Application Sequence Determines the Compatibility and Virulence of Entomopathogenic Nematodes and Entomopathogenic Fungi Against Spodoptera frugiperda.

PURPOSE: The fall armyworm, Spodoptera frugiperda (J.E. Smith), is a destructive invasive pest causing significant crop losses worldwide. This study evaluated the virulence of two entomopathogenic nematodes (EPNs), Steinernema siamkayai and Heterorhabditis indica, against third-instar S. frugiperda larvae and investigated their compatibility with the entomopathogenic fungi (EPF) Metarhizium anisopliae and Beauveria bassiana under laboratory and pot conditions. METHODS: Virulence of S. siamkayai and H. indica was assessed using different infective juvenile (IJ) concentrations (5-100 IJs larva-1), with larval mortality recorded up to 96 h after inoculation. Probit analysis was performed to estimate LC50 and LC90 values. Compatibility between EPNs and EPF was evaluated through simultaneous and sequential inoculations using larval mortality and co-toxicity factor analyses. The most promising combinations were further validated under pot conditions, where larval mortality and co-toxicity factors were determined at 120 h after treatment. RESULTS: Both EPN species exhibited significant dose- and time-dependent virulence against S. frugiperda. S. siamkayai was consistently more virulent than H. indica, achieving 100% larval mortality at 30-40 IJs larva-1 after 96 h, whereas H. indica required 100 IJs larva-1 for complete mortality. At 96 h, S. siamkayai recorded markedly lower LC₅₀ (5.47 IJs larva⁻1) and LC₉₀ (14.23 IJs larva⁻1) values than H. indica (15.14 and 83.52 IJs larva⁻1, respectively). Simultaneous application of EPNs and EPF produced additive interactions in all combinations. The combination of S. siamkayai + M. anisopliae resulted in the highest laboratory mortality (98.55% at 96 h), whereas sequential application of H. indica after EPF inoculation produced antagonistic interactions. In contrast, S. siamkayai maintained additive compatibility with both EPF irrespective of inoculation sequence. Under pot conditions, S. siamkayai alone caused the highest larval mortality (80.3%), followed by H. indica (70.5%). Among the combined treatments, S. siamkayai + M. anisopliae achieved the greatest mortality (71.5%), and all EPN-EPF combinations exhibited additive interactions based on co-toxicity factor analysis. CONCLUSION: Steinernema siamkayai demonstrated superior virulence and greater compatibility with entomopathogenic fungi than H. indica against S. frugiperda. Simultaneous application of EPN and EPF consistently maintained additive interactions under both laboratory and pot conditions, whereas sequential application adversely affected the performance of H. indica. These findings identify S. siamkayai, particularly in combination with M. anisopliae, as a promising microbial biocontrol strategy for the integrated management of fall armyworm.