Pseudomonas parafulva SAPEU-1 as a keystone modulator: reshaping citrus phyllosphere microbiome to suppress citrus canker

Introduction Citrus canker disease, caused by the pathogen Xanthomonas citri subsp. citri ( Xcc ) poses a substantial challenge for citrus production due to the limited efficacy of chemical control and increasing pathogen resistance. Methods In this study, we isolated an endophytic bacterial strain, Endophyte S2, from the phyllosphere of citrus plants in Yunnan Province, China. We evaluated its efficacy both as a biocontrol agent and as a modulator of the citrus leaf microbiome. Results and Discussion In vitro antagonism assays revealed that Endophyte S2 achieved the highest inhibition rate (68.2%) against Xcc among all tested isolates. Molecular identification based on 16S rRNA gene sequencing classified S2 as Pseudomonas parafulva SAPEU-01. In greenhouse trials, citrus plants with Xcc infestation were treated with SAPEU-01, and phyllosphere samples were collected before treatment and one month after, and analyzed by Illumina MiSeq sequencing. Post-treatment, α-diversity (richness and evenness) increased significantly, and β-diversity (PCoA, Bray–Curtis) showed a clear separation of microbial community structure, with reduced intra-group variability. Taxonomic shifts included the enrichment of Proteobacteria (particularly Pseudomonadaceae and Sphingomonadaceae), as well as genera such as Pseudomonas , Sphingomonas, and Methylobacterium, concomitant with a marked decline in Xanthomonadaceae (including X. citri ) and opportunistic taxa such as Escherichia coli O157:H7 and Klebsiella aerogenes . Beneficial taxa, including Leuconostoc tardus , Sphingomonas , and Curtobacterium luteum , also increased. These results suggest Pseudomonas parafulva SAPEU-01 not only suppresses the pathogen but also restructures the phyllosphere microbiome toward greater stability and potential resilience.