary bioassays will be carried out on cut pine shoots to provide initial screening for possible resistance to young Monochamus adults during their maturation feeding phase. The beetles will be obtained from intensive laboratory rearing and P6 will coordinate.
Depending on results from the preliminary screening, further evaluation tests will be carried out on determine whether potential resistance level and identification of resistance markers during the maturation feeding phase is maintained when seedling trees under a range of environmental conditions are subjected to Monochamus spp. feeding pressure and the same methods will be employed to identify putative biochemical markers of pine resistance. P6 will coordinate.
Comparative studies of the evaluation of resistance level and identification of resistance markers during the reproductive phase of host selection will be conducted using mature adults after mating. The preference of the vector to lay eggs in maritime pine logs with and without the presence of PWN will be similarly determined. P6 will coordinate.
The breeding for Resistance to PWN Disease and the estimation of genetic parameters of pine resistance to PWN insect vectors will be estimated to evaluate the relevance of tree breeding as preventive control method of PWN disease.
Hybridizations for resistance to PWN will be evaluated and artificial F1 hybrid progenies within Mediterranean Pinus species will be assessed for expression of PWN resistant, and the Portuguese native Pinus pinaster as sensitive. Foreign pollen of resistant Pinus species, not available in Portugal, will be introduced for artificial crossing using P. pinaster as female genitor. Inoculation tests with PWN will be done for assessment of susceptibility of the progenitors and F1 progenies in the future. P6 will coordinate.
The potential of mosaics of tree species as a measure to reduce PWN impacts in Portugal. The mortality and severity of infection by the PWN will be investigated by field observations of wilt expression in both monoculture stands and in mixed pine-other species forests in Portugal. Wilted trees will be sampled for the presence of the PWN. Plots will be created within the pure and mixed stands, georeferencing all trees and following their sanitary condition through the time, in order to detect differential patterns of PWN infestation and tree mortality. The work will be coordinated by P6.
The principal objective will be the refinement of PHRAME process model, based on ForestETp, for prediction of wilt and to account for latent infestations and delayed expression of wilt.
P1 will carry out refinement of the core model, both to improve and verify its predictions and also to produce a simplified version (PHRAME light) to enable non-specialist users to input parameters to test ‘what-if’ scenarios of regional impacts of PWN under current and future climates. Forward prediction to account for future climates will be a core activity for both the full and ‘light’ models, and will count on the collaboration of P7, following up on the predictive model (scenario) elaborated under PHRAME and working with economists in Forest Research, the model will be extended to assess the economic, as well as environmental, impacts of PWN.
Field experimentation will be undertaken to verify and refine the process models and P1 will work with P6 and also researchers in North America and, using visits by researchers from REPHRAME augmented, if possible by existing research work being carried out by local Japanese research groups which will be investigated as part of international collaboration, Japan and to carry out field inoculations to verify the predictions of the model under conditions where wilt is expected (Japan and Portugal) and where no wilt is expected (North America).
The development of a sub-model to account for latency in wilt expression on both the core model and field experimentation in ‘non-wilt’ and marginal wilt areas will explore the conditions under which either delayed onset of wilt (latency) or non-wilt (eco-climatic resistance) in intrinsically susceptible trees is likely to occur. P1 will work closely with all Partners to develop the sub-model
The analysis of the history of infestation and wilt expression in Portugal will contribute to the refinement of the process model for prediction of wilt; P6 will compile all available information on the spatio-temporal evolution of pine wilt in the affected region south of Lisbon since its appearance in 1999.
The development of a PWN spread model taking into account human influences will be developed at the European scale based upon the previous results taking into account local dispersal (results of WP3) and inadvertent transportation by humans at long-distance (results of WP4). This risk will be calculated using the human population density, and eventually some connection means (such as road network and ports) potentially involved in the transportation of infested materials. Because the transportation of infested material from an infested region to a pest free region is a random process, a stochastic model will be chosen and the probability of occurrence of PWN and PWD will be calculated at different years in the future using several climate scenarios. P4 in collaboration with P1 and P6.
One of the principal objectives will be the synthesis of knowledge from previous EU projects to reflect on the results, conclusions and lessons learned from earlier EU programs, which are directly or indirectly related to PWD, particularly:
This Task will be enhanced by fact that several members of the present consortium have participated in one or both projects (P1, P3, P4, P5, P6, P7, and P8).
Another principal objective will be the Interaction with current EU projects and the final synthesis. Partners of this project will liaise with current EU projects of relevance including:
P7 will coordinate linkages and, where they exist already, those Partners involved in EU topics will ensure a high level of bilateral exchange.
Although all types of nematological research are being undertaken, PWD is the focus given in one Partner institution (P7).
Their task will aim at providing information on PWD to the EUMAINE programme in order to sensitise EU colleagues and students to the seriousness of PWD, providing a major route to disseminate this information more widely amongst scientists, students and the public at large. The inclusion of non-EU teams as collaborators through this WP will also allow for the necessary contacts to obtain material (nematode isolates from centres of origin, in particular), access to field conditions to experiment (inoculation of trees in non-wilt areas in Canada or USA), and transfer of technology already available for testing of rapid and early detection methods (China, Japan, Korea).
The principal objective will be the development of a key platform for provision of consolidated and synthesised information from REPHRAME by means of a
PWN ToolKit (PTK) which will provide a simple and structured electronic interface that is user-friendly and will be a primary source of advice and practical methodology for overall management of the PWN problem.
The design of the PTK interface will evolve throughout the duration of the project, but early attention will be paid to its format and user interface. The core will be a decision-tree expert system to enable users to interrogate the data and obtain general and specific advice and outputs on all aspects of the PWN research and development acquired during REPHRAME.
P1 will work closely with P4 to develop the interface.
After analysis of the synthesis of results and construction of PTK modules the following are likely to be included:
P1 to coordinate, with input from all Partners in this Work Package.
The full public launch of the PTK will take place during the final months of the project to coincide with the International Conference on PWN, although partial versions will be made available once particular modules have been completed. However, the full value of the PTK will be enhanced when all the data gathered during REPHRAME has been analysed and interpreted into suitable outputs.
P1 to coordinate and ensure linkage to WP10.
The principal objective will be the dissemination by means of the appropriate media for project outputs i.e. Website, Project Leaflets, Reports, Scientific Publications. Publication in the peer reviewed literature is the Quality Assurance on the scientific level of the work conducted during the project.
Another means of dissemination will be the setting up of Stakeholder Observer Group (SOG) and establish a specific Web portal for the SOG. This will also be a discussion forum to share information and ideas in parallel with, but independently managed from, the main REPHRAME consortium pages.
Two Themed Workshops during the second and third years of the project will be organised:
The Workshops will be held in Portugal and Spain. P1, P6, P7 and P9 to coordinate.
An International Conference on all aspects of PWN and its vectors research and development will be held during the final three months of the project. The outputs from REPHRAME will form a substantial part of the Conference, but wide international involvement will be actively pursued so that the outcomes of the Conference will provide a state-of-the-art synthesis of knowledge. It is expected that more than 100 scientists from all countries where PWN occurs and others will take part. B5 to coordinate.
The proceedings of the Conference will be published as a printed book (B5 to coordinate). In addition, e-copies of the presentations and a summary of the main findings and outcomes of the Conference will be made available on the REPHRAME website. (B1 assisted by all).
This programme was funded by the European Union – Framework Programme FP7.
This is a small collaborative project involving 11 organisations in 8 countries (Austria, China, France, Germany, Norway, Portugal, Spain, United Kingdom) and international linkages with Canada, Japan and USA.
Hugh Evans coordinated this project.
Although REPHRAME as such has finished, aspects of the project still continue, please contact us for more information.
Research Scientist
Head of Forest Research in Wales /
Pennaeth Ymchwil Coedwigaeth yng Nghymru