Pan-genome and antibiotic resistance insights into Xanthomonas citri pv. punicae pathotypes

Bacterial blight, caused by Xanthomonas citri pv. punicae (Xcp) is a significant threat to pomegranate cultivation in India. Although antibiotics such as streptomycin, streptocycline, and tetracycline are commonly used for its management, their effectiveness is declining due to the emergence of resistant Xcp strains carrying acquired resistance genes. Considering this growing concern, the present study aimed to investigate nine Xcp isolates obtained from pomegranate-growing regions of India by assessing their pathogenicity, followed by in vitro evaluation of their antibiotic sensitivity, and subsequently analyzing their antibiotic resistance gene (ARG) profiles and genome diversity through whole-genome sequencing. Morphological and 16 S rRNA sequencing analyses confirmed that all Xcp strains shared 98.5–100% nucleotide identity with X. citri. Pathogenicity assays identified Xcp1 as the most virulent strain. Antibiotic sensitivity tests showed that Xcp1 was most sensitive to streptomycin, while Xcp3 and Xcp9 were more susceptible to tetracycline. Whole-genome sequencing revealed genome sizes of 4.72–4.93 Mb, a GC content of ~ 65%, and ≥ 99% ANI with X. citri LMG 859. Across the nine Xcp strains, 6,623 to 6,925 genes were annotated with Gene Ontology and grouped into 44 subclasses within three main functional categories. The ARG profiling of nine Xcp strains revealed a conserved set of three chromosomal resistance genes, including vanY and adeF, along with diverse putative ARGs and variable plasmid-associated elements. Prediction of Type III secretion system (T3SS) proteins in nine Xcp strains revealed well-characterized proteins such as XopI, XopA, XopN, XopX, XopL, XopR, XopAV, XopK, XopAE, XopQ, XopP, XopAP, as well as avirulence-associated proteins like AvrBs2 and other LRR-type effectors, highlighting their likely critical role in Xcp pathogenicity. BRIG analysis using Xcp1 as the reference genome revealed near-complete (~ 100%) similarity across most genomic regions of the eight isolates. However, a 500 kb region between 4.5 and 5.0 Mbp showed reduced similarity (~ 70%) in five isolates. Further analysis using Mauve alignments indicated that only Xcp3 and Xcp9 contained additional homologous blocks in this region, suggesting strain-specific structural variations. In contrast, the similarity drop in Xcp2, Xcp4, and Xcp5 corresponded to contig boundaries without changes in gene order, indicating these were likely assembly artifacts rather than true genomic rearrangements. This study highlights genomic and phenotypic variation among Xcp strains, revealing strain-specific virulence, resistance genes, and genomic rearrangements. The findings enhance our understanding of Xcp adaptation and resistance, offering a foundation for improved disease management strategies in India.