Biological Control of Endophytic Bacillus subtilis and Stenotrophomonas rhizophila Against Pyrenophora teres f. teres in Barley

Net form net blotch disease, caused by Pyrenophora teres f. teres (Ptt), is one of the most destructive barley diseases, resulting in severe yield and grain quality losses worldwide. The increasing prevalence of fungicide-resistant Ptt strains, driven by the pathogen’s high genetic variability, highlights the urgent need for sustainable and eco-friendly disease management strategies. The present study provides novel insights into the use of native seed-borne endophytic bacteria naturally associated with barley as biological control agents against Ptt. Two endophytic bacterial strains isolated from healthy barley seeds were identified based on 16S rRNA gene sequencing as Bacillus subtilis PX491551 and Stenotrophomonas rhizophila PX494419. Their biocontrol potential against Ptt was evaluated through in vitro, greenhouse, and field experiments. In the dual-culture assay, B. subtilis and S. rhizophila inhibited the mycelial growth of Pyrenophora teres f. teres by 64.34% and 50.14%, respectively. Under greenhouse conditions, B. subtilis and S. rhizophila significantly reduced disease severity at the seedling stage, with scores of 2.00 and 4.00, respectively, compared to 9.33 in the untreated control. Beyond disease suppression, both endophytic bacteria markedly enhanced the host’s defense system. S. rhizophila induced the highest accumulation of total soluble phenolics, while B. subtilis significantly increased flavonoid content and boosted higher activities of superoxide dismutase and phenylalanine ammonia-lyase. In contrast, S. rhizophila showed the strongest induction of ascorbate peroxidase activity. Notably, field application of both bacteria consistently reduced net blotch severity over two consecutive growing seasons (2023–2024 and 2024–2025) and considerably improved chlorophyll content, 1000-grain weight, and grain yield. Overall, this study demonstrates that native seed-derived endophytic bacteria not only suppress barley net blotch but also enhance host antioxidant and defense responses, highlighting their potential as effective and sustainable biological control agents for barley disease management.