Prior to harvest, maize kernels are invaded by a diverse population of fungal organisms that comprise the microbiome of the grain mass. Poor post-harvest practices and improper drying can lead to the growth of mycotoxigenic storage fungi and deterioration of grain quality. Hermetic storage bags are a low-cost technology for the preservation of grain during storage, which has seen significant adoption in many regions of Sub-Saharan Africa. This study explored the use of high-throughput DNA sequencing of the fungal Internal Transcribed Spacer 2 (ITS2) region for characterization of the fungal microbiome before and after 3 months of storage in hermetic and non-hermetic (woven) bags in the United States and Kenya. Analysis of 1,377,221 and 3,633,944 ITS2 sequences from the United States and Kenya, respectively, resulted in 251 and 164 operational taxonomic units (OTUs). Taxonomic assignment of these OTUs revealed 63 and 34 fungal genera in the US and Kenya samples, respectively, many of which were not detected by traditional plating methods. The most abundant genus was Fusarium, which was identified in all samples. Storage fungi were detected in the grain mass prior to the storage experiments and increased in relative abundance within the woven bags. The results also indicate that storage location had no effect on the fungal microbiome of grain stored in the United States, while storage bag type led to significant changes in fungal composition. The fungal microbiome of the Kenya grain also underwent significant changes in composition during storage and fungal diversity increased during storage regardless of bag type. Our results indicated that extraction of DNA from ground kernels is sufficient for identifying the fungi associated with the maize. The results also indicated that bag type was the most important factor influencing changes in fungal microbiome during storage. The results also support the recommended use of hermetic storage for reducing food safety risks, especially from mycotoxigenic fungi.

BackgroundFusarium verticillioides causes an important seed disease on maize and produces the fumonisin group of mycotoxins, which are toxic to humans and livestock. A previous study discovered that a gene (FST1) in the pathogen affects fumonisin production and virulence. Although the predicted amino acid sequence of FST1 is similar to hexose transporters, previous experimental evidence failed to prove function.ResultsThree new phenotypes were identified that are associated with the FST1 mutant of F. verticillioides (Δfst1), namely reduction in macroconidia production, increased sensitivity to hydrogen peroxide, and reduced mycelial hydrophobicity. A transcriptome comparison of the wild type and strain Δfst1 grown on autoclaved maize kernels for six days identified 2677 genes that were differentially expressed. Through gene ontology analysis, 961 genes were assigned to one of 12 molecular function categories. Sets of down-regulated genes in strain Δfst1 were identified that could account for each of the mutant phenotypes.ConclusionThe study provides evidence that disruption of FST1 causes several metabolic and developmental defects in F. verticillioides. FST1 appears to connect the expression of several gene networks, including those involved in secondary metabolism, cell wall structure, conidiogenesis, virulence, and resistance to reactive oxygen species. The results support our hypothesis that FST1 functions within the framework of environmental sensing.

Fumonisin B1 (FB1), a polyketide mycotoxin produced by Fusarium verticillioides during the colonization of maize kernels, is detrimental to human and animal health. FST1 encodes a putative protein with 12 transmembrane domains; however, its function remains unknown. The FST1 gene is highly expressed by the fungus in the endosperm of maize kernels compared with the levels of expression in germ tissues. Previous research has shown that FST1 affects FB1 production, virulence, hydrogen peroxide resistance, hydrophobicity and macroconidia production. Here, we examine the phylogeny of FST1, its expression in a Saccharomyces cerevisiae strain lacking a functional myo-inositol transporter (ITR1) and the effect of amino acid changes in the central loop and C-terminus regions of FST1 on functionality. The results indicate that expression of FST1 in an ITR1 mutant strain restores growth on myo-inositol medium to wild-type levels and restores the inhibitory effects of FB1, suggesting that FST1 can transport both myo-inositol and FB1 into yeast cells. Our results with engineered FST1 also indicate that amino acids in the central loop and C-terminus regions are important for FST1 functionality in both S. cerevisiae and F. verticillioides. Overall, this research has established the first characterized inositol transporter in filamentous fungi and has advanced our knowledge about the global regulatory functions of FST1.