Divergent Aspergillus flavus corn population is composed of prolific conidium producers: Implications for saprophytic disease cycle.

The ascomycete fungus Aspergillus flavus infects and contaminates corn, peanuts, cottonseed, and tree nuts with toxic and carcinogenic aflatoxins. Subdivision between soil and host plant populations suggests that certain A. flavus strains are specialized to infect peanut, cotton, and corn despite having a broad host range. In this study, the ability of strains isolated from corn and/or soil in 11 Louisiana fields to produce conidia (field inoculum and male gamete) and sclerotia (resting bodies and female gamete) was assessed and compared with genotypic single-nucleotide polymorphism (SNP) differences between whole genomes. Corn strains produced upward of 47× more conidia than strains restricted to soil. Conversely, corn strains produced as much as 3000× fewer sclerotia than soil strains. Aspergillus flavus strains, typified by sclerotium diameter (small S-strains, <400 µm; large L-strains, >400 µm), belonged to separate clades. Several strains produced a mixture (M) of S and L sclerotia, and an intermediate number of conidia and sclerotia, compared with typical S-strains (minimal conidia, copious sclerotia) and L-strains (copious conidia, minimal sclerotia). They also belonged to a unique phylogenetic mixed (M) clade. Migration from soil to corn positively correlated with conidium production and negatively correlated with sclerotium production. Genetic differences correlated with differences in conidium and sclerotium production. Opposite skews in female (sclerotia) or male (conidia) gametic production by soil or corn strains, respectively, resulted in reduced effective breeding population sizes when comparing male:female gamete ratio with mating type distribution. Combining both soil and corn populations increased the effective breeding population, presumably due to contribution of male gametes from corn, which fertilize sclerotia on the soil surface. Incongruencies between aflatoxin clusters, strain morphotype designation, and whole genome phylogenies suggest a history of sexual reproduction within this Louisiana population, demonstrating the importance of conidium production, as infectious propagules and as fertilizers of the A. flavus soil population.

Cumulative Effects of Non-Aflatoxigenic Aspergillus flavus Volatile Organic Compounds to Abate Toxin Production by Mycotoxigenic Aspergilli.

Previously, authors reported that individual volatile organic compounds (VOCs) emitted by non-aflatoxigenic Aspergillus flavus could act as a mechanism of biocontrol to significantly reduce aflatoxins and cyclopiazonic acid (CPA) produced by toxigenic strains. In this study, various combinations and volumes of three mycotoxin-reductive VOCs (2,3-dihydrofuran, 3-octanone and decane) were assessed for their cumulative impacts on four Aspergillus strains (LA1-LA4), which were then analyzed for changes in growth, as well as the production of mycotoxins, including aflatoxins, CPA and multiple indole diterpenes. Fungal growth remained minimally inhibited when exposed to various combinations of VOCs. No single combination was able to consistently, or completely, inhibit aflatoxin or CPA across all toxigenic strains tested. However, the combination of 2,3-dihydrofuran and 3-octanone offered the greatest overall reductions in aflatoxin and CPA production. Despite no elimination of their production, findings showed that combining VOCs produced solely by non-aflatoxigenic A. flavus still inhibited several agriculturally important mycotoxins, including B and G aflatoxins and CPA. Therefore, other VOC combinations are worth testing as post-harvest biocontrol treatments to ensure the prolonged effectiveness of pre-harvest biocontrol efforts.

Small NRPS-like enzymes in Aspergillus sections Flavi and Circumdati selectively form substituted pyrazinone metabolites.

Aspergillus fungi produce mycotoxins that are detrimental to human and animal health. Two sections of aspergilli are of particular importance to cereal food crops such as corn and barley. Aspergillus section Flavi species like A. flavus and A. parasiticus produce aflatoxins, while section Circumdati species like A. ochraceus and A. sclerotiorum produce ochratoxin A. Mitigating these toxins in food and feed is a critical and ongoing worldwide effort. We have previously investigated biosynthetic gene clusters in Aspergillus flavus that are linked to fungal virulence in corn. We found that one such cluster, asa, is responsible for the production of aspergillic acid, an iron-binding, hydroxamic acid-containing pyrazinone metabolite. Furthermore, we found that the asa gene cluster is present in many other aflatoxin- and ochratoxin-producing aspergilli. The core gene in the asa cluster encodes the small nonribosomal peptide synthetase-like (NRPS-like) protein AsaC. We have swapped the asaC ortholog from A. sclerotiorum into A. flavus, replacing its native copy, and have also cloned both asaC orthologs into Saccharomyces cerevisiae. We show that AsaC orthologs in section Flavi and section Circumdati, while only containing adenylation-thiolation-reductase (ATR) domains, can selectively biosynthesize distinct pyrazinone natural products: deoxyaspergillic acid and flavacol, respectively. Because pyrazinone natural products and the gene clusters responsible for their production are implicated in a variety of important microbe-host interactions, uncovering the function and selectivity of the enzymes involved could lead to strategies that ultimately benefit human health.

Flavonoids Modulate the Accumulation of Toxins From Aspergillus flavus in Maize Kernels.

Aspergillus flavus is an opportunistic fungal pathogen capable of producing aflatoxins, potent carcinogenic toxins that accumulate in maize kernels after infection. To better understand the molecular mechanisms of maize resistance to A. flavus growth and aflatoxin accumulation, we performed a high-throughput transcriptomic study in situ using maize kernels infected with A. flavus strain 3357. Three maize lines were evaluated: aflatoxin-contamination resistant line TZAR102, semi-resistant MI82, and susceptible line Va35. A modified genotype-environment association method (GEA) used to detect loci under selection via redundancy analysis (RDA) was used with the transcriptomic data to detect genes significantly influenced by maize line, fungal treatment, and duration of infection. Gene ontology enrichment analysis of genes highly expressed in infected kernels identified molecular pathways associated with defense responses to fungi and other microbes such as production of pathogenesis-related (PR) proteins and lipid bilayer formation. To further identify novel genes of interest, we incorporated genomic and phenotypic field data from a genome wide association analysis with gene expression data, allowing us to detect significantly expressed quantitative trait loci (eQTL). These results identified significant association between flavonoid biosynthetic pathway genes and infection by A. flavus. In planta fungal infections showed that the resistant line, TZAR102, has a higher fold increase of the metabolites naringenin and luteolin than the susceptible line, Va35, when comparing untreated and fungal infected plants. These results suggest flavonoids contribute to plant resistance mechanisms against aflatoxin contamination through modulation of toxin accumulation in maize kernels.

Identification and functional analysis of the aspergillic acid gene cluster in Aspergillus flavus.

Aspergillus flavus can colonize important food staples and produce aflatoxins, a group of toxic and carcinogenic secondary metabolites. Previous in silico analysis of the A. flavus genome revealed 56 gene clusters predicted to be involved in the biosynthesis of secondary metabolites. A. flavus secondary metabolites produced during infection of maize seed are of particular interest, especially with respect to their roles in the biology of the fungus. A predicted nonribosomal peptide synthetase-like (NRPS-like) gene, designated asaC (AFLA_023020), present in the uncharacterized A. flavus secondary metabolite gene cluster 11 was previously shown to be expressed during the earliest stages of maize kernel infection. Cluster 11 is composed of six additional genes encoding a number of putative decorating enzymes as well as a transporter and transcription factor. We generated knock-out mutants of the seven predicted cluster 11 genes. LC-MS analysis of extracts from knockout mutants of these genes showed that they were responsible for the synthesis of the previously characterized antimicrobial mycotoxin aspergillic acid. Extracts of the asaC mutant showed no production of aspergillic acid or its precursors. Knockout of the cluster 11 P450 oxidoreductase afforded a pyrazinone metabolite, the aspergillic acid precursor deoxyaspergillic acid. The formation of hydroxyaspergillic acid was abolished in a desaturase/hydroxylase mutant. The hydroxamic acid functional group in aspergillic acid allows the molecule to bind to iron resulting in the production of a red pigment in A. flavus identified previously as ferriaspergillin. A reduction of aflatoxin B1 and cyclopiazonic acid that correlated with reduced fungal growth was observed in maize kernel infection assays when aspergillic acid biosynthesis in A. flavus is halted.

Inhibition of bacterial and filamentous fungal growth in high moisture, nonsterile corn with intermittent pumping of trans-2-hexenal vapor.

Trans-2-hexenal (T2H), a plant-produced aldehyde, was intermittently pumped over a 7 d period into a small, bench top model of stored corn (nonsterile, moisture content about 23%). Naturally occurring bacteria and fungi, including added Aspergillus flavus, grew rapidly on corn not treated with T2H vapor. However, intermittently pumped T2H (30 min per 2 h or 30 min per 12 h) significantly reduced bacterial and fungal viable populations, with nearly 100% fungal viability loss observed after either (1) one day of pumping at the 30 min per 2 h rate or (2) pumping cycles of 30 min per 12 h period over the initial 48 to 72 h of incubation. Data suggest that short-term intermittent fumigation of stored corn with T2H could prevent growth of bacteria and mycotoxigenic fungi such as A. flavus.

Estrogenic and antiestrogenic activities of phytoalexins from red kidney bean (Phaseolus vulgaris L.).

Legumes are the predominant source of isoflavones considered to be phytoestrogens that mimic the hormone 17ß-estradiol (E2). Due to the risks associated with hormone replacement therapy, there is a growing need for alternative sources of estrogenic formulations for the treatment of menopausal symptoms. Legume phytoalexins (induced isoflavones) are produced under conditions of stress that include insect damage, wounding, or application of elicitors. The estrogenic and antiestrogenic activities of methanolic extracts obtained from red kidney bean treated with the fungus Aspergillus sojae were compared with those of untreated controls using an estrogen responsive element-based (ERE) luciferase reporter assay. A. sojae-treated red kidney bean extracts displayed both estrogenic and antiestrogenic activities. Analysis of elicitor-treated red kidney bean extracts showed that A. sojae treatments achieved maximal levels of kievitone at 1199 ± 101 µg/g and phaseollin at 227.8 ± 44 µg/g. The phytoalexins kievitone and phaseollin were isolated from A. sojae-treated red kidney bean extracts and analyzed for estrogenic activity using ERα and ERß binding, ERE luciferase assays in MCF-7 and HEK 293 cells, and MCF-7 cell proliferation. Kievitone showed the highest relative binding affinity to ERα with kievitone (0.48%) > phaseollin (0.21%), and phaseollin showed the highest relative binding affinity to ERß with phaseollin (0.53%) > kievitone (0.42%). In an ERE luciferase assay in MCF-7 cells, kievitone displayed high ER transactivation at 10 µM; phaseollin displayed low ER transactivation. Both kievitone and phaseollin stimulated MCF-7 cell proliferation, with kievitone displaying agonist activity between 0.1 and 10 µM. Cotransfection reporter assays performed in HEK 293 demonstrated that phaseollin selectively increased ERE transcriptional activity of ERß and kievitone selectively increased ERE transcriptional activity of ERα. Although phaseollin displayed attenuation of ER transactivation in the ERE luciferase assay in MCF-7 cells, both phytoalexins attenuated the effects of E2 in an MCF-7 cell colonial survival assay. This work provides evidence that the red kidney bean phytoalexins kievitone and phaseollin possess both estrogenic and antiestrogenic activities.

Volatile profiles of toxigenic and non-toxigenic Aspergillus flavus using SPME for solid phase extraction.

Toxigenic and atoxigenic strains of Aspergillus flavus were grown on potato dextrose agar (PDA) and wetted (23% moisture) sterile, cracked corn for 14 and 21 days, respectively. Volatile compounds produced by A. flavus, as well as those present in the PDA controls and sterile cracked maize, were collected using solid-phase micro-extraction (SPME) and identified by gas chromatography/mass spectrometry. Results show that growth substrate had a major impact on the number and type of volatiles detected. Growth on sterile cracked maize produced many more volatiles than did potato dextrose agar. There were also differences observed in the type of volatiles produced between toxigenic and non-toxigenic isolates, as well as between isolates of the same toxigenic grouping.

Identification of the potent phytoestrogen glycinol in elicited soybean (Glycine max).

The primary induced isoflavones in soybean, the glyceollins, have been shown to be potent estrogen antagonists in vitro and in vivo. The discovery of the glyceollins' ability to inhibit cancer cell proliferation has led to the analysis of estrogenic activities of other induced isoflavones. In this study, we investigated a novel isoflavone, glycinol, a precursor to glyceollin that is produced in elicited soy. Sensitive and specific in vitro bioassays were used to determine that glycinol exhibits potent estrogenic activity. Estrogen-based reporter assays were performed, and glycinol displayed a marked estrogenic effect on estrogen receptor (ER) signaling between 1 and 10 microM, which correlated with comparable colony formation of MCF-7 cells at 10 microM. Glycinol also induced the expression of estrogen-responsive genes (progesterone receptor and stromal-cell-derived factor-1). Competitive binding assays revealed a high affinity of glycinol for both ER alpha (IC(50) = 13.8 nM) and ER beta (IC(50) = 9.1 nM). In addition, ligand receptor modeling (docking) studies were performed and glycinol was shown to bind similarly to both ER alpha and ER beta. Taken together, these results suggest for the first time that glycinol is estrogenic and may represent an important component of the health effects of soy-based foods.

Effect of soybean lipoxygenase on volatile generation and inhibition of Aspergillus flavus mycelial growth.

Volatiles generated from lipoxygenase (LOX) normal and LOX deficient soybean (Glycine max) varieties with and without added lipase inhibited Aspergillus flavus mycelial growth and aflatoxin production. Soybean volatiles were analyzed using a solid phase microextraction (SPME) method combined with gas chromatography-mass spectrometry (GC-MS). Twenty-one compounds, including 11 aldehydes, three alcohols, four ketones, one furan, one alkane, and one alkene were detected in the LOX normal soybean line. However, only nine volatile compounds were observed in the LOX deficient soybean variety. The antifungal aldehydes hexanal and (E)-2-hexenal were observed in both LOX normal and LOX deficient lines and were detected at significantly higher amounts in soybean homogenate with added lipase. These aldehydes may be formed through alternate pathways, other than the LOX pathway, and may account for the inhibition of A. flavus growth observed. Other volatiles detected, particularly the ketones and alcohols, may contribute to the antifungal activity observed in both LOX normal and LOX deficient soybean lines. These results suggest that other factors, other than LOX activity, may better explain why soybeans are generally not as severely affected by A. flavus and aflatoxin contamination as other oilseed crops.

Feeding and maturation by soybean looper (Lepidoptera: Noctuidae) larvae on soybean affected by weed, fungus, and nematode pests.

Feeding and maturation by the soybean looper, Pseudoplusia includens (Walker) (Lepidoptera: Noctuidae), were investigated in a 2-yr study on 'Davis' soybean, Glycine max (L.) Merr., grown alone and combined with the weed hemp sesbania, Sesbania exaltata (Raf.) Rybd. ex. A. W. Hill, the root-knot nematode, Meloidogyne incognita (Kofoid & White) Chitwood, and the charcoal rot fungus, Macrophomina phaseolina (Tassi) Goid. Of the three pests, hemp sesbania had the greatest effects on plant growth and insect feeding and maturation. When fed foliage from soybean stressed by hemp sesbania, soybean looper larvae remained longer in feeding stages, consumed more foliage, and showed altered weight gain compared with larvae fed control foliage. Results suggest that nutrient (s) critical for proper development of larvae may have been limited in weed-stressed soybean foliage. Less dramatic results were observed when larvae fed on foliage from soybean with roots colonized by the charcoal rot fungus. Such larvae consumed more foliage, weighed more, and showed a slight increase in larval feeding period, but only in 1 yr of the study. Colonization of soybean roots by the root-knot nematode had no consistent effects on either the soybean host or insect.

Identification of volatile compounds in soybean at various developmental stages using solid phase microextraction.

Soybean (Glycine max) seed volatiles were analyzed using a solid phase microextraction (SPME) method combined with gas chromatography-mass spectrometry (GC-MS). Thirty volatile compounds already reported for soybean were recovered, and an additional 19 compounds not previously reported were identified or tentatively identified. The SPME method was utilized to compare the volatile profile of soybean seed at three distinct stages of development. Most of the newly reported compounds in soybean seed were aldehydes and ketones. During early periods of development at maturity stage R6, several volatiles were present at relatively high concentrations, including 3-hexanone, (E)-2-hexenal, 1-hexanol, and 3-octanone. At maturity stage R7 and R8, decreased amounts of 3-hexanone, (E)-2-hexenal, 1-hexanol, and 3-octanone were observed. At maturity stage R8 hexanal, (E)-2-heptenal, (E)-2-octenal, ethanol, 1-hexanol, and 1-octen-3-ol were detected at relatively high concentrations. SPME offers the ability to differentiate between the three soybean developmental stages that yield both fundamental and practical information.

Identification of flavone aglycones and glycosides in soybean pods by liquid chromatography-tandem mass spectrometry.

High-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry was used to identify flavone aglycones and glycosides in soybean pods. Tandem mass spectrometry (MS-MS and MS-MS-MS) and photodiode array detection were also utilized in flavone characterization. A total of seven flavone aglycones and glycosides were identified. Among them three flavone aglycones--apigenin, 7,4'-dihydroxyflavone, and luteolin--and two flavone glycosides--apigenin-7-O-beta-D-glucoside, and luteolin-7-O-beta-D-glucoside--were unambiguously identified based on their abundant (M+H)+ ions, UV spectra, retention time, and tandem mass spectrometric analysis compared with authentic standards. The tentative identification of two flavone glycosides as 7,4'-dihydroxyflavone-7-O-beta-D-glucoside and apigenin-7-O-beta-D-glucoside-6"-O-malonate was based on UV spectra, (M+H)+ ions, and tandem mass spectrometry. This is the first report identifying flavone aglycones and glycosides in soybean pods.

Evaluation of the estrogenic effects of legume extracts containing phytoestrogens.

Seven legume extracts containing phytoestrogens were analyzed for estrogenic activity. Methanol extracts were prepared from soybean (Glycine max L.), green bean (Phaseolus vulgaris L.), alfalfa sprout (Medicago sativa L.), mung bean sprout (Vigna radiata L.), kudzu root (Pueraria lobata L.), and red clover blossom and red clover sprout (Trifolium pratense L.). Extracts of kudzu root and red clover blossom showed significant competitive binding to estrogen receptor beta (ERbeta). Estrogenic activity was determined using an estrogen-dependent MCF-7 breast cancer cell proliferation assay. Kudzu root, red clover blossom and sprout, mung bean sprout, and alfalfa sprout extracts displayed increased cell proliferation above levels observed with estradiol. The pure estrogen antagonist, ICI 182,780, suppressed cell proliferation induced by the extracts, suggesting an ER-related signaling pathway was involved. The ER subtype-selective activities of legume extracts were examined using transiently transfected human embryonic kidney (HEK 293) cells. All seven of the extracts exhibited preferential agonist activity toward ERbeta. Using HPLC to collect fractions and MCF-7 cell proliferation, the active components in kudzu root extract were determined to be the isoflavones puerarin, daidzin, genistin, daidzein, and genistein. These results show that several legumes are a source of phytoestrogens with high levels of estrogenic activity.