Clavijo-Coppens, F. | Search bioRxiv

bioRxiv2026

Ecology, genomics and biocontrol potential of bacteriophages infecting the bacterial wilt pathogen Ralstonia solanacearum species complex in Reunion Island

The Ralstonia solanacearum species complex (RSSC), the causal agent of bacterial wilt, is among the most destructive soil-borne plant pathogens worldwide, yet effective and sustainable control strategies remain limited. Bacteriophages represent promising biocontrol agents, but their efficacy depends on ecological compatibility with local pathogen populations. Here, we combined ecological sampling, comparative genomics, phenotypic characterization and plant assays to investigate RSSC-infecting phages in Reunion Island and evaluate their biocontrol potential. We isolated 45 phages from diverse agricultural matrices and obtained complete genome sequences for 35 novel isolates. Phylogenomic analyses revealed a locally diversified assemblage comprising multiple known taxa and several putative new genera, forming clusters largely distinct from global reference phages. Phage diversity and antibacterial activity were structured primarily by bacterial phylogeny rather than plant host or geographic origin, indicating that plants act mainly as ecological interfaces while environmental bacterial populations shape phage specialization. The community displayed two contrasting evolutionary strategies: expanding virulent lineages associated with strong antibacterial activity and persistent temperate lineages carrying integration and host-interaction functions. Host-range assays confirmed phylotype-dependent susceptibility, and strictly lytic phages showed consistently higher inhibitory activity. Guided by combined genomic and phenotypic screening, we designed a multi-family phage cocktail targeting dominant local RSSC lineages. The cocktail exhibited strong in vitro suppression of bacterial growth and significantly reduced disease severity in tomato plants. Together, our results demonstrate that effective phage biocontrol depends on evolutionary matching between phages and regional pathogen populations. Integrating ecological, genomic and functional characterization provides a robust framework for selecting locally adapted phages and developing durable phage-based strategies for managing bacterial wilt.

microbiology↗

bioRxiv2025

Genomic footprint of a shared Type 5 prophage in 'Candidatus Liberibacter asiaticus' and 'Candidatus Liberibacter africanus', two destructive bacterial pathogens of citrus huanglongbing

Huanglongbing (HLB) is a bacterial disease that affects citrus trees and is considered the most severe citrus disease in the world. The three HLB-associated Ca. Liberibacter species harbor prophage regions which have been described to play critical roles in their evolution and biology. In this study, we assembled and characterized the accessory genome of the first circular de novo Candidatus Liberibacter asiaticus (CLas) assembly (V1R1) from Reunion, one of the sparse areas worldwide hosting CLas and Ca. Liberibacter africanus (CLaf). This 1,272 Mb-long whole-genome harbored 1,129 coding sequences and two complete prophages, including a 37,934 bp-long Type 1 prophage, frequently present in CLas genomes, and a 40,501 bp-long undescribed CLas prophage designated as P-V1R1-5. Comparative genomic approaches suggested that P-V1R1-5 have all the genetic elements to produce new viral particles and revealed that it likely belongs to a new CLas Type 5 prophage. While being reported for the first time in a CLas strain, P-V1R1-5-like prophages were previously identified in CLaf genomes, making it the first evidence of shared prophage-like sequence among HLB-associated Ca, Liberibacter species. Using PCR amplifications targeting P-V1R1-5, we also showed that 85.7% of the tested CLas strains from Reunion and all tested CLaf strains from Madagascar and Reunion harbored a Type 5-like prophage. The identification of CLas Type 5-like prophages not only expanded our knowledge of CLas genomic diversity in Reunion, but also provided new insights into the role of horizontally transferred elements in the evolution of the sympatric HLB-associated bacteria. IMPORTANCEHuanglongbing (HLB) is the most severe citrus disease worldwide. The disease is associated with three Candidatus Liberibacter species harboring prophage regions that have been described to play critical roles in their biology. In this study, we assembled and characterized the accessory genome of the first circular de novo Ca. Liberibacter asiaticus (CLas) assembly from Reunion, one of the very few areas in the world where both of CLas and Ca. Liberibacter africanus (CLaf) coexist. Comparative genomic approaches demonstrated that this genome harbored two complete prophages, including a new CLas Type 5 prophage that was previously identified in CLaf but was reported for the first time in a CLas strain. This first evidence of shared prophage-like sequences among HLB-associated species expands our knowledge of CLas genomic diversity, but also provides new insights into the role of the accessory genome in the evolution of these bacteria.

genomics↗