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.

Bacterial disease management: challenges, experience, innovation and future prospects

“…Multiple NP delivery systems, such as hydrogel, dentrimers, liposomes, carbon nanotubes, micelles, and micro‐ and nanoemulsions, have been studied for the delivery of various active ingredients (De et al ., ). Among them, nanoemulsions have shown promising delivery of an agriculturally important herbicide (Lim et al ., ), and a nanoemulsion formulation was able to enhance the permeability of the antibiotic ampicillin through the citrus cuticle into the phloem via a foliar spray targeted against Huanglongbing disease (Yang et al ., ). As the plant cuticle (wax, cutin and pectin) acts as the major barrier preventing antimicrobial compounds from penetrating into plant tissues, and as many plant‐pathogenic bacteria infect the phloem and xylem tissues, the development of antimicrobial delivery technology which penetrates through the cuticle may have wide applications in the control of bacterial plant diseases.…”

Bio-Based Nanoemulsion Formulations Applicable in Agriculture, Medicine, and Food Industry

“…It could be mentioned that NE containing extract did not induce effect against acetylcholinesterase or mortality in mice. In order to improve the permeability and efficiency of delivery of antibacterial (ampicillin) into the citrus phloem by foliar spray Yang et al (2015) designed a NE using eight adjuvants. Among the adjuvants tested, Brij 35 showed the highest increases (3.33-fold) in the cuticular permeability when compared with control (water).…”

A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation

“…One past project aimed to characterize the optimum parameters for trunk injection of oxytetracycline to achieve uniform antibiotic distribution in planta, and improve tree health, yield and juice quality Project #773). Two past projects focused on enhancing the efficacy of antimicrobials by combining them with adjuvants or delivering them as a nanoemulsion (Yang et al, 2015. "Soft" or non-clumping nanoparticles (SNP) are being explored as vehicles for foliar application of natural biocides to infected trees Projects #771, 909).…”

“…identified optimum parameters for trunk injection of oxytetracycline to achieve uniform antibiotic distribution in planta, thereby enhancing tree productivity . Others showed improved outcomes by adding adjuvants, creating nanoemulsion formulations (Yang et al, 2015, or using "soft" or non-clumping nanoparticles . Moudgil and coworkers (2014) are working to develop SNP nanoemulsions of two essential oils, EO A and EO B, that effectively inhibit bacterial growth.…”