Volatile organic compounds from Bacillus amyloliquefaciens strain D-1 suppress mycelia growth, spore formation and germination, and aflatoxin biosynthesis of Aspergillus flavus on Coix lacryma-jobi

Aspergillus flavus and aflatoxin contamination of coicis semen, a kind of food and medicine homology widely used in China and other Asian countries, leads to serious issues threatening human health and economic loss. Therefore, contamination management of A. flavus and its aflatoxins in coicis semen is of great significance and urgent to ensure its safety. Bacillus amyloliquefaciens strain D-1 was previously obtained from the rhizosphere soil of Pseudostellaria heterophylla with the ability to degrade zearalenone. However, the strain’s inhibitory effects on the mycelium growth, conidia formation, and aflatoxins (AFs) biosynthesis pathway of toxigenic A. flavus have not yet been investigated, and its natural volatile antifungal compounds remain unexplored. In this study, confrontation and dual-culture (FTF) experiments showed that B. amyloliquefaciens strain D-1 exhibited strong inhibitory effects on A. flavus after 5 days of cultivation. It suppressed mycelial growth by 71.28%, reduced spore count by 99.2%, and decreased spore germination by 74.8%. Microscopic observations further revealed that strain D-1 completely blocked the development of A. flavus sporangia and spore peduncles. Gas chromatography-mass spectrometry (GC-MS/MS) profiling identified the key volatile compounds produced by strain D-1, including 2,5-dimethyl-pyrazine (EC 50 = 29.88 μL·L -1 ), phenylethyl alcohol (EC 50 = 4.82 μL·L -1 ), 4-phenyl-2-butanone (EC 50 = 1.09 μL·L -1 ), 2,3,6-trimethyl-phenol (EC 50 = 1.32 μL·L -1 ), and 2,3,5-trimethyl-phenol (EC 50 = 7.23 μL·L -1 ). These compounds significantly inhibited A. flavus mycelial growth, spore formation and germination, as well as AFs biosynthesis. Of which 2,3, 6-trimethyl-phenol, and 2,3,5-trimethyl-phenol were newly discovered compounds with strong inhibitory activities against A. flavus . These findings provide new safe and efficient compounds as fumigants and antifungal agents, which will provide an effective potential management strategy to control the contamination and spread of toxigenic pathogens in traditional Chinese medicine (TCM) and food/feeds during storage. • Bacillus amyloliquefaciens strain D-1 strongly inhibited the mycelia growth, spore production and germination, and aflatoxin biosynthesis of A. flavus via emitting volatiles organic compounds. • 2,5-dimethyl-pyrazine, phenylethyl alcohol, 4-phenyl-2-butanone, 2,3,6-trimethyl-phenol, and 2,3,5-trimethyl-phenol were the dominant inhibitory activity compounds emitted by strain D-1. • Five volatile natural chemicals significantly inhibited the germination of propagule spores at 16 hpi, and made the edge of sporophyte structure rupture or dissolve or even die.