The project was formally completed in 2007 and the final report is available for download:
Summary of the main conclusions from the PHRAME project
The threat of Bursaphelenchus xylophilus
There is no doubt that Bursaphelenchus xylophilus is a significant threat to the pine forests of Europe and this is confirmed by the considerable tree mortality that has already been observed in the affected zone in Portugal.
For example, surveys carried out in the affected zone during the winter of 2006 revealed around 200,000 symptomatic trees and, of the trees sampled for presence of pine wood nematode (PWN), approximately 22% were positive for B. xylophilus (Report of FVO Mission to Portugal, May 2007). Such data indicate that the scale of the problem is large and, therefore, access to definitive information on the interactions between PWN, its vectors and its host trees in a European context is essential. The results presented in this report provide a solid basis from which to determine optimised methods for management of this important new pest to Europe.
The source of the B. xylophilus populations in Portugal
In relation to identification of the source of the B. xylophilus populations in Portugal, a combination of sophisticated molecular techniques have indicated that at least one race of the nematode originating in Asia has arrived in the country. There are indications that two sources of origin are included in the Portuguese population of PWN which suggests either multiple introductions or a single introduction of a mixed population of the nematode.
While of academic interest, this information is valuable in assessing potential future pathways of arrival of further incursions of the pest to Europe. Extensive surveys of trees in the countries represented within the project have revealed no further findings of B. xylophilus outside the affected zone in Portugal. However, the surveys have revealed many other species of Bursaphelenchus, including the closely related B. mucronatus and B. fraudulentus, which are difficult to differentiate morphologically. The data provide encouragement that B. xylophilus itself is still restricted to the affected zone in Portugal. New molecular techniques from this research programme will aid rapid and accurate identification of the members of the genus Bursaphelenchus in future.
Studies of the potential of members of the xylophagous insect fauna to act as vectors of the nematode
A key factor in the survival and spread of PWN in the field is association with a vector insect species. Extensive studies were made of the potential of members of the xylophagous insect fauna associated with PWN-affected trees to act as vectors of the nematode for further spread of the organism. A clear conclusion from the studies is that only M. galloprovincialis has taken on the role of vector in Portugal.
Although there are nematodes associated with a number of bark and wood-boring beetles in Austria, Portugal and Spain, none of these has been shown to successfully transmit B. xylophilus to host trees.
A further significant finding from the project is that despite clear demonstration of the presence of PWN in the roots of affected trees, there was no evidence of transmission of the nematode to adjacent trees either through root grafting or by direct migration through the soil. This information confirms the widely demonstrated results from international literature on PWN that it is only adult beetles in the genus Monochamus that act as effective vectors of the nematode.
The biology and pathogenesis of pine wood nematode in relation to tree species
Information on the biology and pathogenesis of PWN in relation to tree species potentially likely to exhibit wilt expression 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.
Correlative studies in regions of the world where pine wilt disease has been demonstrated all indicate a close relationship with average summer temperatures, including hypotheses that monthly isotherms must exceed 24oC to give rise to extensive tree mortality (Rutherford & Webster, 1987). While of general practical value, such correlations cannot account for local variation in eco-climatic conditions and it was recognised that more detail on the nature of pathogenesis of PWN in living susceptible trees was needed.
Results from studies carried out in Germany, Portugal and Japan within this project have thrown considerable light on the nature of the process of wilt expression when B. xylophilus is introduced to living trees by maturation feeding. Of particular significance is the knowledge that movement of nematodes from the site of introduction is immediate and rapid, with some nematodes being found in the root area within days of inoculation. Such patterns of invasion can be explained by the ability of the nematodes to exploit living tissues both for nutrition and reproduction and also to move into less well defended areas of the affected trees. This matches with previous histopathological examination of nematode invasions that linked their rapid movement downwards to exploitation of the cambial zone, leading to breakdown of tissue integrity and the opening of cavities that facilitated free movement of nematodes.
Lodging of nematode populations in particular parts of the tree was also shown to provoke local defence reactions, which contributed to embolism and cavitation of the xylem. Presence of large numbers of nematodes in xylem cells also confirmed the ability of B. xylophilus to breed successfully in the living trees. Such data are essential building blocks in developing process-based predictive models for wilt expression.
Data on the biology and dispersal of M. galloprovincialis
With the demonstration that M. galloprovincialis is the only known vector in Portugal, data on the biology and dispersal of the vector have been gathered, starting from a low knowledge base on this insect prior to the appearance of PWN in Portugal. Results from the project studies have shown that the vector has a single generation per year, with clearly defined emergence, flight and oviposition peaks around mid-summer in Portugal.
The proportion of adults carrying viable B. xylophilus dauer larvae emerging from trees killed by PWN can be high (ca. 75%), indicating a close relationship between tree mortality from PWN and subsequent exploitation of those trees for breeding by the vector. Such data confirm the cause and effect relationship between PWN, its host trees and the breeding success of the vector, the combination forming a feedback loop leading to increasing tree mortality and availability of breeding resources.
The Portuguese strategy of early identification and removal of PWN affected trees before emergence of vectors insects that may have used them for breeding is, therefore, sound.
Methods to increase the efficacy of sampling for vector insects are part of such an approach and considerable progress has been made in identifying and improving trap and chemical lure designs for use in both monitoring and, potentially, reduction of vector populations. Within this approach, attempts have been made to develop methods for early detection of infestation and potential wilt expression in PWN-affected trees. A simple method to measure oleoresin flow from a punch hole system, proved to be quite reliable for larger trees (> 20cm DBH) and gave early warning of subsequent wilt and tree mortality, but this was dependent also on the time of year and month of infestation by the nematode. Electrical conductivity methods did not provide sufficient warning of likely tree mortality.
Interactions between pine wood nematode and its host trees
Bringing together the detailed and more general information on the interactions between PWN and its host trees in an eco-climatic context, significant progress has been made in predictive modelling of wilt expression.
Two main approaches have been adopted, namely use of correlative heuristic and empirical models to link to tree suitability parameters and a process-based modelling strategy that links directly to tree physiology in relation to local eco-climatic conditions. This complementary approach has started to yield valuable predictive tools for improved risk assessment in the future.
The main conclusions from the correlative modelling approach were:
- Predictions of PWD dispersal over the whole of Portugal using the PWN occurrence data from the affected zone were not successful; however if more data become available, this type of modelling is worth exploring.
- A tree based approach produced useful predictive models of the PWD risk for Portugal - Central Portugal showed up as the area of most concern where there was high pine density growing in poor site conditions.
- High temperatures and low precipitation showed up as the main drivers of tree stress, which indicates climate change as a future central area of research in dealing with the risk of PWN spreading throughout Europe.
- The models can be used to improve the PWN national surveys, focusing field effort at high risk areas.
The process-based models employed and adapted existing tree growth models that have already been validated for pine species in Europe. Addition of PWN as a factor compromising the evapotranspiration models has allowed direct prediction of the effects of the nematode using moisture and temperature as key driving variables. Simulations carried out under a range of conditions in Portugal and in the UK as a contrast, indicated that high mortality can be expected in the Setubal region of Portugal, which confirms the situation observed in P. pinaster trees in this area.
The model also predicted when mortality can be expected in relation to month of infestation in the field and, here, there were some surprising predictions in that some trees in the main affected area are predicted to die in the second year after infestation. This has profound implications for survey strategies based on symptomatic trees; some trees may be infested by the nematode but not be declared symptomatic during a winter survey and could be missed.
Current work includes field trials with inoculated standing trees to verify the predictions of the models. This work is ongoing but early results confirm the mortality and timescales arising from PWN infestation of susceptible trees.
In final conclusion, the combined data from this study have advanced knowledge on the interaction of PWN, its vectors, host trees and eco-climatic factors in both a regional and global context. Much has been learned about the PWN system and there are real prospects of using the advanced knowledge to provide customised Pest Risk Analysis predictions of PWN impact in any location in Europe. This will commence with use of the process-model to provide country and region specific predictions of likelihood of wilt expression.