The Efficacy of Composite Essential Oils against Aflatoxigenic Fungus Aspergillus flavus in Maize.

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.

Genome-wide association study leads to novel genetic insights into resistance to Aspergillus flavus in maize kernels.

BACKGROUND: Fungus infection in staple grains affects the food storage and threatens food security. The Aspergillus flavus is 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 to A. flavus is largely unknown. RESULTS: Here we used corn kernels to investigate resistance genes to A. flavus using genome-wide association study (GWAS) of 313 inbred lines. We characterized the resistance levels of kernels after inoculating with A. 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 with A. 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 to A. 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.

A Novel Site-Specific Integration System for Genetic Modification of Aspergillus flavus.

Aspergillus flavus is a fungus that produces aflatoxin B1, one of the most carcinogenic secondary metabolites. Understanding the regulation mechanism of aflatoxin biosynthesis in this fungus requires precise methods for genomic integration of mutant alleles. To avoid the disadvantage of DNA integration into the genome by non-homologous or ectopic recombination, we developed a novel strategy for site-specific integration of foreign DNA by using a carboxin-resistant sdh2R allele (His 249 Leu). Our results demonstrated that the transformants were generated with a high efficiency (>96%) of correct integration into the sdh2-lcus of the genome of A. flavus NRRL 3357. The advantage of this method is that introduction of the eGFP expression cassette into the sdh2-locus had little effect on fungal growth and virulence while also being rapid and efficient. This system will be a valuable tool for genetic manipulation in A. flavus To the best of our knowledge, this is the first report on the efficient site-specific integration at the sdh2-locus in the genome of Aspergillus.

Differential regulation of mycelial growth and aflatoxin biosynthesis by Aspergillus flavus under different temperatures as revealed by strand-specific RNA-Seq.

Although several regulatory pathways have been reported for Aspergillus flavus, the regulation of aflatoxin production and mycelial growth under different temperatures remains unclear. In this study, A. flavus differentially expressed genes (DEGs) and regulatory pathways were analyzed under three temperatures, by strand-specific RNA-Seq. Results show that a total of 2,428 and 1,474 DEGs were identified in fungal mycelia cultured at 20°C and 37°C, respectively, as compared with the control (28°C). Approximately ~ 79% of DEGs in the 37°C samples were up-regulated genes, while ~ 63% of DEGs in the 20°C samples were down-regulated genes. Most of the DEG pathways enriched by lower temperatures differed from those enriched by higher temperatures, while only a small portion of the pathways were shared by A. flavus grown under different temperatures. Aflatoxin biosynthesis, Butanoate metabolism, oxidation-reduction process, and benzene-containing compound metabolic process were the shared down-regulated pathways, while steroid biosynthesis, oxidoreductase activity, cellular protein modification process, DNA binding, protein complex were the shared up-regulated pathways between lower and higher temperatures. The shared genes and pathways are the key regulatory candidates for aflatoxin biosynthesis with changes of temperature. In addition, the identification of both up-regulated and down-regulated genes provides a useful gene set for further investigation of the aflatoxin biosynthesis among Aspergillus.