An efficient antimicrobial screening system for Candidatus Liberibacter asiaticus and other citrus pathogens

Huanglongbing (HLB) is the most devastating citrus disease worldwide, with no resistant commercial citrus varieties identified and no effective chemical treatments currently available on the market. The main challenge in developing effective strategies for controlling HLB is the inability to culture its associated pathogen, the fastidious Candidatus Liberibacter asiaticus (CLas), in defined media or axenic cultures.

In this study, we developed an effective method for screening antimicrobials against CLas using periwinkle as a model plant. Compared with citrus, periwinkle offers notable advantages, including reduced time, increased efficiency, and lower costs. This method consists of two main components: screening exogenously applied antimicrobials and evaluating overexpressed endogenous antimicrobial peptides. Using this method, we demonstrated that bromothalonil and cyproconazole effectively inhibit the proliferation of CLas in hydroponically grown periwinkle. Further analyses revealed that bromothalonil and cyproconazole were also highly toxic to other citrus pathogens, including Xanthomonas citri subsp. citri (Xcc), Alternaria alternata tangerine pathotype (Z7), and Penicillium digitatum (KH8). In addition, we established an Agrobacterium rhizogenes-mediated hairy root genetic transformation system for periwinkle and demonstrated that the proliferation of CLas was significantly inhibited in transgenic roots overexpressing antimicrobial peptide (SAMP).

An efficient antimicrobial screening system for CLas and several other citrus pathogens has been established. By addressing challenges such as high costs, prolonged experimental periods, and heavy workloads, we anticipate that this developed method will serve as a useful tool for the identification of antimicrobials effective against CLas and other citrus pathogens. © 2025 Society of Chemical Industry.