Potential Advantages of Infrared Spectroscopy in Early Diagnosis of Pine Wilt Disease

Early diagnosis technology is crucial for early warning and control of pine wilt disease (PWD), a devastating forest disease. However, current methods rely largely on external symptoms and are infective during the asymptomatic stage. This study employed 3-year-old Pinus thunbergii seedlings as the experimental materials to compare the timing of anomaly detection in needle water content, relative conductivity (RC), and ATR-FTIR spectroscopy following inoculation with the pine wood nematode (PWN). The feasibility of applying infrared spectroscopy for early PWD diagnosis was further validated through negative controls and chemometric models. The results revealed an approximately two-week asymptomatic diagnostic window following infection. ATR-FTIR spectroscopy enabled earlier detection than measurements of needle water content or relative conductivity. Furthermore, ATR-FTIR spectroscopy distinguished nematode stress from drought stress based on differential trends at 2908 and 2840 cm⁻¹ and in full-spectrum integrated area. Derivative spectra further revealed multiple early-stage characteristics. Exploratory analysis of all stages using PCA and PLS-DA indicated that simultaneous multi-stage discrimination was not feasible due to weak early-stage signals and severe overlap between Stage 0 and Stage I (test-set accuracy: 30.5%). Focusing specially on Stage 0 and Stage I to construct an OPLS-DA model, test-set performance improved to an accuracy of 85.2%, a sensitivity of 85.7%, a specificity of 84.6%, and an AUC of 0.879 (95% CI: 0.741, 0.992). The permutation test was statistically significant (p = 0.0050). VIP analysis identified nine markers with scores ≥ 1.0 indicating that PC1 and raw spectral features drove discrimination synergistically. Univariate testing revealed that only A11_Peak_1278_existence was statistically significant confirming that Stage I was characterized by the superposition of weak multivariate signals. In summary, coordinated changes across multiple peaks and the distinctive decrease-increase-stabilize-decrease pattern observed in certain peaks constitute a unique spectral signature of PWD. Infrared spectroscopy can detect subtle anomalies in 3-year-old P. thunbergii seedlings infected with pine wood nematode while needles remain asymptomatic, water content changes are still insignificant, and cell membrane function has not yet been extensively compromised. This finding demonstrates significant potential for early PWD diagnosis.