Labbé, Frédéric

ABSTRACT 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 circularde novo‘CandidatusLiberibacter asiaticus’ (CLas) assembly (V1R1) from Réunion, 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 Réunion and all tested CLaf strains from Madagascar and Réunion harbored a Type 5-like prophage. The identification of CLas Type 5-like prophages not only expanded our knowledge of CLas genomic diversity in Réunion, but also provided new insights into the role of horizontally transferred elements in the evolution of the sympatric HLB-associated bacteria. IMPORTANCE Huanglongbing (HLB) is the most severe citrus disease worldwide. The disease is associated with three ‘CandidatusLiberibacter’ 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 circularde novo‘Ca.Liberibacter asiaticus’ (CLas) assembly from Réunion, 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.

ABSTRACTIn an era of trade globalization and climate change, crop pathogens and pests are a genuine threat to food security. The detailed characterization of emerging pathogen populations is a prerequisite for managing invasive species pathways and designing sustainable disease control strategies. Huanglongbing is the disease that causes the most damage to citrus, a crop that ranks #1 worldwide in terms of fruit production. Huanglongbing can be caused by three species of the phloem‐limited alpha‐proteobacterium, “Candidatus Liberibacter,” which are transmitted by psyllids. Two of these bacteria are of highest concern, “Ca. Liberibacter asiaticus” and “Ca. Liberibacter africanus,” and have distinct thermal optima. These pathogens are unculturable, which complicates their high‐throughput genetic characterization. In the present study, we used several genotyping techniques and an extensive sample collection to characterize Ca. Liberibacter populations associated with the emergence of huanglongbing in three French outermost regions of the European Union (Guadeloupe, Martinique and Réunion). The outbreaks were primarily caused by “Ca. Liberibacter asiaticus,” as “Ca. Liberibacter africanus” was only found at a single location in Réunion. We emphasize the low diversity and high genetic relatedness between samples from Guadeloupe and Martinique, which suggests the putative movement of the pathogen between the two islands and/or the independent introduction of closely related strains. These samples were markedly different from the samples from Réunion, where the higher genetic diversity revealed by tandem‐repeat markers suggests that the disease was probably overlooked for years before being officially identified in 2015. We show that “Ca. Liberibacter asiaticus” occurs from sea level to an altitude of 950 m above sea level and lacks spatial structure. This suggests the pathogen's medium‐ to long‐distance movement. We also suggest that backyard trees acted as relays for disease spread. We discuss the implications of population biology data for surveillance and management of this threatful disease.