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BACKGROUND Rice spikelet rot disease (RSRD) is an emerging disease that significantly reduces rice yield and quality. In this study, we evaluated the potential use of the broad-spectrum endophytic fungus Phomopsis liquidambaris B3 as a biocontrol agent (BCA) against RSRD. Additionally, we compared the control effects of different methods, including chemical fungicides and treatment with multiple strains and single strains in combination or individually, against RSRD. The objective of this study was to find an effective and environmentally friendly control strategy to reduce the occurrence of RSRD and improve rice yield. RESULTS In the pot experiments, the effect of B3 alone was better than that of fungicide or combined measures. The results showed that root colonization by B3 significantly reduced the incidence and disease index of RSRD, by 41.0% and 53.8%, respectively, which were related to enhanced SOD, POD and PPO activities and significantly upregulated expression levels of OsAOX, OsLOX, OsPAL, and OsPR10 in rice. Moreover, B3 improved the diversity of the bacterial community rather than the fungal community in the rice rhizosphere. In addition, B3 led to the decrease of Fusarium proliferatum colonization and fumonisin content in the grain. Finally, root development was markedly promoted after B3 inoculation, and the yield improved by 48.60%. The result of field experiments showed that the incidence of RSRD and the fumonisin content were observably reduced in rice receiving B3, by 24.41% and 37.87%, respectively. CONCLUSION The endophytic fungus Phomopsis liquidambaris B3 may become an effective tool to relieve rice spikelet rot disease. This article is protected by copyright. All rights reserved.