Genetic responses and aflatoxin inhibition during co-culture of aflatoxigenic and non-aflatoxigenic Aspergillus flavus

Authors: Sweany, Rebecca; Mack, Brian; Moore, Geromy; Gilbert, Matthew; Cary, Jeffrey; Lebar, Matthew; Rajasekaran, Kanniah - Rajah; DAMANN, JR, KENNETH - Retired Non ARS Employee

Submitted to: Toxins
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/5/2021
Publication Date: 11/11/2021
Citation: Sweany, R.R., Mack, B.M., Moore, G.G., Gilbert, M.K., Cary, J.W., Lebar, M.D., Rajasekaran, K., Damann Jr, K.E. 2021. Genetic responses and aflatoxin inhibition during co-culture of aflatoxigenic and non-aflatoxigenic Aspergillus flavus. Toxins. 13:794. https://doi.org/10.3390/toxins13110794.
DOI: https://doi.org/10.3390/toxins13110794

Interpretive Summary: Aspergillus flavus is a mold/fungus that contaminates and produces carcinogenic aflatoxins in corn, peanuts and tree nuts that results in reduced market value and health concerns in animals and human beings. Among several different strategies to prevent pre-harvest aflatoxin contamination, the most commonly applied is biological control. In biological control, A. flavus strains that do not produce aflatoxin (nontoxigenic) are applied to growers’ fields to outcompete toxin producing strains. During competition, nontoxigenic strains are able to reduce aflatoxin production in addition to preventing growth of the toxigenic strains. Differential gene expression analysis was conducted through next generation RNA sequencing to compare gene expression between a nontoxigenic biocontrol strain, toxigenic corn-infecting strain and a co-culture of those strains in a biocontrol interaction. The co-culture biocontrol interaction showed nearly 1000-fold decrease in aflatoxin production. Both growth and gene activation of the toxigenic strain was limited during the biocontrol interaction. Genes in several different secondary metabolite or chemical producing pathways were only activated in the nontoxigenic biological control isolate. Genes in these pathways produced even more RNA during the biocontrol interaction, indicating that the nontoxigenic strain turns on chemical pathway genes in response to the toxigenic fungus. Several of these chemical pathways in nontoxigenic biocontrol strains could be involved in shutting off aflatoxin production by toxigenic strains. This study provides new opportunities to select better biocontrol strains and produce an effective control of aflatoxin contamination.

Technical Abstract: Aflatoxin is a carcinogenic mycotoxin produced by Aspergillus flavus. Non-aflatoxigenic (Non-tox) A. flavus isolates are deployed in corn fields as biocontrol because they substantially reduce af-latoxin contamination via direct replacement and additionally via direct contact or touch with toxigenic (Tox) isolates and secretion of inhibitory/degradative chemicals. To understand touch inhibition, aflatoxin analysis and RNA-seq examined gene expression of Non-tox isolate 17 and Tox isolate 53 monocultures and during their interaction in co-culture. Fewer than expected unique reads were assigned to Tox 53 during co-culture, indicating its growth and/or gene ex-pression was inhibited in response to Non-tox 17. Aflatoxin production was reduced by 99.7% in 72 h co-cultures. Hundreds of predicted secreted proteins and genes involved in oxida-tion/reduction were enriched in Non-tox 17 and co-cultures compared to Tox 53. Five secondary metabolite (SM) gene clusters and kojic acid synthesis genes were upregulated in Non-tox 17 compared to Tox 53 and a few were further upregulated in co-cultures in response to touch. These results suggest Non-tox strains can inhibit growth and aflatoxin gene cluster expression in Tox strains through touch. Additionally, upregulation of other SM genes and redox genes during the biocontrol interaction demonstrates a potential role of inhibitory SMs and antioxidants as addi-tional biocontrol mechanisms and deserves further exploration to improve biocontrol formula-tions.