Antimicrobial Nanoemulsion Formulation with Improved Penetration of Foliar Spray through Citrus Leaf Cuticles to Control Citrus Huanglongbing

Abstract

Huanglongbing (HLB) is the most serious disease affecting the citrus industry worldwide to date. The causal agent, Candidatus Liberibacter asiaticus (Las), resides in citrus phloem, which makes it difficult to effectively treat with chemical compounds. In this study, a transcuticular nanoemulsion formulation was developed to enhance the permeation of an effective antimicrobial compound (ampicillin; Amp) against HLB disease through the citrus cuticle into the phloem via a foliar spray. The results demonstrated that efficiency of cuticle isolation using an enzymatic method (pectinase and cellulase) was dependent on the citrus cultivar and Las-infection, and it was more difficult to isolate cuticles from valencia orange (Citrus sinensis) and HLB-symptomatic leaves. Of eight adjuvants tested, Brij 35 provided the greatest increase in permeability of the HLB-affected cuticle with a 3.33-fold enhancement of cuticular permeability over water control. An in vitro assay using Bacillus subtilis showed that nanoemulsion formulations containing Amp (droplets size = 5.26 ± 0.04 nm and 94 ± 1.48 nm) coupled with Brij 35 resulted in greater inhibitory zone diameters (5.75 mm and 6.66 mm) compared to those of Brij 35 (4.34 mm) and Amp solution (2.83 mm) alone. Furthermore, the nanoemulsion formulations eliminated Las bacteria in HLB-affected citrus in planta more efficiently than controls. Our study shows that a water in oil (W/O) nanoemulsion formulation may provide a useful model for the effective delivery of chemical compounds into citrus phloem via a foliar spray for controlling citrus HLB.

Fig 1. Inhibitory zone diameter of nano-formulations of Nano-1-Amp+Brij 35 and Nano-2-Amp+Brij 35.

All data were analyzed by Duncan’s multiple range tests using SAS software package. Different letters represented significantly differences at the level of 0.05 (p ≤ 0.05).

Fig 2. Ct value detected by qPCR in HLB-affected citrus after foliar spraying by nano-formulations.

Fig 3. HLB-affected citrus treated by nano-formulations.

(A): Amp (left) vs. tap water (CK) (right); (B): Nano-1-Amp+Brij 35 (left) vs. tap water (CK) (right); (C): Nano-2-Amp+Brij 35 (left) vs. tap water (CK) (right).