The pinewood nematode (Bursaphelenchus xylophilus), the causal agent of pine wilt disease, remains one of the most critical threats to coniferous forests worldwide, with major ecological, economic, and territorial impacts. In Europe, surveillance relies on large-scale sampling of wood from both forest stands and wood packaging materials, followed by laboratory incubation at 25 °C for 15 days to enhance nematode detection. However, this step introduces significant logistical constraints, particularly in international trade contexts.

This study integrates experimental evidence from complementary research assessing the relevance of incubation across different wood substrates, including heat-treated (HT) pallet wood and fresh material from Pinus pinaster stands. Wood samples were spiked with B. xylophilus at different population densities and life stages (adult pairs and second-stage juveniles, J2), and the influence of key ecological drivers - moisture and fungal presence (Botrytis cinerea) - was also evaluated.

Results showed a clear substrate-dependent response. In HT pallet wood, incubation did not enhance nematode detection and was associated with a drastic reduction in nematode viability. Only under rare conditions involving fungal colonization did treated wood support nematode persistence. In contrast, incubation of green wood samples resulted in exponential population growth, significantly increasing detection probability - particularly relevant under the zero-tolerance policy applied to forest hosts.

Furthermore, no significant differences were observed between life stages, as J2 individuals retained the capacity to develop and reproduce. Although fungal presence and moisture greatly enhanced population growth, these factors were not strictly required for nematode reproduction under favourable conditions.

These findings provide robust scientific evidence to support a revision of phytosanitary protocols, suggesting that incubation may be safely excluded for treated wood packaging materials, while remaining essential for forest-derived samples. This targeted approach improves diagnostic efficiency, reduces operational delays, and reinforces evidence-based forest protection strategies aligned with sustainable management and trade facilitation.