The efficacy of eleven essential oils (EOs) against Aspergillus flavus NRRL 3357 was investigated. The highest antifungal activity against this aflatoxigenic fungus was exhibited by cinnamon, oregano and lemongrass, which showed low minimum inhibitory concentration (MIC) values under vapor conditions. Interactions of the three EOs were evaluated by the fractional inhibition concentration index (FICI), and the composite essential oils (CEO) showed synergistic inhibitory activities. Chemical analysis of the composite essential oils of cinnamon, oregano, and lemongrass (COL-CEO) revealed that (Z)-citral (33.44%), (E)-citral (32.88%) and carvacrol (19.84%) were the dominant components, followed by limonene (4.29%) and cinnamaldehyde (3.76%). COL-CEO not only inhibited fungal growth but also decreased aflatoxin B1 production by A. flavus. Downregulation of the relative expression of aflatoxin genes in the aflatoxin biosynthetic pathway by COL-CEO revealed its anti-aflatoxigenic mechanism. COL-CEO could also affect the colonization of A. flavus on maize grains. Therefore, COL-CEO may be considered as a potential natural antifungal agent, which could be used for the storage of maize and other grains.

Background: Fungus infection in staple grains affects the food storage and threatens food security. TheAspergillus flavusis known to infect multiple grains and produce mycotoxin Aflatoxin B1, which is mutagenic, teratogenic and causes immunosuppression in animals. However, the molecular mechanism of maize resistance toA. flavusis largely unknown.Results: Here we used corn kernels to investigate resistance genes toA. flavususing genome-wide association study (GWAS) of 313 inbred lines. We characterized the resistance levels of kernels after inoculating withA. flavus. The GWAS with 558,529 SNPs identified four associated loci involving 29 candidate genes that were linked to seed development, resistance or infection, and involved in signal pathways, seed development, germination, dormancy, epigenetic modification, and antimicrobial activity. In addition, a few candidate genes were also associated with several G-protein signaling and phytohormones that might involve in synergistic work conferring different resistance during seed development. Expression of 16 genes out of 29 during kernel development was also associated with resistance levels.Conclusions: We characterized the resistance levels of 313 maize kernels after inoculating withA. flavus, and found four associated loci and 16 candidate maize genes. The expressed 16 genes involved in kernel structure and kernel composition most likely contribute to mature maize kernels’ resistance toA. flavus, and in particular, in the development of pericarp. The linked candidate genes could be experimentally transformed to validate and manipulate fungal resistance. Thus this result adds value to maize kernels in breeding programs.

The basic leucine zipper (bZIP) is an important transcription factor required for fungal development, nutrient utilization, biosynthesis of secondary metabolites, and defense against various stresses. Aspergillus flavus is a major producer of aflatoxin and an opportunistic fungus on a wide range of hosts. However, little is known about the role of most bZIP genes in A. flavus. In this study, we developed a high-throughput gene knockout method based on an Agrobacterium-mediated transformation system. Gene knockout construction by yeast recombinational cloning and screening of the null mutants by double fluorescence provides an efficient way to construct gene-deleted mutants for this multinucleate fungus. We deleted 15 bZIP genes in A. flavus. Twelve of these genes were identified and characterized in this strain for the first time. The phenotypic analysis of these mutants showed that the 15 bZIP genes play a diverse role in mycelial growth (eight genes), conidiation (13 genes), aflatoxin biosynthesis (10 genes), oxidative stress response (11 genes), cell wall stress (five genes), osmotic stress (three genes), acid and alkali stress (four genes), and virulence to kernels (nine genes). Impressively, all 15 genes were involved in the development of sclerotia, and the respective deletion mutants of five of them did not produce sclerotia. Moreover, MetR was involved in this biological process. In addition, HapX and MetR play important roles in the adaptation to excessive iron and sulfur metabolism, respectively. These studies provide comprehensive insights into the role of bZIP transcription factors in this aflatoxigenic fungus of global significance.

Aspergillus flavus often invade many important corps and produce harmful aflatoxins both in preharvest and during storage stages. The regulation mechanism of aflatoxin biosynthesis in this fungus has not been well explored mainly due to the lack of an efficient transformation method for constructing a genome-wide gene mutant library. This challenge was resolved in this study, where a reliable and efficient Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for A. flavus NRRL 3357 was established. The results showed that removal of multinucleate conidia, to collect a homogenous sample of uninucleate conidia for use as the transformation material, is the key step in this procedure. A. tumefaciens strain AGL-1 harboring the ble gene for zeocin resistance under the control of the gpdA promoter from A. nidulans is suitable for genetic transformation of this fungus. We successfully generated A. flavus transformants with an efficiency of ∼ 60 positive transformants per 10⁶ conidia using our protocol. A small-scale insertional mutant library (∼ 1,000 mutants) was constructed using this method and the resulting several mutants lacked both production of conidia and aflatoxin biosynthesis capacity. Southern blotting analysis demonstrated that the majority of the transformants contained a single T-DNA insert on the genome. To the best of our knowledge, this is the first report of genetic transformation of A. flavus via ATMT and our protocol provides an effective tool for construction of genome-wide gene mutant libraries for functional analysis of important genes in A. flavus.