Aspergillus flavus Growth Inhibition and Aflatoxin B1 Decontamination by Streptomyces Isolates and Their Metabolites.

Aflatoxin B1 is a potent carcinogen produced by Aspergillus flavus, mainly during grain storage. As pre-harvest methods are insufficient to avoid mycotoxin presence during storage, diverse curative techniques are being investigated for the inhibition of fungal growth and aflatoxin detoxification. Streptomyces spp. represent an alternative as they are a promising source of detoxifying enzymes. Fifty-nine Streptomyces isolates and a Streptomyces griseoviridis strain from the commercial product Mycostop®, evaluated against Penicillium verrucosum and ochratoxin A during previous work, were screened for their ability to inhibit Aspergillus flavus growth and decrease the aflatoxin amount. The activities of bacterial cells and cell-free extracts (CFEs) from liquid cultures were also evaluated. Fifty-eight isolates were able to inhibit fungal growth during dual culture assays, with a maximal reduction going down to 13% of the control. Aflatoxin-specific production was decreased by all isolates to at least 54% of the control. CFEs were less effective in decreasing fungal growth (down to 40% and 55% for unheated and heated CFEs, respectively) and aflatoxin-specific production, with a few CFEs causing an overproduction of mycotoxins. Nearly all Streptomyces isolates were able to degrade AFB1 when growing in solid and liquid media. A total degradation of AFB1 was achieved by Mycostop® on solid medium, as well as an almost complete degradation by IX20 in liquid medium (6% of the control). CFE maximal degradation went down to 37% of the control for isolate IX09. The search for degradation by-products indicated the presence of a few unknown molecules. The evaluation of residual toxicity of the tested isolates by the SOS chromotest indicated a detoxification of at least 68% of AFB1's genotoxicity.

Minimizing Ochratoxin A Contamination through the Use of Actinobacteria and Their Active Molecules.

Ochratoxin A (OTA) is a secondary metabolite produced by fungal pathogens such as Penicilliumverrucosum, which develops in food commodities during storage such as cereals, grapes, and coffee. It represents public health concerns due to its genotoxicity, carcinogenicity, and teratogenicity. The objective of this study was to evaluate the ability of actinobacteria and their metabolites to degrade OTA and/or to decrease its production. Sixty strains of actinobacteria were tested for their ability to prevent OTA formation by in vitro dual culture assays or with cell free extracts (CFEs). In dual culture, 17 strains strongly inhibited fungal growth, although it was generally associated with an increase in OTA specific production. Seventeen strains inhibited OTA specific production up to 4% of the control. Eleven actinobacteria CFEs reduced OTA specific production up to 62% of the control, while no substantial growth inhibition was observed except for two strains up to 72% of the control. Thirty-three strains were able to degrade OTA almost completely in liquid medium whereas only five were able to decrease it on solid medium, and two of them reduced OTA to an undetectable amount. Our results suggest that OTA decrease could be related to different strategies of degradation/metabolization by actinobacteria, through enzyme activities and secretion of secondary metabolites interfering with the OTA biosynthetic pathway. CFEs appeared to be ineffective at degrading OTA, raising interesting questions about the detoxification mechanisms. Common degradation by-products (e.g., OTα or L-ß-phenylalanine) were searched by HPLC-MS/MS, however, none of them were found, which implies a different mechanism of detoxification and/or a subsequent degradation into unknown products.

Commercial Biocontrol Agents Reveal Contrasting Comportments Against Two Mycotoxigenic Fungi in Cereals: Fusarium Graminearum and Fusarium Verticillioides.

The aim of this study was to investigate the impact of commercialized biological control agents (BCAs) against two major mycotoxigenic fungi in cereals, Fusarium graminearum and Fusarium verticillioides, which are trichothecene and fumonisin producers, respectively. With these objectives in mind, three commercial BCAs were selected with contrasting uses and microorganism types (T. asperellum, S. griseoviridis, P. oligandrum) and a culture medium was identified to develop an optimized dual culture bioassay method. Their comportment was examined in dual culture bioassay in vitro with both fusaria to determine growth and mycotoxin production kinetics. Antagonist activity and variable levels or patterns of mycotoxinogenesis inhibition were observed depending on the microorganism type of BCA or on the culture conditions (e.g., different nutritional sources), suggesting that contrasting biocontrol mechanisms are involved. S. griseoviridis leads to a growth inhibition zone where the pathogen mycelium structure is altered, suggesting the diffusion of antimicrobial compounds. In contrast, T. asperellum and P. oligandrum are able to grow faster than the pathogen. T. asperellum showed the capacity to degrade pathogenic mycelia, involving chitinolytic activities. In dual culture bioassay with F. graminearum, this BCA reduced the growth and mycotoxin concentration by 48% and 72%, respectively, and by 78% and 72% in dual culture bioassay against F. verticillioides. P. oligandrum progressed over the pathogen colony, suggesting a close type of interaction such as mycoparasitism, as confirmed by microscopic observation. In dual culture bioassay with F. graminearum, P. oligandrum reduced the growth and mycotoxin concentration by 79% and 93%, respectively. In the dual culture bioassay with F. verticillioides, P. oligandrum reduced the growth and mycotoxin concentration by 49% and 56%, respectively. In vitro dual culture bioassay with different culture media as well as the nutritional phenotyping of different microorganisms made it possible to explore the path of nutritional competition in order to explain part of the observed inhibition by BCAs.