Citrus huanglongbing (HLB), caused by three species of fastidious, phloem-limited ‘Candidatus Liberibacter’, is one of the most destructive diseases of citrus worldwide. To date, there is no established cure for this century-old and yet, newly emerging disease. As a potential control strategy for citrus HLB, 31 antibiotics were screened for effectiveness and phytotoxicity using the optimized graft-based screening system with ‘Candidatus Liberibacter asiaticus’ (Las)-infected citrus scions. Actidione and Oxytetracycline were the most phytotoxic to citrus with less than 10% of scions surviving and growing; therefore, this data was not used in additional analyses. Results of principal component (PCA) and hierarchical clustering analyses (HCA) demonstrated that 29 antibiotics were clustered into 3 groups: highly effective, partly effective, and not effective. In spite of different modes of actions, a number of antibiotics such as, Ampicillin, Carbenicillin, Penicillin, Cefalexin, Rifampicin and Sulfadimethoxine were all highly effective in eliminating or suppressing Candidatus Liberibacter asiaticus indicated by both the lowest Las infection rate and titers of the treated scions and inoculated rootstock. The non-effective group, including 11 antibiotics alone with three controls, such as Amikacin, Cinoxacin, Gentamicin, Kasugamycin, Lincomycin, Neomycin, Polymixin B and Tobramycin, did not eliminate or suppress Las in the tested concentrations, resulting in plants with increased titers of Las. The other 12 antibiotics partly eliminated or suppressed Las in the treated and graft-inoculated plants. The effective and non-phytotoxic antibiotics could be potential candidates for control of citrus HLB, either for the rescue of infected citrus germplasm or for restricted field application.

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.