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The β-1,6-glucan is the key linker between mannoproteins in the outermost part of the cell wall and β-1,3-glucan/chitin polysaccharide to maintain the rigid structure of the cell wall. The β-1,6-glucanase GluM, which was purified from the fermentation supernatant of Corallococcus sp. EGB, was able to inhibit the germination of Fusarium oxysporum f. sp. cucumerinum conidia at a minimum concentration of 2.0 U/mL (0.08 μg/mL). The survival rates of GluM-treated conidia and monohyphae were 10.4% and 30.7%, respectively, which were significantly lower than that of β-1,3-glucanase treatment (Zymolyase, 20.0 U/mL; equate to 1.0 mg/mL) (72.9% and 73.9%). In contrast to β-1,3-glucanase treatment, the high-osmolarity glycerol (HOG) pathway of F. oxysporum f. sp. cucumerinum cells was activated after GluM treatment, and the intracellular glycerol content was increased by 2.6-fold. Moreover, the accumulation of reactive oxygen species (ROS) in F. oxysporum f. sp. cucumerinum cells after GluM treatment induced apoptosis, but it was not associated with the increased intracellular glycerol content. Together, the results indicate that β-1,6-glucan is a promising target for the development of novel broad-spectrum antifungal agents. [ABSTRACT FROM AUTHOR]