Comprehensive analysis of draft genomes of two closely related Pseudomonas syringae pv. syringae phylogroup 02b strains infecting Citrus plants

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Abstract Pseudomonas syringae pv. syringae ( Pss ) is a cosmopolitan phytopathogen with high genomic plasticity and effector diversity, driving host adaptation and variable virulence. We sequenced, assembled, and annotated the genomes of two Pss strains, LMG 5496 (from Citrus sinensis , Greece, 1962) and EC33 (from C . limon , Tunisia, 2014), to identify genetic determinants underlying their differential virulence. The genomes of LMG 5496 (6.23 Mb; 58.78% GC) and EC33 (5.98 Mb; 58.97% GC) contained 5,563 and 5,312 coding sequences, respectively, and shared highly conserved metabolic and functional profiles. Average Nucleotide Identity (ANI ≈ 100%) and phylogenomic analyses confirmed their close evolutionary relationship within phylogroup 02b. Despite overall similarity, EC33 retained a complete repertoire of Type III secretion system (T3SS) effectors, while LMG 5496 exhibited deletions in two core effectors, correlating with reduced virulence. Functional annotation identified 16 high-confidence T3SS effectors, 12 shared and four strain-specific, reflecting divergent molecular strategies. Enhanced pathogenicity of EC33 was linked to the presence of hypervirulence gene Q4ZSY4 and loss of AvrPph3, suggesting host immune evasion. Both strains possessed largely conserved copper resistance genes, with minor differences in detoxification enzymes. Prophage profiling revealed distinct histories: LMG 5496 carried two intact prophages, EC33 one. This comparative genomic analysis provides new insights into adaptation, phenotypic diversification, and shaping virulence within clonal P . syringae lineages. To our knowledge, this is the first genome-based comparison of Pss strains isolated from Citrus .

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