E. Abdellatif*, A. Rhouma, P. Ferrante, M. Scortichini, J. D. Janse, J. van Vaerenberg, S. Baeyen, B. Bahri, P. Sobiczewski, and M. Kałużna Phylogenetic Relationships of Pseudomonas syringae pv. syringae Isolated from Citrus Blast and Black Pit, Insights into Type III Effector Repertoires and Copper Resistance Genes. Relationships of Pseudomonas syringae pv. syringae

Citrus blast and black pit diseases are the only bacterial diseases reported in the Tunisian citrus orchards. However, there was no study on the bacterial population diversity compared to strains from different origins and hosts. This work provides new information on strains of Pseudomonas syringae pv. syringae (Pss) isolated from trees showing citrus blast and black pit symptoms in Tunisia, which have been characterized previously only by phenotypic features and genetic analysis using fingerprinting methods rep-PCR and PCR MP. In this study, the phylogenetic relationship between Pss strains isolated from citrus in Tunisia was studied based on Multilocus Sequence Typing and Analysis (MLST/MLSA). Five housekeeping genes (rpoD, rpoB, gyrB, cts and pfk) were sequenced for a representative 12 Pss citrus strains and reference citrus strain LMG5496 isolated from Citrus sinensis in 1962 in Greece. Sequence analysis of the five above genes, clearly revealed that all citrus strains studied represented a tight phylogenetic group and that they are closely related to the Pss LMG5496. MLSA analysis showed similar phylogenetic clustering than previous analysis based on cts gene. majority of citrus strains clustered in the Phylogroup02, subgroups “a” and “b” however 5 were placed in a new clade named “f”. Based on two further genes (rpoD and gyrB) and compared to 52 pathotype strains of P. syringae, a robust phylogroup membership has been inferred. To capture the spectrum of the virulence gene repertoire among our strains, we screened 13 representative strains for thirty six TTE families. We determined the presence of six diverse type III-secreted effectors differentiating Pss citrus strains, the model tomato pathogen P. s. pv. tomato strain DC3000, the bean pathogen P. s. pv. phaseolicola Pph1448A and the kiwifruit pathogen P. s. pv. actinidiae 8.43. When studying a more extensive collection of 42 Tunisian Pss strains and the LMG5496, effector repertoires showed to be even more diverse. Frequencies were 100% for hopAN1 and hrpK1; 14% for hopN1, hopR1 and hopQ1-2; and 65% for HopT1-2. Investigation of copper resistance showed that 70% of Pss citrus strains were resistant to copper sulfate in vitro. The copper resistance genes copABCDR were detected in 23% of the strains all of them resistant to CuSO4. Only one strain had additionally the copS gene. Our data collectively presents a better understanding of the disease causal agent and stimulates the need to search for alternative control means to copper bactericidal compounds.