Genomic characterization of intercepted Xanthomonas citri subsp. citri strains: implications for plant health surveillance and biosecurity

Abstract

International trade and the movement of plant material are among the main pathways for the introduction of quarantine pathogens into pest-free regions. In this sense, Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus bacterial canker, represents a major phytosanitary concern. According to the European Union (EU) Plant Health Interceptions System (EUROPHYT), 156 Xcc interceptions have been reported in the EU since 2021, approximately 59% associated with citrus fruit imported from Brazil, followed by China, Uruguay and Vietnam. These data highlight the phytosanitary risk linked to international citrus trade. Intercepted Xcc strains provide a valuable opportunity to investigate introduction pathways, genetic diversity and the epidemiology of populations with the potential to establish within the EU. In this study, whole genome sequencing and analysis were performed on 34 Xcc strains intercepted and isolated by the Spanish National Reference Laboratory for Phytopathogenic Bacteria, using third-generation sequencing technologies. Genome assemblies ranged from 5.23 to 5.55 Mb, with highly conserved GC content (64.4-64.8%). Core-genome analysis, based on 113 Xcc strains, identified 3,760 core genes out of a total of 5,939 genes, revealing a clear geographical population structure: all strains intercepted in Spain clustered within an American clade, mainly associated with Brazil, Argentina and Uruguay, whereas most strains intercepted in the Netherlands grouped within an Asian clade. Five circular plasmids were identified, including three megaplasmids (213,775 271,617 bp) and two additional plasmids (64,920 bp and 34,216 bp). Functional analysis of these plasmids revealed two distinct groups: a virulence-associated group, present in all strains, and a heavy-metal resistance group, particularly associated with copper resistance (including the cop operon and several efflux systems), restricted to the three strains carrying megaplasmids. Both groups also harboured mobility and transfer genes, indicating that these plasmids have the potential to spread horizontally between strains. This association between megaplasmids and resistance was confirmed by phenotypic copper sensitivity assays. Overall, these results demonstrate that intercepted strains constitute a valuable resource for phytosanitary surveillance, enabling the tracing of the origin and dissemination of Xcc lineages and the identification of adaptive mobile genetic elements, such as copper-resistance megaplasmids, which can pose an additional challenge for disease management. These findings underscore the importance of integrating advanced genomic tools into plant health surveillance and biosecurity programs, strengthening prevention strategies against EU quarantine organisms.