Biocontrol with hypocrealean entomopathogenic fungi (EF) is a key tool to develop Integrated Pest Management (IPM) programs for the progressive replacement of synthetic chemical insecticides with more environmentally friendly pest control measures. These fungi stand out among entomopathogens not only for their contact mechanism of infection through the arthropod integument, but also for developing close associations with plants including the endophytic lifestyle and rhizosphere competence that can enable them to make broader contributions to IPM and crop production. Anyhow, the interaction of EF with the plants incorporates multitrophic complexity at different levels including insect pests, plants, and their natural enemies. The aim of the present review was to gather and summarize all available data on multitrophic interactions of EF. These fungi can influence both the chemical ecology of host-plant selection by insect pests and the host or prey selection by parasitoid or predators, respectively. Moreover, EF treatments are compatible with natural enemies in terms of safety and effectiveness, which could allow biocontrol strategies for their synergistic application in IPM programs. A comprehensive understanding of the impact of these multitrophic interactions in longer term, farm-level real-life biocontrol implementation studies will provide new opportunities in plant protection and production.

Entomopathogenic fungi are gaining acceptance in Integrated Pest Management (IPM) systems as effective and environmental safety biological control agents to protect a great variety of crops against pest insects. Many of these insect-pathogenic fungi can establish themselves as endophytes and thereby may induce the plant immune system. The activation of plant defenses by the fungal endophytic colonization can have a direct impact on herbivores and plant pathogens. An integral component of many plant defense responses is also the release of volatile organic compounds, which may serve as an indirect defense by attracting the natural enemies of herbivores. Here we investigated the effect of endophytic colonization by the entomopathogenic fungus Beauveria bassiana on the volatile emission by melon and cotton plants, either unharmed or after being damaged by sap-sucking aphids or leaf chewing caterpillars. We found that when the plants are colonized by B. bassiana they emit a different blend of volatile compounds compared to uncolonized control plants. Some of the emitted compounds have been reported previously to be released in response to herbivory and have been implicated in natural enemy attraction. Several of the compounds are also known to have antimicrobial properties. Therefore, endophytic colonization by B. bassiana might help to not only direct control insect pests but also increase the resistance of plants against agronomically important pests and phytopathogens.

Entomopathogenic fungi are sprayed commercially for aphid control in greenhouses. Recently, their ability to grow endophytically within plants was discovered, offering the opportunity for systemic biological control. Endophytic colonization of host plants could also influence life-table parameters and behavior of herbivores. We investigated lethal and pre-mortality effects of Beauveria bassiana and Metarhizium brunneum on Aphis gossypii; aphids either received inoculum while feeding on recently sprayed leaves (surface inoculum and endophytically-colonized) or while feeding on unsprayed but endophytically-colonized leaves. We used choice assays to identify any preferences for endophytically-colonized or control plants. Volatile emissions from endophytically-colonized plants and control plants were also compared. Aphid mortality rates ranged between 48.2 and 56.9% on sprayed leaves, and between 37.7 and 50.0 on endophytically-colonized leaves. There was a significant effect of endophytic colonization on the rate of nymph production, but this did not result in an overall increase in the aphid population. Endophytic colonization did not influence host-plant selection even though there were qualitative and quantitative differences in the blend of volatiles released by endophytically-colonized and control plants. Although endophytic colonization did not change herbivore behavior, plants still benefit via indirect defense, resistance to plant pathogens or abiotic stress tolerance.

The melon aphid Aphis gossypii is one of the most damaging and economically important crop pests. Entomopathogenic ascomycete (EA) have shown great potential in managing this pest, with several mycoinsecticides available on the market. Recent studies also highlight the systemic efficacy of EA endophytic strains, targeting aphids through the plant and impacting their survival and reproduction. EA endophytic application prevents direct contact between EA and beneficial insects which increases their safety. Indeed, EA endophyte compatibility with other biological control agents, such as entomophagous predators, has been demonstrated under laboratory conditions. However, these results need to be validated using different application methods and under greenhouse conditions. In this study, we evaluated the biocontrol potential of two endophytic EA strains against A. gossypii in melon crops under greenhouse conditions. Based on their known endophytic behavior, Metarhizium brunneum strain EAMa 01/58-Su was applied as a foliar spray while Beauveria bassiana strain EABb 01/33-Su was applied as a seed dressing. Furthermore, the compatibility of these EA applications with the predator Chrysoperla carnea was assessed. Two repetitions were made in a greenhouse in Córdoba (Spain), the first in summer and the second in autumn. It is noteworthy that, in summer, foliar spraying with M. brunneum resulted in 35.0 % aphid mortality and a 20.4 % reduction in total per capita nymph production (TCNP) on sprayed leaves. In contrast, leaves not sprayed with the fungal suspension but endophytically colonized caused 70.0 % aphid mortality and a 12.6 % reduction in TCNP, which demonstrates the systemic potential of this fungus. Compatibility between C. carnea and M. brunneum was also demonstrated; there was a significant increase in prey consumption on endophytically-colonized leaves compared with predation levels observed in control plants. Seed dressing with B. bassiana led to 55.0 % aphid mortality and a 19.2 % reduction in TCNP but had no significant effect on C. carnea predation. The same trends were observed in autumn, although colder temperatures reduced aphid mortality. Our results highlight the potential of fungus-predator biocontrol strategies within an aphid Integrated Pest Management program under greenhouse conditions.

Some strains of entomopathogenic fungi are known to colonize crops endophytically, providing systemic protection against insect pests. This colonization has been revealed to reduce aphid survival and reproductive potential. Understanding the molecular mechanisms underlying these effects could provide insight into what occurs to aphids when they feed on endophytically colonized plants. In this study, an RNA sequencing (RNA-Seq) was conducted for transcriptome analyses of the melon aphid Aphis gossypii and its symbiotic bacteria after feeding on Beauveria bassiana endophytically colonized melon plants. The fungal colonization triggers significant alterations in the aphid transcriptome, reflecting molecular responses to fungal propagules and/or plant-induced systemic resistance. Key changes include the up-regulation of genes involved in host plant recognition by the aphid pest, immune system activation, energy production, and defense mechanisms such as toxin adaptation, detoxification, and cell death markers. Genes associated with escape responses, including alarm pheromone production and wing bud differentiation, were also up-regulated. In contrast, down-regulated genes point to disruptions in homeostasis and neuronal function, further impacting aphid physiology and its symbiotic relationship with Buchnera. These molecular perturbations likely underlie the lethal and sublethal effects observed in aphids feeding on B. bassiana-colonized plants. This study identifies critical pathways involved in aphid-fungal-plant interactions, offering valuable insights into the potential of endophytic entomopathogenic fungi for aphid pest management.

Philaenus spumarius represents the primary vector of the bacterium Xylella fastidiosa in Europe; hence, control measures are directed toward it, with a need for alternatives aligned with the agricultural sustainability criteria. Entomopathogenic ascomycetes (EA) show promise by infecting P. spumarius via endophytic colonization, though their sublethal effects on behavior remain understudied. This study assessed the effects of Metarhizium brunneum strain EAMa 01/58-Su on P. spumarius survival and feeding behavior, using the Electrical Penetration Graph technique. We provide the first evidence of endophytic colonization of olive plants by EA, occurring in both treated and distant untreated leaves. Foliar application enabled the dual establishment of M. brunneum as both epiphytic and endophytic, significantly disrupting vector feeding. Insects fed on M. brunneum sprayed leaves showed a more total and unsuccessful probing events, longer non-probing (np) periods, and reduced xylem contact (Xc) duration. Additionally, successful xylem probing (with xylem ingestion) was significantly delayed. Interestingly, a reduction in waveform N events and their duration in M. brunneum— non-sprayed leaves was detected, suggesting a systemic effect of the fungal colonization on the plant preference by the vector that could ultimately disrupt Xf retention mechanisms. In contrast, the drench application of M. brunneum in the olive saplings did not result in endophytic colonization or significant effects on the insect behavior compared with control plants. These findings provide the basis for future experiments on Xf transmission and support the potential of M. brunneum as a biocontrol agent beyond its conventional use against vector populations on cover crops.