Simple SummaryThis study aimed to explore the effectiveness of a new microbial formulation that utilizes the entomopathogenic fungus,Metarhizium anisopliae, in controlling the red palm weevil (RPW),Rhynchophorus ferrugineus, which is the most destructive insect pest of major cultivated palms. Previously, we published the development of an oil-emulsion formulation system to enhance the biological performance of this particular fungal isolate. The system improved the stability, replicability, shelf life, and resistance to heat stress and UV irradiation. The current results reveal that the formulation has improved fungal bioefficacy and pathogenicity through direct and indirect contact bioassays. The lethal infection of formulated conidia against RPW was more effective and dispersed better than the aqueous suspension of non-formulated conidia. The spread of disease can be observed from a treated individual to a healthy RPW upon contact, which explains the disease-spreading ability of formulated conidia. Additionally, the infections were confirmed through both morphological analysis and DNA sequencing. The results showed that the RPWs were exclusively infected byM. anisopliaethroughout the experiments. Therefore, this oil-emulsion formulated conidia could be incorporated into current integrated pest management programs to achieve more promising biocontrol outcomes in the long-term application of sustainable agricultural practices.AbstractThe red palm weevil (RPW),Rhynchophorus ferrugineus, poses a severe threat to agro-industrial crops, particularly major cultivated palm species. Infestations result in economic losses due to reduced fruit quality and yield. The entomopathogenic fungus,Metarhizium anisopliae, has shown promise as a potential biocontrol agent against the RPW. However, the use of an emulsion formulation ofM. anisopliaefor managing this serious insect pest has yet to be fully explored. The oil-emulsion formulation containing this entomopathogen may enhance the conidia’s stability, prolong its lifetime, and reduce the impact of heat stress or UV irradiation on the fungus. Therefore, this study aimed to investigate the bioefficacy of a new oil-in-glycerol emulsion formulation on mycoinsecticidal activity against RPW adults by direct and indirect bioassays. Results showed that conidia concentration was directly proportional to the RPW mortality percentage. The LT50of 8.183 days was achieved by the conidial formulation against RPW, with a significantly lower LC50(1.910 × 105conidia mL−1) compared to the aqueous conidia suspension (LT50= 8.716 days; LC50= 7.671 × 105conidia mL−1). Indirect bioassays revealed that the oil-in-glycerol emulsion had a disease-spreading ability that resulted in up to 56.67% RPW mortality. A zero E-value reading indicates that the DNA sequence being studied is highly similar to that of the fungal speciesM. anisopliae, which has been identified in the NCBI database. Although the new emulsion formulation has improved the efficacy and pathogenicity ofM. anisopliaein vitro, it is important to also consider the fungal pathogen’s compatibility with other agricultural practices to prevent any loss of control efficiency in the actual usage environment.