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Work Packages REPHRAME: detection, control and eradication of pine wood nematodes

REPHRAME work packages

Note: P1 to P11 refer to the 11 project Partners

Work Package 1: Management and co-ordination

The following will need to be implemented so as to ensure the smooth operation of the project:

A consortium Management Committee consisting of the Principal Investigators from each Partner Participant,

The administrative requirements, technology and management tools necessary for the Project.

Appropriate processes and procedures for ongoing monitoring with Work Package (WP) leaders, and establish task and activity lists, deliverables and milestones for all project members in accordance with the Grant Agreement.

Gather information, conduct progress reviews and integrate information from project WPs to form the mid-term assessment and report of the project

Establishment of a sound financial management system with appropriate processes and reporting requirements including arrangements for distribution of EU pre-financing.

Monitor consortium member financial record keeping.

Work Package 2: Behaviour and dynamics of pine wood nematodes (PWN) in infested trees

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 characterization 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.

Work Package 3:Assessing phenology and dispersal capacities of PWN vectors

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 Monochamusspecies, 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.

Work Package 4:Development of new methods for monitoring and control of Monochamus spp. and PWN

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 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.

Work Package 5:Determine risk of non-vector spread of PWN through various pathways to healthy forests

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 ofB. 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 infestation 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.

Work Package 6:Host tree resistance to PWN and its vectors for future planting

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, characterize 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 infestation 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.

Work Package 7:Prediction of pine wilt expression across eco-climatic zones, taking account of latency

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.

Work Package 8:EU and international cooperation and collaboration

One of the principal objectives will be the synthesis of knowledge from previous EU projects so as 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:

COST872, a COST project on plant-nematode interactions (coordinated by SCRI, Scotland, UK)

PRATIQUE (Enhancements of Pest Risk Analysis Techniques, coordinated by FERA), P4 participating

QBOL (KBBE-2008-1-4-01: Development of new diagnostic methods in support of plant health policy, coordinated by The Netherlands, Switzerland and INRA), P4 participating

EUPHRESCO (ERA Net on Plant Protection)

ISEFOR (Increasing Sustainability of European Forests: Modelling for Security Against Invasive Pests and Pathogens under Climate Change, coordinated by Aberdeen University), P1 and P4 participating

Other bilateral ongoing collaborations have been established between Partners and several EU or neighbouring countries, such as the Czech Republic, Slovenia, Russia, Turkey.

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 among 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).

Work Package 9:Synthesis and development of PWN Tool Kit for monitoring and management of PWN

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:

Statistically reliable survey methods for PWN in symptomatic and asymptomatic trees
Rapid and accurate diagnostic procedures for PWN
Statistically reliable improved survey methods for Monochamus spp.
Options for the management of vector populations
Quantification of the potential for non-vector transmission of PWN to host trees
Recommendations for pathway-specific risk reduction
Recommendations for choice of conifer species choice in relation to susceptibility to PWN
Prediction of pine wilt expression by eco-climatic zone
Factors to be accounted for in dealing with new PWN infestations, including optimised and statistically reliable survey techniques.

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.

Work Package 10:Stakeholder engagement and dissemination

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:

Describe and enable users to use the PWN Tool Kit and to provide training and hands-on experience in laboratory techniques (Workshop 1)

Field sampling and vector trapping techniques (Workshop 2).

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).
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