Simultaneous exposure of nematophagous fungi, entomopathogenic nematodes and entomopathogenic fungi can modulate belowground insect pest control.

Entomopathogenic nematodes (EPNs) and fungi (EPF) are well known biological control agents (BCAs) against insect pests. Similarly, the nematophagous fungi (NF) are considered good BCA candidates for controlling plant parasitic nematodes. Because NF can employ EPNs as food and interact with EPF, we speculate that the simultaneous application of EPNs and EPF might result in higher insect mortality, whereas the triple species combination with NF will reduce the EPN and EPF activity by predation or inhibition. Here we evaluated single, dual (EPN + EPF, EPF + NF, EPN + NF) and triple (EPN + EPF + NF) combinations of one EPN, Steinernema feltiae (Rhabditida: Steinernematidae), one EPF, Beauveria bassiana (Hypocreales: Clavicipitaceae), and two NF, Arthrobotrys musiformis (Orbiliales: Orbiliaceae) and Purpureocillium lilacinum (Hypocreales: Ophiocordycipitaceae) under laboratory conditions. First, we showed that EPF reduced the growth rate of NF and vice versa when combined in both rich and limiting media, suggesting a negative interaction when combining both fungi. Three different fungal applications (contact with mycelia-conidia, immersion in conidial suspension, and injection of conidial suspension) were tested in single, dual and triple species combinations, evaluating Galleria mellonella (Lepidoptera: Pyralidae) larval mortality and time to kill. When mycelia was presented, the EPF appeared to be the dominant in combined treatments, whereas in immersion exposure was the EPN. In both types of exposure, NF alone did not produce any effect on larvae. However, when A. musiformis was injected, it produced larval mortalities >70% in the same time span as EPN. Overall, additive effects dominated the dual and triple combinations, with the exception of injection method, where synergisms occurred for both NF species combined with EPN + EPF. This study illustrates how differences in species combination and timing of fungal arrival can modulate the action of BCAs when augmented in the soil. Further studies are required to fine-tune these multitrophic interactions to provide successful, sustainable and resilient pest management in agroecosystems.