As primary producers, plants play a central role in mediating interactions across trophic levels. Although plants are the primary food source for herbivorous insects, they can protect themselves from herbivore damage. Many plants produce toxic compounds that directly reduce herbivore feeding, but plants also protect themselves indirectly by attracting natural enemies of the attacking herbivore through volatile signaling. These so-called tritrophic interactions have historically been documented aboveground in aerial plant parts but are also known to occur belowground in root systems. In addition to herbivores, plants directly interact with other organisms, which can influence the outcomes of tri-trophic interactions. Arbuscular mycorrhizal fungi (AMF) are symbiotic soil microbes that colonize the roots of plants and facilitate nutrient uptake. These microbes can alter plant chemistry and subsequent resistance to herbivores. Few studies, however, have shown how AMF affect tri-trophic interactions above- or belowground. This study examines how AMF colonization affects the emission of root volatiles when plants are under attack by western corn rootworm, a problematic pest of corn, and subsequent attraction of entomopathogenic nematodes, a natural enemy of western corn rootworm. Mycorrhizal fungi increased rootworm survival but decreased larval weight. Differences were detected across root volatile profiles, but there was not a clear link between volatile signaling and nematode behavior. Nematodes were more attracted to non-mycorrhizal plants without rootworms and AMF alone in soil, suggesting that AMF may interfere with cues that are used in combination with volatiles which nematodes use to locate prey.

We explored the activation of defense genes and the changes in volatile profiles in olive (Olea europaea var. Picual) plants subjected to mechanical wounding and prior soil inoculation with the fungus Trichoderma afroharzianum T22. Our findings indicate a sustained effect of the inoculant in olive plants, which shifted the constitutive volatile emission more significantly towards an aldehyde-dominated blend than the mechanical damage alone. Furthermore, we found that wounding alone did not alter the expression of hydroperoxide lyase genes associated with aldehyde biosynthesis. However, this expression was significantly enhanced when combined with prior T22 inoculation. Mechanical wounding amplified the plant's immediate defensive response by enhancing the upregulation of the direct defense enzyme acetone cyanohydrin lyase. Trichoderma afroharzianum T22 also modulated direct defense, although to a lesser extent, and its effect persisted 9 months after inoculation. Metagenomic analyses revealed that aerial mechanical damage did influence specific root bacterial functions. Specifically, an upregulation of predicted bacterial functions related to various metabolic processes, including responses to biotic and abiotic stresses, was observed. On the contrary, T22's impact on bacterial functional traits was minor and/or transient.