Screening, isolation and characterization of endophytic bacteria from upland Rice for antagonism against Fusarium graminearum

Endophytic bacteria play an important role in inhibiting plant pathogens. This study aimed to screen endophytic bacteria from upland rice with antagonistic activity against <i>Fusarium graminearum</i>, evaluated their antagonistic potential against <i>F. graminearum</i>, assessed their anti-fungal substances, and elucidated the underlying mechanisms. Some methods were performed, including dual-culture antagonism assays, lipopeptide extraction, identification of antifungal compounds via LC-MS and HS-GC-MS, metabolomic analysis, and microscopic observation. Two endophytic bacterial strains, URR1 and URR2, were identified as Pseudomonas sp. and Bacillus subtilis, respectively. Dual-culture antagonism assays demonstrated that both strains exhibited strong inhibitory activity against <i>F. graminearum</i>, with inhibition rates of 69.73% and 76.33%, respectively. In vitro experiments further revealed that bacterial suspensions at approximately 3.3 × 10 8 CFU·mL^-1 significantly alleviated stress in upland rice seedlings infected with <i>F. graminearum</i> after 7 days. Both crude lipopeptides and volatile organic compounds (VOCs) markedly suppressed the hyphal growth of the pathogen. The maximum inhibition rate of crude lipopeptides reached 63.86% after 96 hours of treatment, while VOCs showed a peak hyphal inhibition rate of 30.38% after 48 hours of exposure. Antimicrobial lipopeptides, comprising 10 distinct surfactin isoforms and 7 fengycin variants, as well as VOCs such as acetone, ethanol, trichloromethane, pyruvic acid, and propadiene, were identified. After antagonism with lipopeptides extracted from endophytic bacteria URR2, the fungal hyphae of <i>F. graminearum</i> exhibited morphological abnormalities. Notably, treatment with URR2 resulted in the upregulation of metabolites and activation of key metabolic pathways. Metabolomic analysis indicated that the differentially upregulated metabolites encompassed a wide range of classes, including organic acids and derivatives, lipids and lipid-like molecules, organoheterocyclic compounds, organic oxygen compounds, benzenoids, organic nitrogen compounds, nucleosides, nucleotides and analogues, phenylpropanoids and polyketides, as well as lignans and neolignans. The analysis revealed that the associated molecules were significantly concentrated in multiple metabolic pathways, primarily involving ABC transporters, protein digestion and absorption, amino acid biosynthesis, aminoacyl-tRNA biosynthesis, the phosphotransferase system (PTS), starch and sucrose metabolism, among others. These results conclusively demonstrate the strong antifungal activity of strain URR2 against <i>F. graminearum</i>. The antagonistic mechanism of B. subtilis against <i>F. graminearum</i> appears to be multifaceted. Overall, our findings indicate that URR2 has promising potential to be developed as a novel biocontrol agent for the development of sustainable agriculture.