In vivo volatile organic compounds analysis in pinus pinaster after infection with bursaphelenchus xylophilus

The pine wilt disease (PWD), caused by the nematode Bursaphelenchus xylophilus, is devastating for Pinus pinaster plantations, leading to the loss of thousands of trees every year worldwide. Despite the important research efforts of the past decades, no effective strategies against the PWN have been developed and the physiological aspects related to disease resistance remain largely unknown. In this work, 1-yr-old P. pinaster plants were inoculated with ca. 1000 B. xylophilus nematodes or with water (controls). Three and 21 days post inoculation (dpi), volatile organic compounds (VOCs) were analysed through a non-invasive methodology: VOCs were concentrated in a personalized acrylic chamber for 60 min and subsequently collected with a DVB/CAR/PDMS fibre for 30 min, after which they were identified by gas chromatography/ mass spectrometry (GC/MS). An additional set of inoculated plants was sacrificed three and 21 dpi for total stem nematode counting. Results showed that nematodes successfully reproduced throughout the experimental period, reaching ca. 3000 nematodes per plant just 21 dpi. Compared with control plants, total VOCs, especially monoterpenes, significantly increased three dpi in inoculated plants, probably as part of P. pinaster chemical defence mechanisms against the pathogen. VOCs dramatically decreased 21 dpi, most likely due to tissue damage induced by B. xylophilus, demonstrating the susceptible character of P. pinaster. At 3 dpi, there was increased biosynthesis of almost all VOCs, specially α-pinene and ß-myrcene (ca. 4-fold), known to accumulate in leaf tissues to repel herbivores and pathogens. Sabinene and 3-carene also increased significantly in infected plants (4.1- and 5-fold, respectively), 21 dpi, which suggests that the biosynthesis of different defence-related VOCS is triggered at different stages of the disease. 4-hexen- 1-ol, a known allelopathic agent, was only found in inoculated plants, perhaps as a strategy to attract B. xylophilus predators. These findings could be of utmost importance for the precocious diagnostic of the PWD.