Huanglongbing (HLB) is an important citrus disease associated with the phloem-limited, uncultured bacterium ‘Candidatus Liberibacter asiaticus’(CLas). Effective treatments against CLas have to be validated in the field, however, methods for the field assessment of treatment effectiveness are time-consuming, in part because DNA-based assays, including quantitative PCR (qPCR), cannot differentiate between live and dead bacterial DNA. The aim of this study was to develop a method for rapid the evaluation of HLB therapies in field experiments. To this aim, a DNA extraction method from citrus leaf tissues with propidum monoazide (PMA), a dye that binds covalently to dsDNA making it unavailable for amplification in subsequent qPCR reactions, was optimized. The results indicated that the efficacy of PMA-qPCR was highly dependent on the primer set used. Primers targeting the 16S region of CLas showed a clear distinction between qPCR from PMA-treated and non-treated samples, while the RNR and LJ900 primers did not show significant differences between the DNA extraction methods. The PMA-qPCR viability analysis of CLas from citrus cuttings treated with different ampicillin (Amp) concentrations showed that all concentrations reduced CLas titers significantly starting 4 days after the initial treatment, unlike the water treatment, which did not show any change. This method was used for assessing the antibacterial activity of Amp, Streptomycin, Oxytetracycline (OTC), and a water control in field tests. The PMA-qPCR results indicated that Amp and OTC displayed significant antibacterial activity against CLas by 8 days post-injection, which was not detected in the non-PMA qPCR analysis. This method could allow the rapid validation of treatments against CLas in field experiments and facilitate the implementation of effective management strategies against HLB.
Citrus Huanglongbing (HLB) or citrus greening, is the most destructive disease for citrus worldwide. It is caused by the psyllid-transmitted, phloem-limited bacteria “Candidatus Liberibacter asiaticus” (CLas). To date, there are still no effective practical strategies for curing citrus HLB. Understanding the mechanisms against CLas can contribute to the development of effective approaches for combatting HLB. However, the unculturable nature of CLas has hindered elucidating mechanisms against CLas. In this review, we summarize the main aspects that contribute to the understanding about the mechanisms against CLas, including (1) CLas virulence targets, focusing on inhibition of virulence genes; (2) activation of citrus host defense genes and metabolites of HLB-tolerant citrus triggered by CLas, and by agents; and (3) we also review the role of citrus microbiome in combatting CLas. Finally, we discuss novel strategies to continue studying mechanisms against CLas and the relationship of above aspects.
Citrus huanglongbing (HLB) is a devastating disease for the citrus industry. The previous studies demonstrated that oxytetracycline and penicillin are effective antibiotics against Candidatus Liberibacter asiaticus (CLas). However, since CLas is uncultured, the mechanisms of action of antibiotics against CLas are still unclear. It was recently reported that the endophytic microbial communities are associated with the progression of citrus HLB after oxytetracycline and penicillin treatment. Therefore, we hypothesize that penicillin has greater antibacterial activity against CLas than oxytetracycline, which may be associated with the alteration of the structure and function of endophytic microbial communities in HLB-affected citrus in response to these antibiotics. To test this hypothesis, the microbiome of HLB-affected citrus leaves treated with these two antibiotics was analyzed using a metagenomic method. Our results indicate that the microbial structure and function in HLB-affected citrus were altered by these two antibiotics. The relative abundance of beneficial bacterial species, including Streptomyces avermitilis and Bradyrhizobium, was higher in penicillin-treated plants compared to those treated with oxytetracycline, and the relative abundance of the bacterial species (such as Propionibacterium acnes and Synechocystis sp PCC 6803) associated with CLas survival was lower for penicillin-treated plants compared to oxytetracycline-treated plants. These results indicate that penicillin has greater antibacterial activity against CLas. Based on the metagenomic analysis, this study elucidated the mechanism for the observed increase in antibacterial activity of penicillin against CLas. The data presented here are not only invaluable for developing eco-friendly and effective biocontrol strategies to combat citrus HLB, but also provide a method for revealing mechanism of antimicrobial against uncultured bacteria in host.