Evaluation of Bacillus subtilis Czk1 Metabolites by LC-MS/MS and Their Antifungal Potential against Pyrrhoderma noxium Causing Brow Rot Disease

Brown rot disease caused by Pyrrhoderma noxium is a widespread disease that severely affects the roots of rubber trees (Hevea brasiliensis Muell. Arg.). The economic losses, along with environmental and health problems arising from the use of disease control chemicals, have raised the interest of scholars to explore the use of biological control agents for the effective control of fungal pathogen P. noxium. Here, the inhibition effect of the culture filtrate of B. subtilis Czk1 on P. noxium was demonstrated. The findings indicate that the antifungal activity of this strain is mediated wholly or partly by compounds produced in the culture filtrate. The combined use of liquid chromatography-tandem mass spectrometry and antifungal activity assays rapidly identified compounds produced by B. subtilis Czk1. Metabolic profiles were assessed and used to identify major metabolites based on the scores of variable importance in the projection and the plot scores of principal component analysis. A total of 296 differential metabolites were screened, including 208 in positive ion mode and 88 in negative ion mode. Two key metabolites, diacetyl and trans-2-octenoic acid, were screened from 29 metabolites by antifungal activity assays. The median effective concentration (EC50) of trans-2-octenoic acid and diacetyl were 0.9075 mg/mL and 4.8213 mg/mL, respectively. The antifungal metabolites can disrupt the internal structure of the pathogenic fungal mycelium, thereby impeding its growth. This study is expected to contribute to the existing knowledge of Czk1-produced metabolites and their future antifungal applications. This study is also expected to provide a new biopreservative perspective on unexplored antifungal metabolites produced by Czk1 as a biocontrol agent.