Effective antibiotics against 'Candidatus Liberibacter asiaticus' in HLB-affected citrus plants identified via the graft-based evaluation

Abstract

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.

Figure 1. Huanglongbing (HLB)-affected grapefruit (‘Duncan’) plants with graft-inoculation of Las-infected lemon scions treated with different antibiotics.

I-CAR: Highly effective compounds; II-RIF: Partly effective compounds; III-KSM: non-effective compounds; CK: water control. Photograph was taken 6 months after graft inoculation. Typical HLB symptom of leaf curl and corky veins on grapefruit rootstock and blotchy mottle or yellow shoots on leaves of lemon scion were apparent.

Figure 2. Principal component analysis (PCA) plots.

A) Cumulative variance; B) Loading plots for different variables on PC₁ (antibacterial activity) and PC₂ (phytotoxicity). CTPr: Ct before treatment of infected scion; CTS-1 and CTR-1: Ct value in the scion and rootstock of the graft-inoculated at DAT 120, respectively; CTS-2 and CTR-2: Ct value in the scion and rootstock of the graft-inoculated at DAT 180, respectively; SI36 and LT36: Scion infection percentage (SI) and Las transmission rate (LT) were calculated when Ct value was cut off at 36.0, respectively; SS and SG: Scion survival and scion growth, respectively; C) PCA scores plot for different antibiotics on PC₁ (antibacterial activity) and PC₂ (phytotoxicity).

Figure 3. Dendrogram of the hierarchical cluster analysis for antibacterial activity against Las with 29 antibiotics.