Genomic and Functional Characterization of Pseudomonas shiyinii sp. nov. ST4 Reveals Conserved Biocontrol Mechanisms Against Sugarcane Smut

Introduction Pseudomonas sp. ST4 is a potent biocontrol agent against sugarcane smut disease, acting primarily by inhibiting the sexual mating of the fungal pathogen Sporisorium scitamineum . However, the genetic basis of its antifungal activity has remained unexplored. Methods To elucidate the genetic basis of ST4’s biocontrol activity, we obtained its complete genome sequence and conducted comprehensive genomic, phylogenetic, and chemotaxonomic analyses. We additionally performed a genome-wide Tn5 transposon mutagenesis screen to identify genes critical for its inhibitory activity, followed by a broader phylogenomic analysis to assess the evolutionary conservation of these traits across the Pseudomonadaceae family. Results Based on our analyses, we delineated ST4 as a novel species, Pseudomonas shiyinii sp. nov. The 5.14-Mb genome harbors numerous genes and biosynthetic gene clusters associated with plant-beneficial traits. The Tn5 mutagenesis screen revealed that the pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (Gcd) pathway is essential for the inhibitory activity of ST4, with 78% of loss-of-function mutants mapping to the pqq or gcd genes. We further demonstrated that acidification of the environment mediated by the PQQ-Gcd system in ST4 inhibits S. scitamineum mating—a critical step in pathogenicity—without compromising haploid sporidial viability. In addition to this primary mechanism, the ST4 genome contains complete biosynthesis pathways for the phytohormone indole-3-acetic acid (IAA), which may also serve as a precursor for the previously identified active metabolite indole-3-carbaldehyde (ICHO), a specific inhibitor of fungal sexual mating. Phylogenomic analysis showed that while the PQQ-Gcd system is broadly conserved across Pseudomonadaceae , the capacity for IAA/I3C production is more specialized, confined to approximately 21% of Pseudomonas species. Discussion Together, our findings decipher the molecular mechanism of a widely conserved biocontrol trait and establish P. shiyinii ST4 as a promising, ecologically adaptable candidate for sustainable crop protection.