Characterization of the pinewood nematode, bursaphelenchus xylophilus-pinus system in Portugal: phytochemical, histopathological, molecular, and biotechnological approaches

The pinewood nematode, Bursaphelenchus xylophilus, is one of the main threats affecting Portuguese maritime pine (Pinus pinaster). Several research teams have joined efforts to better understand the plant-nematode system. Over 150 essential oils (EO), as well as several EO fractions and decoction waters have been evaluated, Ruta graveolens, Satureja montana, Thymbra capitata, Thymus pulegioides, and T. vulgaris EOs being the most nematotoxic. Two-year-old P. pinaster, P. pinea, P. sylvestris, and P. halepensis were inoculated with a virulent PWN Portuguese isolate, and comparatively evaluated with non-inoculated and wounded plants to understand the plant-nematode interaction and the role of plant volatiles. Histological studies showed that the number of nematodes increased in P. pinaster and P. sylvestris with disease progression, and 7 weeks after inoculation all pine tissues were severely damaged. PWN distribution in P. pinea and P. halepensis was nearly restricted to the inoculated area; no clear change was observed in the stem tissues. Pine species volatiles showed the existence of chemotypes in some cases. Key volatile organic compounds, such as 4-hexen-1-ol, involved in P. pinaster response against the nematode have also been identified using non-destructive methods, with the potential to be used as biomarkers for early detection of infected trees. In vitro co-cultures of the host with parasite have also been established as a biotechnological tool to evaluate the effect of nematotoxic addition and assess their phytotoxicity to the host. Molecular approaches have addressed the changes in α-pinene synthase gene expression in susceptible P. pinaster and non-susceptible P. pinea, following nematode invasion. Preliminary results showed an increased expression of this gene in P. pinea, contrary to P. pinaster which revealed the same expression level in infected and non-infected controls.