Support to scientific activities to enhance the efficiency and reliability of surveillance for Pine wood nematode in particular in the buffer zone in Portugal

1.Aim: To determine the relative efficiency of sampling at breast height compared with sampling in the upper trunk and tree canopy.

• Background: The vector beetle has a long flight period which may extend for several months (May to October/November) and during this period feeds in small branches and lays eggs only in the thin bark of the upper trunk and branches, it is expected that, in some trees, mainly at the end of the flight period pine wood nematode will be found only near such higher sections of the tree. In such cases it is possible that sampling at breast height during winter will give a false negative result.
  
  
• Methodology: Work will be done in PWN-infested pine forests in Portugal. During the winter trees, with and without “typical symptoms” will be felled, and an intensive sampling for the presence of PWN and its vector will be done in all parts of the tree, with samples being analysed separately. These data will enable the insects’ and nematode’s abundance and distribution to be related to visual symptoms on the host . The results will determine the relative efficiency of sampling at breast height versus sampling in the upper part of the tree. Expected outcome: Development of a fully quantified sampling regime taking account of the possibility that some PWN infestations may be found only in the upper parts of the tree. This will optimise sampling effort and increase reliability.

2.Aim To assess the reliability of models of expression of pine wilt symptoms developed during the REPHRAME project, in Portugal and Spain.

• Background: The ETpN simulation model developed in REPHRAME predicts where, and to what extent, wilt expression arising from infestation by pine wood nematode can be expected. It has been validated partly by results from Japan and partly from some areas of Portugal. The model now needs to be run for more locations in Portugal and, if resources allow, Spain in order to predict where wilt, latency or no wilt can be expected.
  
  
• Methodology: A detailed database of climatic conditions and observations of wilt expression from Portugal will be created to enable running of the model under a range of environmental and site criteria. This will allow the selection of pine stands in different regions as defined by the model (with different wilting expression expected). Some trees will be selected for breast height versus crown assessment and vector activity. Other trees left to observe symptom development in relation to model validation and latency Expected outcome: Detailed mapping of Portugal with confidence limits based on field observation of actual wilt expression. The information will be used to aid risk-based sampling to detect pine wood nematode in field surveys as part of an improved strategy for survey and management of the pine wood nematode problem in Portugal and Spain.

3.Aim: To evaluate the risk of asymptomatic pines in winter developing pine wilt disease in the following spring in relation to spread of the disease.

• Background: Predictions from the ETpN model and field observations indicate that live trees infested by PWN during vector maturation feeding do not always die in the year of infestation. This latency in the development of visual symptoms can result in surveys under-recording the actual rate of infestation and also could make the trees vulnerable to vector breeding even though the trees look healthy.
  
  
• Methodology: The ETpN model outcomes will be used to select sites for inoculation and observation to determine whether latent expression of wilt takes place. This can be done in late 2015 to select sites for field inoculation and observation throughout 2016 and 2017. Field experiments will be done by inoculation of nematodes in healthy trees in different periods of the year related to expected activity of the vector. Periodically, visual symptoms and resin flow (this will stop or reduce long before the first visual symptoms appear) will be evaluated along with detailed local climatic conditions. Additionally a detailed photographic record will be made from defined positions to provide ‘time lapse’ of any change in tree condition. Two weeks after a tree shows the first visual wilting symptoms, it will be felled and analyzed to detect B. xylophilus and Monochamus presence (oviposition activity). In October of the subsequent year, all remaining inoculated trees without visual symptoms will be felled and analyzed. This will be supplemented by general field observation and sampling of symptomatic and asymptomatic trees in areas predicted to have latent PWN wilt expression. This will provide data to validate and refine the model and also indicate where latency in expression of wilt can be expected. Expected outcome: An increase in knowledge of the likelihood of latently infested trees becoming vulnerable to vector breeding. The outcome will be used to refine sampling regimes, particularly if it is shown that latently infested trees could have successful vector breeding and be missed during spring surveys.

The three topics will provide information for targeting the surveillance and eradication programme to reduce the likelihood of outbreaks of PWN in the Portuguese Active Containment Zone and Buffer Zone.