Chemical diversity analysis identifies inhibitory compounds in the secretomes of citrus-associated Bacillus spp. to the Huanglongbing pathogen, “ Candidatus Liberibacter asiaticus,” and citrus root pathogens

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.