Maloney, Katherine N.

ABSTRACT Huanglongbing (HLB) is a devastating and incurable citrus disease associated with the gram-negative, phloem-limited, and unculturable bacterium “ Candidatus Liberibacter asiaticus” ( C Las), transmitted by the psyllid Diaphorina citri . Our goal was to identify high-performing microbial candidates from a previously established citrus-associated microbial collection for the management of HLB and associated root pathogens. Candidate selection was based on bioactivity, genomic, and metabolomic profiles. Eighteen Bacillus isolates were screened for antagonistic activity against Liberibacter crescens , C Las, and citrus-associated Phytophthora and Fusarium spp. Phylogenetic analysis grouped the isolates into four species groups: Bacillus velezensis , B. subtilis , B. pumilus , and B. cereus . Among these, B. velezensis exhibited the strongest and broadest inhibitory activity, followed by B. subtilis . This enhanced antagonistic potential is likely associated with the greater number and diversity of biosynthetic gene clusters (BGCs) identified. Notably, C Las-inhibitory isolates included B. velezensis (CB0021 and CB0909) and B. altitudinis (CB0086). Bacilysin and surfactin BGCs were conserved across all C Las-inhibitory isolates, suggesting a potential role in C Las suppression. Accordingly, surfactins were detected in bioactive fractions of B. velezensis (CB0027 and CB0909) and B. subtilis (CB0893), supporting their likely contribution to bioactivity and inhibiting L. crescens growth in vitro , highlighting their potential as candidate compounds for C Las management. Overall, this study underscores the importance of microbial discovery, screening, and characterization as an effective strategy for identifying sustainable solutions to manage HLB. IMPORTANCE Huanglongbing (HLB) is a severe disease of citrus that has altered the citrus landscape worldwide and currently has no cure. It is caused by the bacterium “ Candidatus Liberibacter asiaticus” ( C Las) and transmitted by the psyllid Diaphorina citri . Currently, the most effective control strategy for this disease relies on trunk injection of antibiotics oxytetracycline and streptomycin, and there is a need to find alternative solutions to complement or replace chemical control. Here, we aimed to select high-performing candidates from our previously established citrus-associated microbial collection to manage HLB and root-associated pathogens. We screened 18 Bacillus isolates for antagonistic activity against Liberibacter crescens , C Las, and citrus-associated Phytophthora and Fusarium spp., and we generated their genomic and metabolomic profiles. Our research centers on selecting microbial candidates for their bioactivity and antimicrobial metabolite production, aiming to develop effective plant-protective bioinoculants for mitigation of HLB.

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.

ABSTRACT Huanglongbing (HLB) is a devastating citrus disease associated with the gram-negative, phloem-limited, and unculturable bacterium “ Candidatus Liberibacter asiaticus ( C Las),” which is transmitted by the Asian citrus psyllid Diaphorina citri . Despite extensive research, effective, long-term, and sustainable solutions for managing HLB remain elusive. Oxytetracycline (OTC) is currently used as an emergency measure, but there is an urgent need for alternative compounds to complement or replace OTC. In this study, we identified amicoumacins, a class of antimicrobial compounds produced by the bacterium Bacillus safensis CB729 isolated from the citrus microbiome, and demonstrated their ability to suppress C Las. Genome mining of B. safensis CB729, combined with metabolomic analysis and bioassay-guided fractionation, revealed the presence of amicoumacins and related derivatives in fractions inhibitory to Liberibacter crescens , a culturable surrogate for C Las. We tested commercially available synthetic amicoumacins A and B, along with a B. safensis -derived amicoumacin mixture, against L. crescens and C Las. We determined the MICs of amicoumacin A (1.25 µg/mL) and amicoumacin B (10 µg/mL) against L. crescens . Furthermore, amicoumacin B and the amicoumacin mixture significantly reduced C Las populations in ex vivo citrus hairy root assays. This study highlights the potential of amicoumacins as a promising group of natural products for the management of HLB, offering valuable insights for the development of novel and sustainable disease control strategies. IMPORTANCE For two decades, the citrus industry has been severely impacted by Huanglongbing (HLB), a devastating disease caused by “ Candidatus Liberibacter asiaticus ( C Las)” and transmitted by the Asian citrus psyllid ( Diaphorina citri ). Despite extensive research, effective, long-term, and sustainable solutions remain unavailable for growers. Currently, medically relevant antibiotics, such as oxytetracycline (OTC), are used as an emergency response to combat HLB in Florida, the most affected citrus-producing state in the U.S. This underscores the urgent need for alternative treatments that can be used in rotation or as replacements for OTC. Here, we present amicoumacins, a group of bioactive secondary metabolites with antibiotic properties. We identified amicoumacin B and its derivatives from the culture broth of a Bacillus safensis isolate, native to citrus, and demonstrated their ability to inhibit Liberibacter spp. and reduce C Las populations in citrus tissue. This study highlights how microbial discovery can lead to the identification of antimicrobial compounds with potential applications in plant disease management.

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 .