The saprophytic soil fungus Aspergillus flavus infects crops and produces aflatoxin. Pichia anomala, which is a biocontrol yeast and produces the major volatile 2-phenylethanol (2-PE), is able to reduce growth of A. flavus and aflatoxin production when applied onto pistachio trees. High levels of 2-PE are lethal to A. flavus and other fungi. However, at low levels, the underlying mechanism of 2-PE to inhibit aflatoxin production remains unclear. In this study, we characterized the temporal transcriptome response of A. flavus to 2-PE at a subinhibitory level (1 μL/mL) using RNA-Seq technology and bioinformatics tools. The treatment during the entire 72 h experimental period resulted in 131 of the total A. flavus 13,485 genes to be significantly impacted, of which 82 genes exhibited decreased expression. They included those encoding conidiation proteins and involved in cyclopiazonic acid biosynthesis. All genes in the aflatoxin gene cluster were also significantly decreased during the first 48 h treatment. Gene Ontology (GO) analyses showed that biological processes with GO terms related to catabolism of propionate and branched-chain amino acids (valine, leucine and isoleucine) were significantly enriched in the down-regulated gene group, while those associated with ribosome biogenesis, translation, and biosynthesis of α-amino acids were over-represented among the up-regulated genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that metabolic pathways negatively impacted among the down-regulated genes parallel to those active at 30 °C, a condition conducive to aflatoxin biosynthesis. In contrast, metabolic pathways positively related to the up-regulated gene group resembled those at 37 °C, which favors rapid fungal growth and is inhibitory to aflatoxin biosynthesis. The results showed that 2-PE at a low level stimulated active growth of A. flavus but concomitantly rendered decreased activities in branched-chain amino acid degradation. Since secondary metabolism occurs after active growth has ceased, this growth stimulation resulted in suppression of expression of aflatoxin biosynthesis genes. On the other hand, increased activities in degradation pathways for branched-chain amino acids probably are required for the activation of the aflatoxin pathway by providing building blocks and energy regeneration. Metabolic flux in primary metabolism apparently has an important role in the expression of genes of secondary metabolism.

Penicillium is a large genus of common molds with over 400 described species; however, identification of individual species is difficult, including for those species that cause postharvest rots. In this study, blue rot fungi from stored apples and pears were isolated from a variety of hosts, locations, and years. Based on morphological and cultural characteristics and partial amplification of the β-tubulin locus, the isolates were provisionally identified as several different species of Penicillium. These isolates were investigated further using a suite of molecular DNA markers and compared to sequences of the ex-type for cognate species in GenBank, and were identified as P. expansum (3 isolates), P. solitum (3 isolates), P. carneum (1 isolate), and P. paneum (1 isolate). Three of the markers we used (ITS, internal transcribed spacer rDNA sequence; benA, β-tubulin; CaM, calmodulin) were suitable for distinguishing most of our isolates from one another at the species level. In contrast, we were unable to amplify RPB2 sequences from four of the isolates. Comparison of our sequences with cognate sequences in GenBank from isolates with the same species names did not always give coherent data, reinforcing earlier studies that have shown large intraspecific variability in many Penicillium species, as well as possible errors in some sequence data deposited in GenBank.

Aspergillus flavus is a ubiquitous saprophytic fungus found in soils across the world. The fungus is the major producer of aflatoxin (AF) B₁, which is toxic and a potent carcinogen to humans. Aflatoxin B₁ (AFB₁) is often detected in agricultural crops such as corn, peanut, almond, and pistachio. It is a serious and recurrent problem and causes substantial economic losses. Wickerhamomyces anomalus WRL-076 was identified as an effective biocontrol yeast against A. flavus. In this study, the associated molecular mechanisms of biocontrol were investigated. We found that the expression levels of eight genes, aflR, aflJ, norA, omtA, omtB, pksA, vbs, and ver-1 in the aflatoxin biosynthetic pathway cluster were suppressed. The decreases ranged from several to 10,000 fold in fungal samples co-cultured with W. anomalus. Expression levels of conidiation regulatory genes brlA, abaA, and wetA as well as sclerotial regulatory gene (sclR) were all down regulated. Consistent with the decreased gene expression levels, aflatoxin concentrations in cultural medium were reduced to barely detectable. Furthermore, fungal biomass and conidial number were significantly reduced by 60% and more than 95%, respectively. The results validate the biocontrol efficacy of W. anomalus WRL-076 observed in the field experiments.