Formulation optimization of D-limonene-loaded nanoemulsions as a natural and efficient biopesticide

D-limonene is an important constituent of citrus fruits, such as oranges and lemons, and can be extracted as a natural essential oil for controlling the agricultural pests because of its antifungal and insecticidal activities. However, the hydrophobicity of D-limonene hinders its full potential and excellent performance. In this research, the effect of emulsifier type, dosage, and emulsification method on the formation and stability of D-limoneneloaded nanoemulsions was investigated by sample appearance, microstructural observations, droplet size measurement, and multiple light scattering analysis. Nanoemulsions prepared with emulsifier (EL-40) exhibited the highest stability, with an appropriate HLB (Hydrophilic Lipophilic Balance) value of 13.5. The stability of nanoemulsions increased at higher emulsifier dosage from 2% to 6%. Also, nanoemulsions fabricated by dropwise addition of aqueous phase (deionized water) into D-limonene as the oil phase premixed with emulsifier were the most stable samples. The optimized formulation of D-limonene-loaded nanoemulsions was determined as: 10 % D-limonene, 6% EL-40, and 84 % deionized water. The quality indicators of this formulation were acceptable in terms of FAO standards. Finally, D-limonene-loaded nanoemulsions could inhibit the growth of various fungal pathogens, including Pyricularia oryzae (rice blast), Rhizoctonia solani (rice sheath blight), Colletortrichum gloeosporiodes (pepper anthracnose), and Phomopsis amygdali (peach shoot blight). This research provides a practical solution for the formulation of stable and effective nanoemulsions containing D-limonene for the control of agricultural crop diseases.