In an attempt to develop a biological control agent against mycotoxigenic Fusarium species, we isolated Bacillus amyloliquefaciens strain DA12 from soil and explored its antimicrobial activities. DA12 was active against the growth of mycotoxigenic F. asiaticum, F. graminearum, F. proliferatum, and F. verticillioides both in vitro and in planta (maize). Further screening using dual culture extended the activity range of strain DA12 against other fungal pathogens including Botrytis cinerea, Colletotrichum coccodes, Endothia parasitica, Fusarium oxysporum, Raffaelea quercus-mongolicae, and Rhizoctonia solani. The butanol extract of the culture filtrate of B. amyloliquefaciens DA12 highly inhibited the germination of F. graminearum macroconidia with inhibition rate 83% at a concentration of 31.3 μg/ml and 100% at a concentration of 250 μg/ml. The antifungal metabolite from the butanol extract was identified as iturin A by thin layer chromatography-bioautography. In addition, volatile organic compounds produced by DA12 were able to inhibit mycelial growth of various phytopathogenic fungi. The volatile compounds were identified as 2-heptanone, 5-methyl heptanone and 6-methyl heptanone by gas chromatography-mass spectrometry (GC-MS) analysis. These results indicate that the antagonistic activity of Bacillus amyloliquefaciens DA12 was attributable to iturin A and volatile heptanones, and the strain could be used as a biocontrol agent to reduce the development of Fusarium diseases and mycotoxin contamination of crops.
To investigate contamination of ground red pepper with fungi and mycotoxin, we obtained 30 ground red pepper samples from 15 manufacturers in the main chili-pepper-producing areas in Korea. Fungal contamination was evaluated by spreading diluted samples on potato dextrose agar plates. The total fungi counts ranged from 0 to 7.3 × 10 CFU/g. In the samples, the genus Aspergillus had the highest incidence, while Paecilomyces was isolated most frequently. The next most frequent genera were Rhizopus, Penicillium, Cladosporium, and Alternaria. Within Aspergillus, A. ruber was predominant, followed by A. niger, A. amstelodami, A. ochraceus, A. terreus, A. versicolor, A. flavus, and A. fumigatus. The samples were analyzed for aflatoxins, ochratoxin A, and citrinin by ultra-perfomance liquid chromatography (UPLC) with a fluorescence detector. Ochratoxin A was detected from three samples at 1.03‒2.08 μg/kg, whereas no aflatoxins or citrinin were detected. To test the potential of fungal isolates to produce aflatoxin, we performed a PCR assay that screened for the norB-cypA gene for 64 Aspergillus isolates. As a result, a single 800-bp band was amplified from 10 A. flavus isolates, and one Aspergillus sp. isolate. UPLC analyses confirmed aflatoxin production by nine A. flavus isolates and one Aspergillus sp. isolate, which produced total aflatoxins at 146.88‒909.53 μg/kg. This indicates that continuous monitoring of ground red pepper for toxigenic fungi is necessary to minimize mycotoxin contamination.
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