Turpin, Patrick

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, using infected insect vectors, we assembled and characterized the accessory genome of the first circular de novo “ Candidatus Liberibacter 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 has all the genetic elements to produce new viral particles and revealed that it likely belongs to a new CLas Type 5 prophage. While P-V1R1-5-like prophages had been identified previously in CLaf genomes, our study demonstrates the first lysogenic conversion of these prophages in CLas, suggesting a positive selection of prophage-like sequences 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 “ Candidatus Liberibacter” species harboring prophage regions that have been described to play critical roles in their biology. In this study, using infected insect vectors, we assembled and characterized the accessory genome of the first circular de 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.

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.

AbstractPlant pathogens can alter the behavior of their insect vectors as well as their survival and reproduction. The African psyllid, Trioza erytreae, is one of the vectors of Huanglongbing, a citrus disease caused mainly by “Candidatus Liberibacter asiaticus” (CLas). The purpose of this study was to characterize the effects of CLas on the psyllid, T. erytreae using Citrus volkamerina plants as the study system. The study focused more specifically on the CLas effects prior to and after its acquisition by the psyllid T. erytreae. Our results did not support the hypothesis that CLas effects psyllid probing behavior prior to acquisition; few differences were observed between uninfected T. erytrea feeding on CLas‐infected versus control plants. On the other hand, compared to psyllids that had completed their development on control plants, the ones that had completed their development on a CLas‐infected plant exhibited changes in their behavior (greater velocity), physiology (smaller mass) and biochemistry (lower water and lipid content). Altogether, our results confirm the existence of a marked postacquisition effect on the vector locomotor behavior and a minor preacquisition effect of CLas on the vector behavior, which can be partially explained by physiological and biochemical changes.

IntroductionHuanglonbing (HLB) is the most serious disease of citrus in the world, associated with three non-cultivable phloem-restricted bacteria Candidatus Liberibacter asiaticus (CLas), Ca L. africanus (CLaf) and Ca L. americanus (CLam). CLas is transmitted by the Asian citrus psyllid Diaphorina citri, and has spread to several countries. The African psyllid Trioza erytreae, the vector of CLaf occurs in Africa and neighbouring islands. Only two major citrus-growing regions - Australia/New Zealand and the Mediterranean Basin - are still HLB-free in the world. However, T. erytreae has recently been introduced into continental Europe (Portugal and Spain) and has become a potential threat to citrus production. The transmission of CLas by T. erytreae had been postulated but never tested. To evaluate the risk of T. erytreae transmitting CLas, comparative transmissions of CLas by T. erytreae and D. citri were assessed.MethodsTransmission tests were performed on excised leaves and seedlings of Citrus volkameriana with different inoculation access periods (in series) for both insect species. Quantifications of bacterial titers were made in excised leaves, seedlings three and six months after inoculation and on individual insects.ResultsOur results showed that T. erytreae was able to efficiently acquire CLas. Furthermore, T. erytreae carried significantly higher bacterial titers than D. citri, and was able to efficiently transmit the bacteria to seedlings at a similar rate that D. citri highlighting the high risk of spread of the most aggressive variant of HLB (CLas) by T. erytreae in Europe.DiscussionThus, extreme precautions to prevent any entry of CLas into Europe should be adopted.