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The pinewood nematode (PWN) is the causal agent of pine wilt disease, a pathology that affects conifer forests, mainly Pinus spp. PWN infection can induce the expression of phytohormone-related genes; however, changes at the early phytohormone level have not yet been explored. Phytohormones are low-abundance metabolites, and thus, difficult to quantify. Moreover, most methodologies focus mainly on Arabidopsis or crop species. This work aimed to validate a fast (run time 6.6 min) liquid chromatography-triple quadrupole tandem mass spectrometry (LC-QqQ-MS/MS) analytical method to quantify 14 phytohormones in Pinus pinaster stem tissues. This method was further applied to evaluate, for the first time, early phytohormone changes in susceptible and resistant phenotypes of P. pinaster 24, 48 and 72 h after inoculation (HAI) with PWN. A significant increase in salicylic acid (SA, 48 and 72 HAI) and jasmonic acid methyl ester (JA-ME, 72 HAI) was observed in susceptible phenotypes. Results indicate that the higher susceptibility of P. pinaster to PWN infection might result from an inefficient trigger of hypersensitive responses, with the involvement of JA and SA pathways. This work provides an important update in forest research, and adds to the current knowledge of Pinus spp. defence responses to PWN infection. [ABSTRACT FROM AUTHOR]