Drozd, Christopher

ABSTRACT Plant microbiomes are a rich source of secondary metabolites, often with pathogen- or disease-suppressive traits. Huanglongbing (HLB) is a serious disease of citrus for which there is currently no cure. Here, we tested the hypothesis that bacteria native to the citrus microbiome may possess inhibitory activity against the associated causal agent of HLB, Candidatus Liberibacter asiaticus ( C Las). We coupled a robotic microfluidic isolation platform with a high-throughput antimicrobial screening assay using Liberibacter crescens , a culturable surrogate for the unculturable C Las, to identify isolates with anti- Liberibacter activity. We identified 35 bacterial isolates with activity using this pipeline. One strongly inhibitory citrus root isolate, Pseudomonas chlororaphis (SRAM11), was selected for bioassay-guided fractionation. We determined a significant driver of in vitro Liberibacter inhibition was attributed to the production of phenazine-1-carboxamide (PCN) by P. chlororaphis . We also demonstrated significant anti- C Las activity of PCN, comparable to that of oxytetracycline, using an ex vivo citrus hairy root assay. P. chlororaphis and phenazines have been implicated in successful biocontrol of other diseases and represent a new tool with possible application to HLB mitigation. IMPORTANCE Huanglongbing (HLB) is a devastating citrus disease that causes millions of dollars in crop loss each year in the United States alone. In this study, we mined the citrus microbiome for bacteria that produce antimicrobials with suppressive activity against Candidatus Liberibacter asiaticus, the causal agent of HLB. We demonstrated that the citrus microbiome is a rich source of microorganisms that inhibit Liberibacter spp. We selected a Pseudomonas chlororaphis strain for further characterization and assigned anti- Liberibacter activity to the production of phenazine-1-carboxamide. Phenazine-producing P. chlororaphis strains are known biocontrols in other pathosystems, and this study highlights their potential for mitigation of HLB.

Globally, citrus is threatened by huanglongbing (HLB), and the lack of effective control measures is a major concern of farmers, markets, and consumers. There is compelling evidence that plant health is a function of the activities of the plant's associated microbiome. Using Liberibacter crescens , a culturable surrogate for the unculturable HLB-associated bacterium “ Candidatus Liberibacter asiaticus,” we tested the hypothesis that members of the citrus microbiome produce potential anti-“ Ca . Liberibacter asiaticus” natural products with potential anti-“ Ca . Liberibacter asiaticus” activity. A subset of isolates obtained from the microbiome inhibited L. crescens growth in an agar diffusion inhibition assay. Further fractionation experiments linked the inhibitory activity of the fungus Cladosporium cladosporioides to the fungus-produced natural products cladosporols A, C, and D, demonstrating dose-dependent antagonism to L. crescens .