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.

Soil Biodegradation Resistance of Cotton Fiber Doped with Interior and Exterior Silver Nanoparticles.

Engineering fibers with nanomaterials is an effective way to modify their properties and responses to external stimuli. In this study, we doped cotton fibers with silver nanoparticles, both on the surface (126 ± 17 nm) and throughout the fiber cross section (18 ± 4 nm), and examined the resistance to soil biodegradation. A reagent-free one-pot treatment of a raw cotton fabric, where noncellulosic constituents of the raw cotton fiber and starch sizing served as reducing agents, produced silver nanoparticles with a total concentration of 11 g/kg. In a soil burial study spanning 16 weeks, untreated cotton underwent a sequential degradation process-fibrillation, fractionation, and merging-corresponding to the length of the soil burial period, whereas treated cotton did not exhibit significant degradation. The remarkable biodegradation resistance of the treated cotton was attributed to the antimicrobial properties of silver nanoparticles, as demonstrated through a test involving the soil-borne fungus Aspergillus flavus. The nonlinear loss behavior of silver from the treated cotton suggests that nanoparticle depletion in the soil depends on their location, with interior nanoparticles proving durable against environmental exposure.

Microbiota of maize kernels as influenced by Aspergillus flavus infection in susceptible and resistant inbreds.

Background: Nearly everything on Earth harbors a microbiome. A microbiome is a community of microbes (bacteria, fungi, and viruses) with potential to form complex networks that involve mutualistic and antagonistic interactions. Resident microbiota on/in an organism are determined by the external environment, both biotic and abiotic, and the intrinsic adaptability of each organism. Although the maize microbiome has been characterized, community changes that result from the application of fungal biocontrol strains, such as non-aflatoxigenic Aspergillus flavus, have not. Methods: We silk channel inoculated field-grown maize separately with a non-aflatoxigenic biocontrol strain (K49), a highly toxigenic strain (Tox4), and a combination of both A. flavus strains. Two maize inbreds were treated, A. flavus-susceptible B73 and A. flavus-resistant CML322. We then assessed the impacts of A. flavus introduction on the epibiota and endobiota of their maize kernels. Results: We found that the native microbial communities were significantly affected, irrespective of genotype or sampled tissue. Overall, bacteriomes exhibited greater diversity of genera than mycobiomes. The abundance of certain genera was unchanged by treatment, including genera of bacteria (e.g., Enterobacter, Pantoea) and fungi (e.g., Sarocladium, Meyerozyma) that are known to be beneficial, antagonistic, or both on plant growth and health. Conclusion: Beneficial microbes like Sarocladium that responded well to A. flavus biocontrol strains are expected to enhance biocontrol efficacy, while also displacing/antagonizing harmful microbes.

Vibrio gazogenes-dependent disruption of aflatoxin biosynthesis in Aspergillus flavus: the connection with endosomal uptake and hyphal morphogenesis.

Aflatoxins, a family of fungal secondary metabolites, are toxic and carcinogenic compounds that pose an enormous threat to global food safety and agricultural sustainability. Specifically agricultural products in African, Southeast Asian and hot and humid regions of American countries suffer most damage from aflatoxin producing molds due to the ideal climate conditions promoting their growth. Our recent studies suggest that Vibrio gazogenes (Vg), an estuarine bacterium non-pathogenic to plants and humans, can significantly inhibit aflatoxin biosynthesis in the producers. In this study, we investigated the mechanism underlying Vg-dependent aflatoxin inhibition using the prominent aflatoxin producer, Aspergillus flavus. We show that aflatoxin inhibition upon Vg treatment was associated with fungal uptake of Vg-prodigiosin, a red pigment, which was consistently visible inside fungal hyphae during treatment. The association of prodigiosin with aflatoxin inhibition was further evident as Serratia marcescens, another prodigiosin producer, significantly inhibited aflatoxin, while non-producers like Escherichia coli, Staphylococcus aureus, Vibrio harveyi, and Vibrio fischeri did not. Also, pure prodigiosin significantly inhibited aflatoxin biosynthesis. Endocytosis inhibitors, filipin and natamycin, reduced the Vg-prodigiosin uptake by the fungus leading to a significant increase in aflatoxin production, suggesting that uptake is endocytosis-dependent. The Vg treatment also reduced hyphal fusion (>98% inhibition) and branching, which are both endosome-dependent processes. Our results, therefore, collectively support our theory that Vg-associated aflatoxin inhibition is mediated by an endocytosis-dependent uptake of Vg-prodigiosin, which possibly leads to a disruption of normal endosomal functions.

Targeting the Aspergillus flavus p2c gene through host-induced gene silencing reduces A. flavus infection and aflatoxin contamination in transgenic maize.

Aspergillus flavus is an opportunistic fungal pathogen that infects maize and produces aflatoxins. Using biocontrol or developing resistant cultivars to reduce aflatoxin contamination has only achieved limited success. Here, the A. flavus polygalacturonase gene (p2c) was targeted for suppression through host-induced gene silencing (HIGS) to reduce aflatoxin contamination in maize. An RNAi vector carrying a portion of the p2c gene was constructed and transformed into maize B104. Thirteen out of fifteen independent transformation events were confirmed to contain p2c. The T2 generation kernels containing the p2c transgene had less aflatoxin than those without the transgene in six out of eleven events we examined. Homozygous T3 transgenic kernels from four events produced significantly less aflatoxins (P ≤ 0.02) than the kernels from the null or B104 controls under field inoculation conditions. The F1 kernels from the crosses between six elite inbred lines with P2c5 and P2c13 also supported significantly less aflatoxins (P ≤ 0.02) than those from the crosses with null plants. The reduction in aflatoxin ranged from 93.7% to 30.3%. Transgenic leaf (T0 and T3) and kernel tissues (T4) were also found to have significantly higher levels of p2c gene-specific small RNAs. Further, homozygous transgenic maize kernels had significantly less fungal growth (27~40 fold) than the null control kernels 10 days after fungal inoculation in the field. The calculated suppression of p2c gene expression based on RNAseq data was 57.6% and 83.0% in P2c5 and P2c13 events, respectively. These results indicate clearly that the reduced aflatoxin production in the transgenic kernels is due to RNAi-based suppression of p2c expression, which results in reduced fungal growth and toxin production.

Multiplexed Host-Induced Gene Silencing of Aspergillus flavus Genes Confers Aflatoxin Resistance in Groundnut.

Aflatoxins are immunosuppressive and carcinogenic secondary metabolites, produced by the filamentous ascomycete Aspergillus flavus, that are hazardous to animal and human health. In this study, we show that multiplexed host-induced gene silencing (HIGS) of Aspergillus flavus genes essential for fungal sporulation and aflatoxin production (nsdC, veA, aflR, and aflM) confers enhanced resistance to Aspergillus infection and aflatoxin contamination in groundnut (<20 ppb). Comparative proteomic analysis of contrasting groundnut genotypes (WT and near-isogenic HIGS lines) supported a better understanding of the molecular processes underlying the induced resistance and identified several groundnut metabolites that might play a significant role in resistance to Aspergillus infection and aflatoxin contamination. Fungal differentiation and pathogenicity proteins, including calmodulin, transcriptional activator-HacA, kynurenine 3-monooxygenase 2, VeA, VelC, and several aflatoxin pathway biosynthetic enzymes, were downregulated in Aspergillus infecting the HIGS lines. Additionally, in the resistant HIGS lines, a number of host resistance proteins associated with fatty acid metabolism were strongly induced, including phosphatidylinositol phosphate kinase, lysophosphatidic acyltransferase-5, palmitoyl-monogalactosyldiacylglycerol Δ-7 desaturase, ceramide kinase-related protein, sphingolipid Δ-8 desaturase, and phospholipase-D. Combined, this knowledge can be used for groundnut pre-breeding and breeding programs to provide a safe and secure food supply.

Broad-Spectrum Antimicrobial Activity of Synthetic Peptides GV185 and GV187.

Optimizing synthetic antimicrobial peptides for safe and enhanced activity against fungal and bacterial pathogens is useful for genetic engineering of plants for resistance to plant pathogens and their associated mycotoxins. Nine synthetic peptides modeled after lytic peptides tachyplesin 1, D4E1 from cecropin A, and protegrin 1 were added to germinated spores of fungal species Aspergillus flavus, Rhizopus stolonifer, Fusarium oxysporum f. sp. vasinfectum, F. verticillioides, F. graminearum, Claviceps purpurea, Verticillium dahliae, and Thielaviopsis basicola and bacterial cultures of Pseudomonas syringae pv. tabaci and Xanthomonas campestris pv. campestris at different doses and inhibitory dose response curves, and were modeled to assess antimicrobial activity. Peptides GV185 and GV187, modified from tachyplesin 1, had superior abilities to inhibit fungal and bacterial growth (50% inhibitory concentrations [IC50] ranging from 0.1 to 8.7 µM). R. stolonifer (IC50 = 8.1 µM), A. flavus (IC50 = 3.1 µM), and F. graminearum (IC50 = 2.2 µM) were less inhibited by GV185 and GV187 than all the remaining fungi (IC50 = 1.4 µM) and bacteria (IC50 = 0.1 µM). Of the remaining peptides, GV193, GV195, and GV196 (IC50 range of 0.9 to 6.6 µM) inhibited fungal growth of A. flavus, F. verticillioides, and F. graminearum less than GV185 and GV187 (IC50 range of 0.8 to 3.9 µM), followed by GV197 (IC50 range of 0.8 to 9.1 µM), whereas GV190 and GV192 inhibited poorly (IC50 range of 28.2 to 36.6 µM and 15.5 to 19.4 µM, respectively) and GV198 stimulated growth. GV185 and GV187 had slightly weaker hydrophobic and cationic residues than other tachyplesin 1 modified peptides but still had unexpectedly high lytic activity. Germinated fungal spores of R. stolonifer and F. graminearum exposed to these two peptides and D4E1 and AGM182 appeared wrinkled, with perforations near potential cytoplasmic leakage, which provided evidence of plasma membrane and cell wall lysis. We conclude that peptides GV185 and GV187 are promising candidates for genetic engineering of crops for resistance to plant-pathogenic bacteria and fungi, including A. flavus and aflatoxin contamination.

Putative Core Transcription Factors Affecting Virulence in Aspergillus flavus during Infection of Maize.

Aspergillus flavus is an opportunistic pathogen responsible for millions of dollars in crop losses annually and negative health impacts on crop consumers globally. A. flavus strains have the potential to produce aflatoxin and other toxic secondary metabolites, which often increase during plant colonization. To mitigate the impacts of this international issue, we employ a range of strategies to directly impact fungal physiology, growth and development, thus requiring knowledge on the underlying molecular mechanisms driving these processes. Here we utilize RNA-sequencing data that are obtained from in situ assays, whereby Zea mays kernels are inoculated with A. flavus strains, to select transcription factors putatively driving virulence-related gene networks. We demonstrate, through growth, sporulation, oxidative stress-response and aflatoxin/CPA analysis, that three A. flavus strains with knockout mutations for the putative transcription factors AFLA_089270, AFLA_112760, and AFLA_031450 demonstrate characteristics such as reduced growth capacity and decreased aflatoxin/CPA accumulation in kernels consistent with decreased fungal pathogenicity. Furthermore, AFLA_089270, also known as HacA, eliminates CPA production and impacts the fungus's capacity to respond to highly oxidative conditions, indicating an impact on plant colonization. Taken together, these data provide a sound foundation for elucidating the downstream molecular pathways potentially contributing to fungal virulence.

Dataset for transcriptomic profiles associated with development of sexual structures in Aspergillus flavus.

Information on the transcriptomic changes that occur within sclerotia of Aspergillus flavus during its sexual cycle is very limited and warrants further research. The findings will broaden our knowledge of the biology of A. flavus and can provide valuable insights in the development or deployment of non-toxigenic strains as biocontrol agents against aflatoxigenic strains. This article presents transcriptomic datasets included in our research article entitled, "Development of sexual structures influences metabolomic and transcriptomic profiles in Aspergillus flavus" [1], which utilized transcriptomics to identify possible genes and gene clusters associated with sexual reproduction and fertilization in A. flavus. RNA was extracted from sclerotia of a high fertility cross (Hi-Fert-Mated), a low fertility cross (Lo-Fert-Mated), and unmated strains (Hi-Fert-Unmated and Lo-Fert-Unmated) of A. flavus collected immediately after crossing and at every two weeks until eight weeks of incubation on mixed cereal agar at 30 °C in continuous darkness (n = 4 replicates from each treatment for each time point; 80 total). Raw sequencing reads obtained on an Illumina NovaSeq 6000 were deposited in NCBI's Sequence Read Archive (SRA) repository under BioProject accession number PRJNA789260. Reads were mapped to the A. flavus NRRL 3357 genome (assembly JCVI-afl1-v2.0; GCA_000006275.2) using STAR software. Differential gene expression analyses, functional analyses, and weighted gene co-expression network analysis were performed using DESeq2 R packages. The raw and analyzed data presented in this article could be reused for comparisons with other datasets to obtain transcriptional differences among strains of A. flavus or closely related species. The data can also be used for further investigation of the molecular basis of different processes involved in sexual reproduction and sclerotia fertility in A. flavus.

Development of sexual structures influences metabolomic and transcriptomic profiles in Aspergillus flavus.

Sclerotium (female) fertility, the ability of a strain to produce ascocarps, influences internal morphological changes during sexual reproduction in Aspergillus flavus. Although sclerotial morphogenesis has been linked to secondary metabolite (SM) biosynthesis, metabolic and transcriptomic changes within A. flavus sclerotia during sexual development are not known. Successful mating between compatible strains may result in relatively high or low numbers of ascocarps being produced. Sclerotia from a high fertility cross (Hi-Fert-Mated), a low fertility cross (Lo-Fert-Mated), unmated strains (Hi-Fert-Unmated and Lo-Fert-Unmated) were harvested immediately after crosses were made and every two weeks until 8 weeks of incubation, then subjected to targeted metabolomics (n = 106) and transcriptomics analyses (n = 80). Aflatoxin B1 production varied between Hi-Fert-Mated and Hi-Fert-Unmated sclerotia, while it remained low or was undetected in Lo-Fert-Mated and Lo-Fert-Unmated sclerotia. Profiling of 14 SMs showed elevated production of an aflavazole analog, an aflavinine isomer, and hydroxyaflavinine in Hi-Fert-Mated sclerotia at 4 to 8 weeks. Similarly, genes ayg1, hxtA, MAT1, asd-3, preA and preB, and genes in uncharacterized SM gene clusters 30 and 44 showed increased expression in Hi-Fert-Mated sclerotia at these time points. These results broaden our knowledge of the biochemical and transcriptional processes during sexual development in A. flavus.

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.

Resistance to Aflatoxin Accumulation in Maize Mediated by Host-Induced Silencing of the Aspergillus flavus Alkaline Protease (alk) Gene.

Aspergillus flavus is a fungal pathogen that infects maize and produces aflatoxins. Host-Induced Gene Silencing (HIGS) has been shown to reduce host infection by various fungal pathogens. Here, the A. flavus alkaline protease (alk) gene was targeted for silencing through HIGS. An RNAi vector carrying a portion of the alk gene was incorporated into the B104 maize genome. Four out of eight transformation events containing the alk gene, Alk-3, Alk-4, Alk-7 and Alk-9, were self-pollinated to T4/T6 generations. At T3, the Alk-transgenic lines showed up to 87% reduction in aflatoxin accumulation under laboratory conditions. T4 transgenic Alk-3 and Alk-7 lines, and T5 and T6 Alk-4 and Alk-9 showed an average of 84% reduction in aflatoxin accumulation compared to their null controls under field inoculations (p < 0.05). F1 hybrids of three elite maize inbred lines and the transgenic lines also showed significant improvement in aflatoxin resistance (p < 0.006 to p < 0.045). Reduced A. flavus growth and levels of fungal ß-tubulin DNA were observed in transgenic kernels during in vitro inoculation. Alk-4 transgenic leaf and immature kernel tissues also contained about 1000-fold higher levels of alk-specific small RNAs compared to null controls, indicating that the enhanced aflatoxin resistance in the transgenic maize kernels is due to suppression of A. flavus infection through HIGS of alk gene.

Genetic Responses and Aflatoxin Inhibition during Co-Culture of Aflatoxigenic and Non-Aflatoxigenic Aspergillus flavus.

Aflatoxin is a carcinogenic mycotoxin produced by Aspergillus flavus. Non-aflatoxigenic (Non-tox) A. flavus isolates are deployed in corn fields as biocontrol because they substantially reduce aflatoxin contamination via direct replacement and additionally via direct contact or touch with toxigenic (Tox) isolates and secretion of inhibitory/degradative chemicals. To understand touch inhibition, HPLC analysis and RNA sequencing examined aflatoxin production and gene expression of Non-tox isolate 17 and Tox isolate 53 mono-cultures and during their interaction in co-culture. Aflatoxin production was reduced by 99.7% in 72 h co-cultures. Fewer than expected unique reads were assigned to Tox 53 during co-culture, indicating its growth and/or gene expression was inhibited in response to Non-tox 17. Predicted secreted proteins and genes involved in oxidation/reduction were enriched in Non-tox 17 and co-cultures compared to Tox 53. Five secondary metabolite (SM) gene clusters and kojic acid synthesis genes were upregulated in Non-tox 17 compared to Tox 53 and a few were further upregulated in co-cultures in response to touch. These results suggest Non-tox strains can inhibit growth and aflatoxin gene cluster expression in Tox strains through touch. Additionally, upregulation of other SM genes and redox genes during the biocontrol interaction demonstrates a potential role of inhibitory SMs and antioxidants as additional biocontrol mechanisms and deserves further exploration to improve biocontrol formulations.

Changes in Bacterial Endophyte Community Following Aspergillus flavus Infection in Resistant and Susceptible Maize Kernels.

Aspergillus flavus (A. flavus)-mediated aflatoxin contamination in maize is a major global economic and health concern. As A. flavus is an opportunistic seed pathogen, the identification of factors contributing to kernel resistance will be of great importance in the development of novel mitigation strategies. Using V3-V4 bacterial rRNA sequencing and seeds of A. flavus-resistant maize breeding lines TZAR102 and MI82 and a susceptible line, SC212, we investigated kernel-specific changes in bacterial endophytes during infection. A total of 81 bacterial genera belonging to 10 phyla were detected. Bacteria belonging to the phylum Tenericutes comprised 86-99% of the detected phyla, followed by Proteobacteria (14%) and others (<5%) that changed with treatments and/or genotypes. Higher basal levels (without infection) of Streptomyces and Microbacterium in TZAR102 and increases in the abundance of Stenotrophomonas and Sphingomonas in MI82 following infection may suggest their role in resistance. Functional profiling of bacteria using 16S rRNA sequencing data revealed the presence of bacteria associated with the production of putative type II polyketides and sesquiterpenoids in the resistant vs. susceptible lines. Future characterization of endophytes predicted to possess antifungal/ anti-aflatoxigenic properties will aid in their development as effective biocontrol agents or microbiome markers for maize aflatoxin resistance.

Chemical repertoire and biosynthetic machinery of the Aspergillus flavus secondary metabolome: A review.

Filamentous fungi represent a rich source of extrolites, including secondary metabolites (SMs) comprising a great variety of astonishing structures and interesting bioactivities. State-of-the-art techniques in genome mining, genetic manipulation, and secondary metabolomics have enabled the scientific community to better elucidate and more deeply appreciate the genetic and biosynthetic chemical arsenal of these microorganisms. Aspergillus flavus is best known as a contaminant of food and feed commodities and a producer of the carcinogenic family of SMs, aflatoxins. This fungus produces many SMs including polyketides, ribosomal and nonribosomal peptides, terpenoids, and other hybrid molecules. This review will discuss the chemical diversity, biosynthetic pathways, and biological/ecological role of A. flavus SMs, as well as their significance concerning food safety and security.

Draft Genome Sequences of 20 Aspergillus flavus Isolates from Corn Kernels and Cornfield Soils in Louisiana.

Aspergillus flavus is a common saprophyte and opportunistic fungal pathogen that infects plants, animals, and humans. It also produces numerous toxic and nontoxic secondary metabolites. Here, we report the draft genome sequences of 20 A. flavus isolates, belonging to 16 vegetative compatibility groups, from Louisiana corn kernels and cornfield soils.

Characterization of morphological changes within stromata during sexual reproduction in Aspergillus flavus.

Aspergillus flavus contaminates agricultural products worldwide with carcinogenic aflatoxins that pose a serious health risk to humans and animals. The fungus survives adverse environmental conditions through production of sclerotia. When fertilized by a compatible conidium of an opposite mating type, a sclerotium transforms into a stroma within which ascocarps, asci, and ascospores are formed. However, the transition from a sclerotium to a stroma during sexual reproduction in A. flavus is not well understood. Early events during the interaction between sexually compatible strains of A. flavus were visualized using conidia of a green fluorescent protein (GFP)-labeled MAT1-1 strain and sclerotia of an mCherry-labeled MAT1-2 strain. Both conidia and sclerotia of transformed strains germinated to produce hyphae within 24 h of incubation. Hyphal growth of these two strains produced what appeared to be a network of interlocking hyphal strands that were observed at the base of the mCherry-labeled sclerotia (i.e., region in contact with agar surface) after 72 h of incubation. At 5 wk following incubation, intracellular green-fluorescent hyphal strands were observed within the stromatal matrix of the mCherry-labeled strain. Scanning electron microscopy of stromata from a high- and low-fertility cross and unmated sclerotia was used to visualize the formation and development of sexual structures within the stromatal and sclerotial matrices, starting at the time of crossing and thereafter every 2 wk until 8 wk of incubation. Morphological differences between sclerotia and stromata became apparent at 4 wk of incubation. Internal hyphae and croziers were detected inside multiple ascocarps that developed within the stromatal matrix of the high-fertility cross but were not detected in the matrix of the low-fertility cross or the unmated sclerotia. At 6 to 8 wk of incubation, hyphal tips produced numerous asci, each containing one to eight ascospores that emerged out of an ascus following the breakdown of the ascus wall. These observations broaden our knowledge of early events during sexual reproduction and suggest that hyphae from the conidium-producing strain may be involved in the early stages of sexual reproduction in A. flavus. When combined with omics data, these findings could be useful in further exploration of the molecular and biochemical mechanisms underlying sexual reproduction in A. flavus.

Host Induced Gene Silencing Targeting Aspergillus flavus aflM Reduced Aflatoxin Contamination in Transgenic Maize Under Field Conditions.

Maize (Zea mays L.) is one of the major crops susceptible to Aspergillus flavus infection and subsequent contamination with aflatoxins, the most potent naturally produced carcinogenic secondary metabolites. This pathogen can pose serious health concerns and cause severe economic losses due to the Food and Drug Administration (FDA) regulations on permissible levels of aflatoxins in food and feed. Although biocontrol has yielded some successes in managing aflatoxin contamination, enhancing crop resistance is still the preferred choice of management for long-term sustainability. Hence, host induced gene silencing (HIGS) strategy was explored in this study. The A. flavus gene aflM encoding versicolorin dehydrogenase, a key enzyme involved in the aflatoxin biosynthetic pathway, was selected as a possible target for suppression through HIGS. An RNAi vector containing a portion of the aflM gene was constructed and introduced into immature B104 maize zygotic embryos through Agrobacterium transformation. PCR analysis of the genomic DNA from T0 leaf tissue confirmed the presence of the transgene in six out of the seven events. The seeds from the lines that showed reduced aflatoxin production in laboratory aflatoxin kernel screening assay (KSA) have been increased from T1 to T4 generation in the past four years. Changes in aflatoxin resistance in these transgenic kernels have been evaluated under both field and laboratory conditions. The T2 generation kernels containing the transgene from two events out of four examined had less aflatoxin (P ≤ 0.01 and P ≤ 0.08) than those without the transgene. Field-inoculated homozygous T3 and T4 transgenic kernels also revealed lower levels of aflatoxins (P ≤ 0.04) than kernels from the null (segregated non-transgenic samples) or B104 controls. A similar result was observed when the harvested T3 and T4 homozygous transgenic kernels were evaluated under KSA conditions without inoculation (P ≤ 0.003-0.05). These two events were crossed with LH195, LH197, LH210, and PHW79 elite breeding lines and the resulting crosses supported less aflatoxin (P ≤ 0.02) than the crosses made with non-transgenic lines. In addition, significantly higher levels of aflM gene-specific small RNAs were detected in the transgenic leaf and kernel tissues, indicating that the enhanced aflatoxin resistance in the homozygous transgenic kernels is likely due to suppression of aflM expression through HIGS.

Biosynthesis of conidial and sclerotial pigments in Aspergillus species.

Fungal pigments, which are classified as secondary metabolites, are polymerized products derived mostly from phenolic precursors with remarkable structural diversity. Pigments of conidia and sclerotia serve myriad functions. They provide tolerance against various environmental stresses such as ultraviolet light, oxidizing agents, and ionizing radiation. Some pigments even play a role in fungal pathogenesis. This review gathers available research and discusses current knowledge on the formation of conidial and sclerotial pigments in aspergilli. It examines organization of genes involved in pigment production, biosynthetic pathways, and biological functions and reevaluates some of the current dogma, especially with respect to the DHN-melanin pathway, on the production of these enigmatic polymers. A better understanding of the structure and biosynthesis of melanins and other pigments could facilitate strategies to mitigate fungal pathogenesis.