| Summary: | <i>Dickeya zeae</i> is the causal agent of bacterial soft rot disease, with a wide range of hosts all over the world. At present, chemical agents, especially agricultural antibiotics, are commonly used in the prevention and control of bacterial soft rot, causing the emergence of resistant pathogens and therefore increasing the difficulty of disease prevention and control. This study aims to provide a safer and more effective biocontrol method for soft rot disease caused by <i>D. zeae</i>. The spot-on-lawn assay was used to screen antagonistic bacteria, and three strains including SC3, SC11 and 3-10 revealed strong antagonistic effects and were identified as <i>Pseudomonas fluorescens</i>, <i>P. parafulva</i> and <i>Bacillus velezensis</i>, respectively, using multi-locus sequence analysis (MLSA) based on the sequences of 16S rRNA and other housekeeping genes. <i>In vitro</i> antimicrobial activity showed that two <i>Pseudomonas</i> strains SC3 and SC11 were only antagonistic to some pathogenic bacteria, while strain 3-10 had broad-spectrum antimicrobial activity on both pathogenic bacteria and fungi. Evaluation of control efficacy in greenhouse trials showed that they all restrained the occurrence and development of soft rot disease caused by <i>D. zeae</i> MS2 or EC1. Among them, strain SC3 had the most impressive biocontrol efficacy on alleviating the soft rot symptoms on both monocotyledonous and dicotyledonous hosts, and strain 3-10 additionally reduced the occurrence of banana wilt disease caused by <i>Fusarium oxysporum</i> f. sp. <i>cubensis</i>. This is the first report of <i>P. fluorescens</i>, <i>P. parafulva</i> and <i>B. velezensis</i> as potential bio-reagents on controlling soft rot disease caused by <i>D. zeae</i>.
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