A high-throughput pipeline for isolation and screening of the citrus bacteriome identifies
Pseudomonas chlororaphis
with anti-
Candidatus
Liberibacter asiaticus activity
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.