Effects of Temperature-Moisture Interactions on Storage Survival and Virulence in Two Entomopathogenic Nematode Species

Entomopathogenic nematodes (EPNs) are among the most promising biocontrol agents; however, their short shelf life constrains commercial application. This study evaluated the effects of storage temperature (6 °C and 25 °C) and sponge substrate moisture content (42%, 48%, and 55%) on the 18-week survival and post-storage virulence of two indigenous EPN species from Gansu Province, China: Heterorhabditis megidis 0627M and Steinernema feltiae 0619HT. A Generalized Linear Mixed Model (GLMM) revealed that storage duration, temperature, moisture content, and species all significantly affected survival of H. megidis 0627M and S. feltiae 0619HT (all p < 0.001). Low-temperature storage (6 °C) reduced mortality odds by 82.4% compared with room temperature (OR = 0.176, 95% CI: 0.117–0.263, p < 0.001). High moisture content (55%) increased mortality odds by 10.1-fold relative to moderate moisture (48%; OR = 10.114, 95% CI: 6.155–16.618, p < 0.001), whereas low moisture (42%) showed no significant difference from 48% (OR = 0.942, p = 0.810). The two species exhibited distinct adaptation strategies: S. feltiae 0619HT achieved the highest survival under low-temperature storage at 48% moisture content (56.48% at week 18), whereas H. megidis 0627M demonstrated a delayed competitive advantage under room-temperature, low-moisture conditions. Virulence assays revealed that low-temperature storage better preserved infectivity under most conditions. Notably, survival and virulence were not always concordant, necessitating their evaluation as complementary metrics. Species-specific storage protocols are proposed, providing a scientific basis for the future development of regionally targeted and cost-effective native EPN formulations, as well as for regionally targeted biocontrol applications.