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.

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.

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.

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.

The Aspergillus flavus rtfA Gene Regulates Plant and Animal Pathogenesis and Secondary Metabolism.

Aspergillus flavus is an opportunistic fungal plant and human pathogen and a producer of mycotoxins, including aflatoxin B1 (AFB1). As part of our ongoing studies to elucidate the biological functions of the A. flavusrtfA gene, we examined its role in the pathogenicity of both plant and animal model systems. rtfA encodes a putative RNA polymerase II (Pol II) transcription elongation factor previously characterized in Saccharomyces cerevisiae, Aspergillus nidulans, and Aspergillus fumigatus, where it was shown to regulate several important cellular processes, including morphogenesis and secondary metabolism. In addition, an initial study in A. flavus indicated that rtfA also influences development and production of AFB1; however, its effect on virulence is unknown. The current study reveals that the rtfA gene is indispensable for normal pathogenicity in plants when using peanut seed as an infection model, as well as in animals, as shown in the Galleria mellonella infection model. Interestingly, rtfA positively regulates several processes known to be necessary for successful fungal invasion and colonization of host tissue, such as adhesion to surfaces, protease and lipase activity, cell wall composition and integrity, and tolerance to oxidative stress. In addition, metabolomic analysis revealed that A. flavusrtfA affects the production of several secondary metabolites, including AFB1, aflatrem, leporins, aspirochlorine, ditryptophenaline, and aflavinines, supporting a role of rtfA as a global regulator of secondary metabolism. Heterologous complementation of an A. flavusrtfA deletion strain with rtfA homologs from A. nidulans or S. cerevisiae fully rescued the wild-type phenotype, indicating that these rtfA homologs are functionally conserved among these three species.IMPORTANCE In this study, the epigenetic global regulator rtfA, which encodes a putative RNA-Pol II transcription elongation factor-like protein, was characterized in the mycotoxigenic and opportunistic pathogen A. flavus Specifically, its involvement in A. flavus pathogenesis in plant and animal models was studied. Here, we show that rtfA positively regulates A. flavus virulence in both models. Furthermore, rtfA-dependent effects on factors necessary for successful invasion and colonization of host tissue by A. flavus were also assessed. Our study indicates that rtfA plays a role in A. flavus adherence to surfaces, hydrolytic activity, normal cell wall formation, and response to oxidative stress. This study also revealed a profound effect of rtfA on the metabolome of A. flavus, including the production of potent mycotoxins.

Identification of a copper-transporting ATPase involved in biosynthesis of A. flavus conidial pigment.

Conidia are asexual spores and play a crucial role in fungal dissemination. Conidial pigmentation is important for tolerance against UV radiation and contributes to survival of fungi. The molecular basis of conidial pigmentation has been studied in several fungal species. In spite of sharing the initial common step of polyketide formation, other steps for pigment biosynthesis appear to be species-dependent. In this study, we isolated an Aspergillus flavus spontaneous mutant that produced yellow conidia. The underlying genetic defect, a three-nucleotide in-frame deletion in the gene, AFLA_051390, that encodes a copper-transporting ATPase, was identified by a comparative genomics approach. This genetic association was confirmed by disruption of the wild-type gene. When yellow mutants were grown on medium supplemented with copper ions or chloride ions, green conidial color was partially and nearly completely restored, respectively. Further disruption of AFLA_045660, an orthologue of Aspergillus nidulans yA (yellow pigment) that encodes a multicopper oxidase, in wild type and a derived strain producing dark green conidia showed that it yielded mutants that produced gold conidia. The results placed formation of the gold pigment after that of the yellow pigment and before that of the dark green pigment. Using reported inhibitors of DHN-melanin (tricyclazole and phthalide) and DOPA-melanin (tropolone and kojic acid) pathways on a set of conidial color mutants, we investigated the involvement of melanin biosynthesis in A. flavus conidial pigment formation. Results imply that both pathways have no bearing on conidial pigment biosynthesis of A. flavus.

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.

RNA interference-based silencing of the alpha-amylase (amy1) gene in Aspergillus flavus decreases fungal growth and aflatoxin production in maize kernels.

MAIN CONCLUSION: Expressing an RNAi construct in maize kernels that targets the gene for alpha-amylase in Aspergillus flavus resulted in suppression of alpha-amylase (amy1) gene expression and decreased fungal growth during in situ infection resulting in decreased aflatoxin production. Aspergillus flavus is a saprophytic fungus and pathogen to several important food and feed crops, including maize. Once the fungus colonizes lipid-rich seed tissues, it has the potential to produce toxic secondary metabolites, the most dangerous of which is aflatoxin. The pre-harvest control of A. flavus contamination and aflatoxin production is an area of intense research, which includes breeding strategies, biological control, and the use of genetically-modified crops. Host-induced gene silencing, whereby the host crop produces siRNA molecules targeting crucial genes in the invading fungus and targeting the gene for degradation, has shown to be promising in its ability to inhibit fungal growth and decrease aflatoxin contamination. Here, we demonstrate that maize inbred B104 expressing an RNAi construct targeting the A. flavus alpha-amylase gene amy1 effectively reduces amy1 gene expression resulting in decreased fungal colonization and aflatoxin accumulation in kernels. This work contributes to the development of a promising technology for reducing the negative economic and health impacts of A. flavus growth and aflatoxin contamination in food and feed crops.

Peanuts that keep aflatoxin at bay: a threshold that matters.

Aflatoxin contamination in peanuts poses major challenges for vulnerable populations of sub-Saharan Africa and South Asia. Developing peanut varieties to combat preharvest Aspergillus flavus infection and resulting aflatoxin contamination has thus far remained a major challenge, confounded by highly complex peanut-Aspergilli pathosystem. Our study reports achieving a high level of resistance in peanut by overexpressing (OE) antifungal plant defensins MsDef1 and MtDef4.2, and through host-induced gene silencing (HIGS) of aflM and aflP genes from the aflatoxin biosynthetic pathway. While the former improves genetic resistance to A. flavus infection, the latter inhibits aflatoxin production in the event of infection providing durable resistance against different Aspergillus flavus morphotypes and negligible aflatoxin content in several peanut events/lines well. A strong positive correlation was observed between aflatoxin accumulation and decline in transcription of the aflatoxin biosynthetic pathway genes in both OE-Def and HIGS lines. Transcriptomic signatures in the resistant lines revealed key mechanisms such as regulation of aflatoxin synthesis, its packaging and export control, besides the role of reactive oxygen species-scavenging enzymes that render enhanced protection in the OE and HIGS lines. This is the first study to demonstrate highly effective biotechnological strategies for successfully generating peanuts that are near-immune to aflatoxin contamination, offering a panacea for serious food safety, health and trade issues in the semi-arid regions.

The 14-3-3 Protein Homolog ArtA Regulates Development and Secondary Metabolism in the Opportunistic Plant Pathogen Aspergillus flavus.

The opportunistic plant-pathogenic fungus Aspergillus flavus produces carcinogenic mycotoxins termed aflatoxins (AF). Aflatoxin contamination of agriculturally important crops, such as maize, peanut, sorghum, and tree nuts, is responsible for serious adverse health and economic impacts worldwide. In order to identify possible genetic targets to reduce AF contamination, we have characterized the artA gene, encoding a putative 14-3-3 homolog in A. flavus The artA deletion mutant presents a slight decrease in vegetative growth and alterations in morphological development and secondary metabolism. Specifically, artA affects conidiation, and this effect is influenced by the type of substrate and culture condition. In addition, normal levels of artA are required for sclerotial development. Importantly, artA negatively regulates AF production as well as the concomitant expression of genes in the AF gene cluster. An increase in AF is also observed in seeds infected with the A. flavus strain lacking artA Furthermore, the expression of other secondary metabolite genes is also artA dependent, including genes in the cyclopiazonic acid (CPA) and ustiloxin gene clusters, in this agriculturally important fungus.IMPORTANCE In the current study, artA, which encodes a 14-3-3 homolog, was characterized in the agriculturally and medically important fungus Aspergillus flavus, specifically, its possible role governing sporulation, formation of resistant structures, and secondary metabolism. The highly conserved artA is necessary for normal fungal morphogenesis in an environment-dependent manner, affecting the balance between production of conidiophores and the formation of resistant structures that are necessary for the dissemination and survival of this opportunistic pathogen. This study reports a 14-3-3 protein affecting secondary metabolism in filamentous fungi. Importantly, artA regulates the biosynthesis of the potent carcinogenic compound aflatoxin B1 (AFB1) as well as the production of other secondary metabolites.

Cultural and Genetic Approaches to Manage Aflatoxin Contamination: Recent Insights Provide Opportunities for Improved Control.

Aspergillus flavus is a morphologically complex species that can produce the group of polyketide derived carcinogenic and mutagenic secondary metabolites, aflatoxins, as well as other secondary metabolites such as cyclopiazonic acid and aflatrem. Aflatoxin causes aflatoxicosis when aflatoxins are ingested through contaminated food and feed. In addition, aflatoxin contamination is a major problem, from both an economic and health aspect, in developing countries, especially Asia and Africa, where cereals and peanuts are important food crops. Earlier measures for control of A. flavus infection and consequent aflatoxin contamination centered on creating unfavorable environments for the pathogen and destroying contaminated products. While development of atoxigenic (nonaflatoxin producing) strains of A. flavus as viable commercial biocontrol agents has marked a unique advance for control of aflatoxin contamination, particularly in Africa, new insights into the biology and sexuality of A. flavus are now providing opportunities to design improved atoxigenic strains for sustainable biological control of aflatoxin. Further, progress in the use of molecular technologies such as incorporation of antifungal genes in the host and host-induced gene silencing, is providing knowledge that could be harnessed to develop germplasm that is resistant to infection by A. flavus and aflatoxin contamination. This review summarizes the substantial progress that has been made to understand the biology of A. flavus and mitigate aflatoxin contamination with emphasis on maize. Concepts developed to date can provide a basis for future research efforts on the sustainable management of aflatoxin contamination.

rtfA, a putative RNA-Pol II transcription elongation factor gene, is necessary for normal morphological and chemical development in Aspergillus flavus.

The filamentous fungus Aspergillus flavus is an agriculturally important opportunistic plant pathogen that produces potent carcinogenic compounds called aflatoxins. We identified the A. flavus rtfA gene, the ortholog of rtf1 in Saccharomyces cerevisiae and rtfA in Aspergillus nidulans. Interestingly, rtfA has multiple cellular roles in this mycotoxin-producing fungus. In this study, we show that rtfA regulates conidiation. The rtfA deletion mutant presented smaller conidiophores with significantly reduced conidial production compared to the wild-type strain. The absence of rtfA also resulted in a significant decrease or lack of sclerotial production under conditions that allowed abundant production of these resistance structures in the wild type. Importantly, the deletion of rtfA notably reduced the production of aflatoxin B1, indicating that rtfA is a regulator of mycotoxin biosynthesis in A. flavus. In addition, the deletion rtfA also altered the production of several unknown secondary metabolites indicating a broader regulatory scope. Furthermore, our study revealed that rtfA controls the expression of the global regulators veA and laeA, which further influence morphogenesis and secondary metabolism in A. flavus.

An Aspergillus flavus secondary metabolic gene cluster containing a hybrid PKS-NRPS is necessary for synthesis of the 2-pyridones, leporins.

The genome of the filamentous fungus, Aspergillus flavus, has been shown to harbor as many as 56 putative secondary metabolic gene clusters including the one responsible for production of the toxic and carcinogenic, polyketide synthase (PKS)-derived aflatoxins. Except for the production of aflatoxins, cyclopiazonic acid and several other metabolites the capability for metabolite production of most of these putative clusters is unknown. We investigated the regulation of expression of the PKS-non-ribosomal peptide synthetase (NRPS) containing cluster 23 and determined that it produces homologs of the known 2-pyridone leporin A. Inactivation and overexpression of a cluster 23 gene encoding a putative Zn(2)-Cys(6) transcription factor (AFLA_066900, lepE) resulted in downregulation of nine and up-regulation of 8, respectively, of the fifteen SMURF-predicted cluster 23 genes thus allowing delineation of the cluster. Overexpression of lepE (OE::lepE) resulted in transformants displaying orange-red pigmented hyphae. Mass spectral analysis of A. flavus OE::lepE extracts identified the known 2-pyridone metabolite, leporin B, as well as the previously unreported dehydroxy-precursor, leporin C. We provide strong evidence that leporin B forms a unique trimeric complex with iron, not found previously for other 2-pyridones. This iron complex demonstrated antiinsectan and antifeedant properties similar to those previously found for leporin A. The OE::lepE strain showed reduced levels of conidia and sclerotia suggesting that unscheduled leporin production affects fungal developmental programs.

VeA is associated with the response to oxidative stress in the aflatoxin producer Aspergillus flavus.

Survival of fungal species depends on the ability of these organisms to respond to environmental stresses. Osmotic stress or high levels of reactive oxygen species (ROS) can cause stress in fungi resulting in growth inhibition. Both eukaryotic and prokaryotic cells have developed numerous mechanisms to counteract and survive the stress in the presence of ROS. In many fungi, the HOG signaling pathway is crucial for the oxidative stress response as well as for osmotic stress response. This study revealed that while the osmotic stress response is only slightly affected by the master regulator veA, this gene, also known to control morphological development and secondary metabolism in numerous fungal species, has a profound effect on the oxidative stress response in the aflatoxin-producing fungus Aspergillus flavus. We found that the expression of A. flavus homolog genes involved in the HOG signaling pathway is regulated by veA. Deletion of veA resulted in a reduction in transcription levels of oxidative stress response genes after exposure to hydrogen peroxide. Furthermore, analyses of the effect of VeA on the promoters of cat1 and trxB indicate that the presence of VeA alters DNA-protein complex formation. This is particularly notable in the cat1 promoter, where the absence of VeA results in abnormally stronger complex formation with reduced cat1 expression and more sensitivity to ROS in a veA deletion mutant, suggesting that VeA might prevent binding of negative transcription regulators to the cat1 promoter. Our study also revealed that veA positively influences the expression of the transcription factor gene atfB and that normal formation of DNA-protein complexes in the cat1 promoter is dependent on AtfB.

Functional characterization of a veA-dependent polyketide synthase gene in Aspergillus flavus necessary for the synthesis of asparasone, a sclerotium-specific pigment.

The filamentous fungus, Aspergillus flavus, produces the toxic and carcinogenic, polyketide synthase (PKS)-derived family of secondary metabolites termed aflatoxins. While analysis of the A. flavus genome has identified many other PKSs capable of producing secondary metabolites, to date, only a few other metabolites have been identified. In the process of studying how the developmental regulator, VeA, affects A. flavus secondary metabolism we discovered that mutation of veA caused a dramatic down-regulation of transcription of a polyketide synthase gene belonging to cluster 27 and the loss of the ability of the fungi to produce sclerotia. Inactivation of the cluster 27 pks (pks27) resulted in formation of greyish-yellow sclerotia rather than the dark brown sclerotia normally produced by A. flavus while conidial pigmentation was unaffected. One metabolite produced by Pks27 was identified by thin layer chromatography and mass spectral analysis as the known anthraquinone, asparasone A. Sclerotia produced by pks27 mutants were significantly less resistant to insect predation than were the sclerotia produced by the wild-type and more susceptible to the deleterious effects of ultraviolet light and heat. Normal sclerotia were previously thought to be resistant to damage because of a process of melanization similar to that known for pigmentation of conidia. Our results show that the dark brown pigments in sclerotia derive from anthraquinones produced by Pks27 rather than from the typical tetrahydronapthalene melanin production pathway. To our knowledge this is the first report on the genes involved in the biosynthesis of pigments important for sclerotial survival.

The role of Aspergillus flavus veA in the production of extracellular proteins during growth on starch substrates.

The aflatoxin-producer and opportunistic plant pathogenic, filamentous fungus Aspergillus flavus is responsible for the contamination of corn and other important agricultural commodities. In order to obtain nutrients from the host A. flavus produces a variety of extracellular hydrolytic enzymes. Interestingly, A. flavus amylase and protease activity are dependent on the global regulator veA, a gene known to regulate morphogenesis and secondary metabolism in numerous fungi. Analysis of starch degradation by fungal enzymes secreted into broths of starch- or corn kernel-based media showed a notable accumulation of glucose in samples of the A. flavus control strain while the deletion veA sample accumulated high levels of maltose and maltotriose and only a small amount of glucose. Furthermore, SDS-PAGE and proteomics analysis of culture broths from starch- or corn kernel-based media demonstrated differential production of a number of proteins that included a reduction in the amount of a glucoamylase protein in the veA mutant compared to the control strain, while an alpha-amylase was produced in greater quantities in the veA mutant. Quantitative real-time PCR and western blot analyses using anti-glucoamylase or alpha-amylase antisera supported the proteomics results. Additionally, an overall reduction in protease activity was observed in the veA mutant including production of the alkaline protease, oryzin, compared to the control strain. These findings contribute to our knowledge of mechanisms controlling production of hydrolases and other extracellular proteins during growth of A. flavus on natural starch-based substrates.

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.

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.

Dothistromin genes at multiple separate loci are regulated by AflR.

In fungi, genes involved in the production of secondary metabolites are generally clustered at one location. There are some exceptions, such as genes required for synthesis of dothistromin, a toxin that is a chemical analog of the aflatoxin precursor versicolorin A and made by the pine needle pathogen Dothistroma septosporum. The availability of the D. septosporum genome sequence enabled identification of putative dothistromin genes, including an ortholog of the aflatoxin regulatory gene AflR, and revealed that most of the genes are spread over six separate regions (loci) on chromosome 12 (1.3 Mb). Here we show that levels of expression of the widely dispersed genes in D. septosporum are not correlated with gene location with respect to their distance from a telomere, but that AflR regulates them. The production of dothistromin by D. septosporum in which the AflR gene was knocked out (ΔDsAflR) was drastically reduced, but still detectable. This is in contrast to orthologous ΔAflR mutants in Aspergillus species that lack any aflatoxin production. Expression patterns in ΔDsAflR mutants helped to predict the complete set of genes involved in dothistromin production. This included a short-chain aryl alcohol dehydrogenase (NorB), which is located on chromosome 11 rather than chromosome 12, but was 24-fold down regulated in ΔDsAflR. An orthologous set of dothistromin genes, organized in a similar fragmented cluster arrangement to that seen in D. septosporum, was found in the closely related tomato pathogen Cladosporium fulvum even though this species does not produce dothistromin. In C. fulvum, pseudogenization of key biosynthetic genes explains the lack of dothistromin production. The fragmented arrangement of dothistromin genes provides an example of coordinated control of a dispersed set of secondary metabolite genes; it also provides an example where loss of dothistromin production might have allowed adaptation to a new pathogenic lifestyle.

NsdC and NsdD affect Aspergillus flavus morphogenesis and aflatoxin production.

The transcription factors NsdC and NsdD are required for sexual development in Aspergillus nidulans. We now show these proteins also play a role in asexual development in the agriculturally important aflatoxin (AF)-producing fungus Aspergillus flavus. We found that both NsdC and NsdD are required for production of asexual sclerotia, normal aflatoxin biosynthesis, and conidiophore development. Conidiophores in nsdC and nsdD deletion mutants had shortened stipes and altered conidial heads compared to those of wild-type A. flavus. Our results suggest that NsdC and NsdD regulate transcription of genes required for early processes in conidiophore development preceding conidium formation. As the cultures aged, the ΔnsdC and ΔnsdD mutants produced a dark pigment that was not observed in the wild type. Gene expression data showed that although AflR is expressed at normal levels, a number of aflatoxin biosynthesis genes are expressed at reduced levels in both nsd mutants. Expression of aflD, aflM, and aflP was greatly reduced in nsdC mutants, and neither aflatoxin nor the proteins for these genes could be detected. Our results support previous studies showing that there is a strong association between conidiophore and sclerotium development and aflatoxin production in A. flavus.