Aspergillus flavus Exploits Maize Kernels Using an "Orphan" Secondary Metabolite Cluster.

Aspergillus flavus is a saprophytic cosmopolitan fungus, capable of infecting crops both pre- and post-harvest and exploiting different secondary metabolites, including aflatoxins. Aflatoxins are known carcinogens to animals and humans, but display no clear effect in host plants such as maize. In a previous study, we mined the genome of A. flavus to identify secondary metabolite clusters putatively involving the pathogenesis process in maize. We now focus on cluster 32, encoding for fungal effectors such as salicylate hydroxylase (SalOH), and necrosis- and ethylene-inducing proteins (npp1 domain protein) whose expression is triggered upon kernel contact. In order to understand the role of this genetic cluster in maize kernel infection, mutants of A. flavus, impaired or enhanced in specific functions (e.g., cluster 32 overexpression), were studied for their ability to cause disease. Within this frame, we conducted histological and histochemical experiments to verify the expression of specific genes within the cluster (e.g., SalOH, npp1), the production of salicylate, and the presence of its dehydroxylated form. Results suggest that the initial phase of fungal infection (2 days) of the living tissues of maize kernels (e.g., aleuron) coincides with a significant increase of fungal effectors such as SalOH and Npp1 that appear to be instrumental in eluding host defences and colonising the starch-enriched tissues, and therefore suggest a role of cluster 32 to the onset of infection.

Transcriptome Sequencing Revealed an Inhibitory Mechanism of Aspergillus flavus Asexual Development and Aflatoxin Metabolism by Soy-Fermenting Non-Aflatoxigenic Aspergillus.

Aflatoxins (AFs) have always been regarded as the most effective carcinogens, posing a great threat to agriculture, food safety, and human health. Aspergillus flavus is the major producer of aflatoxin contamination in crops. The prevention and control of A. flavus and aflatoxin continues to be a global problem. In this study, we demonstrated that the cell-free culture filtrate of Aspergillus oryzae and a non-aflatoxigenic A. flavus can effectively inhibit the production of AFB1 and the growth and reproduction of A. flavus, indicating that both of the non-aflatoxigenic Aspergillus strains secrete inhibitory compounds. Further transcriptome sequencing was performed to analyze the inhibitory mechanism of A. flavus treated with fermenting cultures, and the results revealed that genes involved in the AF biosynthesis pathway and other biosynthetic gene clusters were significantly downregulated, which might be caused by the reduced expression of specific regulators, such as AflS, FarB, and MtfA. The WGCNA results further revealed that genes involved in the TCA cycle and glycolysis were potentially involved in aflatoxin biosynthesis. Our comparative transcriptomics also revealed that two conidia transcriptional factors, brlA and abaA, were found to be significantly downregulated, which might lead to the downregulation of conidiation-specific genes, such as the conidial hydrophobins genes rodA and rodB. In summary, our research provides new insights for the molecular mechanism of controlling AF synthesis to control the proliferation of A. flavus and AF pollution.

Antimicrobial Activities of Rhopalaea-Associated Fungus Aspergillus flavus strain MFABU9.

BACKGROUND AND OBJECTIVE: Rhopalaea is a genus of ascidian belonging to the family Diazonidae. Ascidians provide niches for various microorganisms including fungi. This present study describes the potential new source for natural bioactive compounds from Rhopalaea-associated fungi obtained from Bunaken marine park. MATERIALS AND METHODS: As part of an on-going research program to explore the chemical diversity of marine derived fungi, we performed an antimicrobial bioactivity-guided screening of EtOAc extracts of the fungi isolated from ascidian Rhopalaea sp. RESULTS: The study confirms that the ascidian obtained from Bunaken marine park was Rhopalaea sp. The fungus isolated from the ascidian was Aspergillus flavus which showed antimicrobial activity against bacteria Escherichia coli, Staphylococcus aereus, Aeromonas hydrophila and antifungal against the human pathogenic fungus Candida albicans. CONCLUSION: Aspergillus flavus isolated from ascidian Rhopalaea sp. has the potential as antibacterial and antifungal.

The bZIP Transcription Factor AflRsmA Regulates Aflatoxin B1 Biosynthesis, Oxidative Stress Response and Sclerotium Formation in Aspergillus flavus.

Fungal secondary metabolites play important roles not only in fungal ecology but also in humans living as beneficial medicine or harmful toxins. In filamentous fungi, bZIP-type transcription factors (TFs) are associated with the proteins involved in oxidative stress response and secondary metabolism. In this study, a connection between a bZIP TF and oxidative stress induction of secondary metabolism is uncovered in an opportunistic pathogen Aspergillus flavus, which produces carcinogenic and mutagenic aflatoxins. The bZIP transcription factor AflRsmA was identified by a homology research of A. flavus genome with the bZIP protein RsmA, involved in secondary metabolites production in Aspergillusnidulans. The AflrsmA deletion strain (ΔAflrsmA) displayed less sensitivity to the oxidative reagents tert-Butyl hydroperoxide (tBOOH) in comparison with wild type (WT) and AflrsmA overexpression strain (AflrsmAOE), while AflrsmAOE strain increased sensitivity to the oxidative reagents menadione sodium bisulfite (MSB) compared to WT and ΔAflrsmA strains. Without oxidative treatment, aflatoxin B1 (AFB1) production of ΔAflrsmA strains was consistent with that of WT, but AflrsmAOE strain produced more AFB1 than WT; tBOOH and MSB treatment decreased AFB1 production of ΔAflrsmA compared to WT. Besides, relative to WT, ΔAflrsmA strain decreased sclerotia, while AflrsmAOE strain increased sclerotia. The decrease of AFB1 by ΔAflrsmA but increase of AFB1 by AflrsmAOE was on corn. Our results suggest that AFB1 biosynthesis is regulated by AflRsmA by oxidative stress pathways and provide insights into a possible function of AflRsmA in mediating AFB1 biosynthesis response host defense in pathogen A. flavus.

Inactivation Efficacies and Mechanisms of Gas Plasma and Plasma-Activated Water against Aspergillus flavus Spores and Biofilms: a Comparative Study.

Atmospheric cold plasma (ACP) treatment is an emerging food technology for product safety and quality retention, shelf-life extension, and sustainable processing. The activated chemical species of ACP can act rapidly against microorganisms without leaving chemical residues on food surfaces. The main objectives of this study were to investigate the efficiency and mechanisms of inactivation of fungal spores and biofilms by ACP and to understand the effects of the gas-mediated and liquid-mediated modes of application against important fungal contaminants. Aspergillus flavus was selected as the model microorganism. A. flavus spores were exposed to either gas plasma (GP) or plasma-activated water (PAW), whereas gas plasma alone was used to treat A. flavus biofilms. This study demonstrated that both GP and PAW treatments independently resulted in significant decreases of A. flavus metabolic activity and spore counts, with maximal reductions of 2.2 and 0.6 log10 units for GP and PAW, respectively. The characterization of the reactive oxygen and nitrogen species in PAW and spore suspensions indicated that the concentration of secondary reactive species was an important factor influencing the antimicrobial activity of the treatment. The biofilm study showed that GP had detrimental effects on biofilm structure; however, the initial inoculum concentration prior to biofilm formation can be a crucial factor influencing the fungicidal effects of ACP.IMPORTANCE The production of mycotoxin-free food remains a challenge in both human and animal food chains. A. flavus, a mycotoxin-producing contaminant of economically important crops, was selected as the model microorganism to investigate the efficiency and mechanisms of ACP technology against fungal contaminants of food. Our study directly compares the antifungal properties of gas plasma (GP) and plasma-activated water (PAW) against fungi as well as reporting the effects of ACP treatment on biofilms produced by A. flavus.

Variation in Occurrence and Aflatoxigenicity of Aspergillus flavus from Two Climatically Varied Regions in Kenya.

Aflatoxins are carcinogenic chemical metabolites produced by Aspergillus spp. of the section Flavi. In Kenya, Aspergillus flavus is the most prevalent and has been associated with several acute and chronic aflatoxin outbreaks in the past. In this study, we evaluated the occurrence of A. flavus in soils from two agro-ecological regions with contrasting climatic conditions, aflatoxin contamination histories and cropping systems. Aspergillus spp. were first isolated from soils before the identification and determination of their aflatoxigenicity. Further, we determined the occurrence of Pseudomonas and Bacillus spp. in soils from the two regions. These bacterial species have long been associated with biological control of several plant pathogens including Aspergillus spp. Our results show that A. flavus occurred widely and produced comparatively higher total aflatoxin levels in all (100%) study sites from the eastern to the western regions of Kenya. For the western region, A. flavus was detected in 4 locations (66.7%) that were previously under maize cultivation with the isolates showing low aflatoxigenicity. A. flavus was not isolated from soils under sugarcane cultivation. Distribution of the two bacterial species varied across the regions but we detected a weak relationship between occurrence of bacterial species and A. flavus. We discuss these findings in the context of the influence of climate, microbial profiles, cropping systems and applicability in the deployment of biological control remedies against aflatoxin contamination.

Cellular, physiological and molecular approaches to investigate the antifungal and anti-aflatoxigenic effects of thyme essential oil on Aspergillus flavus.

Several approaches, including the detection of apoptotic-like cell death, aflatoxin B1 (AFB1) production and gene expression analysis, were carried out to provide insights into the antifungal and anti-aflatoxigenic effects of thyme essential oil (EO) on Aspergillus flavus. At 0.5 µL mL-1, thyme EO completely inhibited A. flavus growth. Furthermore, this antifungal activity triggered significant apoptosis, via nuclear condensation (87.5% of nuclei analyzed) and plasma membrane damage (in 100% of treated hyphae). Further analysis of AFB1 production and gene expression related to secondary metabolism (laeA) and the mechanism of virulence (lipA and meT) of A. flavus in the presence of thyme EO indicated important physiological changes related to its anti-aflatoxigenic property. These results highlight the potent antifungal abilities of thyme EO in controlling A. flavus and AFB1 production, especially the abilities that operate by exerting changes at the molecular level and inducing significant apoptotic-like cell death.

Transcriptome and proteome analyses of resistant preharvest peanut seed coat in response to Aspergillus flavus infection

BACKGROUND: The infection of peanut (Arachis hypogaea L.) seed coat by the pathogenic fungus Aspergillus flavus has highly negative economic and health impacts. However, the molecular mechanism underlying such defense response remains poorly understood. This study aims to address this issue by profiling the transcriptomic and proteomic changes that occur during the infection of the resistant peanut cultivar J11 by A. flavus. RESULTS: Transcriptomic study led to the detection of 13,539 genes, among which 663 exhibited differential expression. Further functional analysis found the differentially expressed genes to encode a wide range of pathogenesis- and/or defense-related proteins such as transcription factors, pathogenesis-related proteins, and chitinases. Changes in the expression patterns of these genes might contribute to peanut resistance to A. flavus. On the other hand, the proteomic profiling showed that 314 of the 1382 detected protein candidates were aberrantly expressed as a result of A. flavus invasion. However, the correlation between the transcriptomic and proteomic data was poor. We further demonstrated by in vitro fungistasis tests that hevamine-A, which was enriched at both transcript and protein levels, could directly inhibit the growth of A. flavus. Conclusions: The results demonstrate the power of complementary transcriptomic and proteomic analyses in the study of pathogen defense and resistance in plants and the chitinase could play an important role in the defense response of peanut to A. flavus. The current study also constitutes the first step toward building an integrated omics data platform for the development of Aspergillus-resistant peanut cultivars

The bZIP transcription factor Afap1 mediates the oxidative stress response and aflatoxin biosynthesis in Aspergillus flavus.

Aflatoxin is a carcinogenic secondary metabolite produced mainly by Aspergillus flavus and Aspergillus parasiticus, which can seriously endanger the health of humans and animals. Oxidative stress is a common defense response, and it is known that reactive oxygen species (ROS) can induce the synthesis of a series of secondary metabolites, including aflatoxin. By using mutants lacking the afap 1 gene, the role of afap1 gene in oxidative stress and aflatoxin synthesis was assessed. The growth of the mutant strains was significantly inhibited by the increase in the concentration of H2O2, inhibition was complete at 40mmol/l. However, in the quantitative analysis by HPLC, the concentration of AFB1 increased with the increased H2O2 until 10mmol/l. Following an analysis based on the information provided by the NCBI BLAST analysis, it was assumed that Afap1, a basic leucine zipper (bZIP) transcription factor, was associated with the oxidative stress in this fungus. Treatment with 5mmol/l H2O2 completely inhibited the growth of the mutant strains in afap 1 but did not affect the growth of the CA14PTs strain (non-mutant strain). In addition, the concentration of AFB1 in the mutant strains was approximately » of that observed in the CA14PTs strain. These results suggested that Afap1 plays a key role in the regulation of oxidative stress and aflatoxin production in A. flavus.

Transcriptional Regulation of Aflatoxin Biosynthesis and Conidiation in Aspergillus flavus by Wickerhamomyces anomalus WRL-076 for Reduction of Aflatoxin Contamination.

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.

Antioxidant, Antimicrobial and the Phenolic Content of Infusion, Decoction and Methanolic Extracts of Thyme and Rosmarinus Species.

BACKGROUND: The plant species Rosmarinus officinalis (RO), Thymus algeriensis (TA) and Thymus capitatus (TC) are widely used in traditional medicine in Tunisia. Their bioactivities have been reported before and particularly referred to their essential oils. The main objective of this work was to assess the phytochemical composition, the antioxidant activity, the antibacterial, antifungal, and cytotoxic potential of these 3 plants. METHOD: The High Performance Liquid Chromatography (HPLC), chemical tests and spectrophotometric methods were used for screening, quantification of phytochemicals and for antioxidant activities. Extracts were evaluated for antibacterial potential by the microdilution method. Antifungal activities were tested using the Poisoned food method against: Aspergillus niger and Aspergillus flavus. The cytotoxic potential of the plant extracts was checked using HCT 116 cultures. RESULTS: Results revealed that aqueous extracts are not toxic compared to the methanolic extracts. Phenolic compounds were detected and these extracts showed excellent antioxidant activity presenting dose-dependent relationship. For antibacterial potential, all tested strains are more sensitive to Thymus extracts than Rosmarinus extracts. However, for antifungal activities, only Rosmarinus extracts inhibited mycelial growth. HPLC analysis allowed the identification of ten compounds with the abundance of gallic acid. CONCLUSION: This study showed important bioactivities (antioxidant, antimicrobial, and safety potential) of the plant species RO, TA and TC used in traditional medicine.

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.

G Protein α Subunit GpaB is Required for Asexual Development, Aflatoxin Biosynthesis and Pathogenicity by Regulating cAMP Signaling in Aspergillus flavus.

The heterotrimeric G proteins are critical for signal transduction and function in numerous biological processes including vegetative growth, asexual development and fungal virulence in fungi. Here, we identified four G protein alpha subunits (GanA, GpaB, FadA and GaoC) in the notorious Aflatoxin-producing fungus Aspergillus flavus. GanA, GpaB and FadA have homologues in other fungal species, while GaoC is a novel one. Here, we showed that the loss function of gpaB displayed a defect in conidiophore formation and considerably reduced expression levels of conidia-specific genes brlA and abaA. A decreased viability of cell wall integrity stress and oxidative stress were also found in the ∆gpaB mutant. More importantly, aflatoxin (AF) biosynthesis and infection on crop seeds were severely impaired in the gpaB-deficient mutant. Further analyses demonstrated that the intracellular cAMP levels significantly reduced in the gpaB-deficient mutant compared to wildtype strains. Additionally, an alteration of PKA activities in the ∆gpaB mutant was also found. Overall, our results indicated that GpaB played diverse roles in asexual sporulation, AF biosynthesis and virulence by regulating cAMP signaling in Aspergillus flavus.

Phenotypic and functional profile of Th17 and Treg cells in allergic fungal sinusitis.

Interleukin-17 producing T helper (Th17) and regulatory T cells (Treg) cells have been identified to play a critical role in atopic inflammation. However, conflicting reports on the role of Th17/Treg cells in allergic fungal rhinosinusitis (AFRS) patients of different ethnicities has mystified its pathogenesis. To better understand the pathophysiological mechanisms involved in AFRS, we conducted a prospective, analytical, case-control study involving 40 confirmed immunocompetent AFRS patients and 20 healthy controls. The distribution of Th17 and Treg cells in PBMC, intracellular mRNA expression of retinoid orphan nuclear receptor (RORγt) in Th17 and forkhead transcription factor (FoxP3) in Treg cells, and serum cytokine levels were investigated. Aspergillus flavus was identified from majority (85%) of patient tissue biopsies. Total serum IgE level along with cytokines IL-17, IL-21, IL-1ß and TGF-ß were comparatively elevated in AFRS. Nevertheless, IL-2 and IL-10 were reduced. Higher percentages of CD3+CD4+ T cells in AFRS with increased expression of CD161 and/or IL-23R markers were observed. Though, lower percentages of CD4+CD25+ Treg cells with elevated expression of GITR were patent. Transcription factor RORγt mRNA was upregulated, whereas FoxP3 mRNA was downregulated in AFRS patients. This inclination of Th17/Treg balance towards Th17, and the proposed role of Tregs on Th1 and Th2 cells in AFRS, directed us to conclude that Aspergillus infestation may lead to development of atopy and immunological dysbalance inciting a Th17 driven response, thereby, promoting aggravation of nasal polyposis. The observation may provide new insight into the molecular mechanisms leading to revision of the classical paradigm.

Aflatoxins Contamination in Maize Products from Rural Communities in San Luis Potosi, Mexico.

BACKGROUND: Aflatoxins are a group of mycotoxins that have been associated with hepatic damage and cancer. Aflatoxins B1 and B2 are secondary metabolites produced by fungi Aspergillus. These toxins can be found in a variety of commodities, especially in maize, and have been studied around the world due to their effects in human health. The Latin American population is especially exposed to aflatoxins given that maize products can be found in traditional diets all over the continent. Interestingly, in Mexico, chronic hepatic diseases and cirrhosis are leading causes of death in adult population. METHODS: In order to observe the effect of physical variables like temperature and humidity, this study was conducted collecting samples in four different seasons, in two communities in the State of San Luis Potosi, in Mexico. The content of aflatoxins in tortillas was measured using immunoaffinity columns prior to HPLC-FLD analysis. FINDINGS: Results showed that 18% of samples exceeded the Mexican limits for AFB1; whereas, 26% of the samples exceeded the limits of the European Union for AFB1. The AFB1 was detected in 80% of samples in one site and higher concentrations were found in samples collected during fall and winter seasons. CONCLUSIONS: Lack of control in storing practices is the principal cause for the contamination of maize. Considering that maize products are part of the staple diet of Mexican population, our results show that AFB1 detection has to be declared a public health priority. Detection and prevention of aflatoxins through a surveillance program, may avoid chronic health effects.

Biodiversity of mycobiota throughout the Brazil nut supply chain: From rainforest to consumer.

A total of 172 Brazil nut samples (114 in shell and 58 shelled) from the Amazon rainforest region and São Paulo state, Brazil was collected at different stages of the Brazil nut production chain: rainforest, street markets, processing plants and supermarkets. The mycobiota of the Brazil nut samples were evaluated and also compared in relation to water activity. A huge diversity of Aspergillus and Penicillium species were found, besides Eurotium spp., Zygomycetes and dematiaceous fungi. A polyphasic approach using morphological and physiological characteristics, as well as molecular and extrolite profiles, were studied to distinguish species among the more important toxigenic ones in Aspergillus section Flavi and A. section Nigri. Several metabolites and toxins were found in these two sections. Ochratoxin A (OTA) was found in 3% of A. niger and 100% of A. carbonarius. Production of aflatoxins B and G were found in all isolates of A. arachidicola, A. bombycis, A. nomius, A. pseudocaelatus and A. pseudonomius, while aflatoxin B was found in 38% of A. flavus and all isolates of A. pseudotamarii. Cyclopiazonic acid (CPA) was found in A. bertholletius (94%), A. tamarii (100%), A. caelatus (54%) and A. flavus (41%). Tenuazonic acid, a toxin commonly found in Alternaria species was produced by A. bertholletius (47%), A. caelatus (77%), A. nomius (55%), A. pseudonomius (75%), A. arachidicola (50%) and A. bombycis (100%). This work shows the changes of Brazil nut mycobiota and the potential of mycotoxin production from rainforest to consumer, considering the different environments which exist until the nuts are consumed.

RNA Sequencing of Contaminated Seeds Reveals the State of the Seed Permissive for Pre-Harvest Aflatoxin Contamination and Points to a Potential Susceptibility Factor.

Pre-harvest aflatoxin contamination (PAC) is a major problem facing peanut production worldwide. Produced by the ubiquitous soil fungus, Aspergillus flavus, aflatoxin is the most naturally occurring known carcinogen. The interaction between fungus and host resulting in PAC is complex, and breeding for PAC resistance has been slow. It has been shown that aflatoxin production can be induced by applying drought stress as peanut seeds mature. We have implemented an automated rainout shelter that controls temperature and moisture in the root and peg zone to induce aflatoxin production. Using polymerase chain reaction (PCR) and high performance liquid chromatography (HPLC), seeds meeting the following conditions were selected: infected with Aspergillus flavus and contaminated with aflatoxin; and not contaminated with aflatoxin. RNA sequencing analysis revealed groups of genes that describe the transcriptional state of contaminated vs. uncontaminated seed. These data suggest that fatty acid biosynthesis and abscisic acid (ABA) signaling are altered in contaminated seeds and point to a potential susceptibility factor, ABR1, as a repressor of ABA signaling that may play a role in permitting PAC.

The Master Transcription Factor mtfA Governs Aflatoxin Production, Morphological Development and Pathogenicity in the Fungus Aspergillus flavus.

Aspergillus flavus produces a variety of toxic secondary metabolites; among them, the aflatoxins (AFs) are the most well known. These compounds are highly mutagenic and carcinogenic, particularly AFB1. A. flavus is capable of colonizing a number of economically-important crops, such as corn, cotton, peanut and tree nuts, and contaminating them with AFs. Molecular genetic studies in A. flavus could identify novel gene targets for use in strategies to reduce AF contamination and its adverse impact on food and feed supplies worldwide. In the current study, we investigated the role of the master transcription factor gene mtfA in A. flavus. Our results revealed that forced overexpression of mtfA results in a drastic decrease or elimination of several secondary metabolites, among them AFB1. The reduction in AFB1 was accompanied by a decrease in aflR expression. Furthermore, mtfA also regulates development; conidiation was influenced differently by this gene depending on the type of colonized substrate. In addition to its effect on conidiation, mtfA is necessary for the normal maturation of sclerotia. Importantly, mtfA positively affects the pathogenicity of A. flavus when colonizing peanut seeds. AF production in colonized seeds was decreased in the deletion mtfA strain and particularly in the overexpression strain, where only trace amounts were detected. Interestingly, a more rapid colonization of the seed tissue occurred when mtfA was overexpressed, coinciding with an increase in lipase activity and faster maceration of the oily part of the seed.

Adenylate Cyclase AcyA Regulates Development, Aflatoxin Biosynthesis and Fungal Virulence in Aspergillus flavus.

Aspergillus flavus is one of the most important opportunistic pathogens of crops and animals. The carcinogenic mycotoxin, aflatoxins produced by this pathogen cause a health problem to human and animals. Since cyclic AMP signaling controls a range of physiological processes, like fungal development and infection when responding to extracellular stimuli in fungal pathogens, in this study, we investigated the function of adenylate cyclase, a core component of cAMP signaling, in aflatoxins biosynthesis and virulence on plant seeds in A. flavus. A gene replacement strategy was used to generate the deletion mutant of acyA that encodes the adenylate cyclase. Severe defects in fungal growth, sporulation and sclerotia formation were observed in the acyA deletion mutant. The defect in radical growth could be partially rescued by exogenous cAMP analog. The acyA mutant was also significantly reduced in aflatoxins production and virulence. Similar to the former studies in other fungi, The acyA mutant showed enhancing tolerance to oxidative stress, but more sensitive to heat stress. Overall, the pleiotropic defects of the acyA deletion mutant indicates that the cAMP-PKA pathway is involved in fungal development, aflatoxins biosynthesis and plant seed invasion in A. flavus.

Larval Preference and Performance of Amyelois transitella (Navel Orangeworm, Lepidoptera: Pyralidae) in Relation to the Fungus Aspergillus flavus.

The navel orangeworm, Amyelois transitella (Walker), is a polyphagous pest of California nut crops and is responsible for extensive losses in the United States. It directly damages crops by feeding and contaminating nuts with frass and webbing and vectors saprophytic fungi that infect crops. The navel orangeworm is commonly associated with Aspergillus species, including the toxigenic Aspergillus flavus, which causes crop loss by producing carcinogens, including aflatoxin B1. This lepidopteran-fungus association is the most economically serious pest complex in Central Valley orchards, and evidence indicates that this relationship is mutualistic. We assessed preference and performance of navel orangeworm larvae associated with A. flavus in behavioral bioassays in which neonates were allowed to orient within arenas to media with or without fungal tissue, and performance bioassays in which larvae were reared with and without A. flavus on potato dextrose agar (PDA) and a semidefined almond PDA diet to evaluate effects on development and pupal weight. Navel orangeworm larvae were attracted to A. flavus and developed faster in its presence, indicating a nutritional benefit to the caterpillars. Larvae reached pupation ∼33% faster on diet containing A. flavus, and pupal weights were ∼18% higher for males and ∼13% higher for females on this diet. Our findings indicate that A. flavus plays an important role in larval orientation and development on infected hosts. The preference-performance relationship between navel orangeworms and Aspergillus flavus is consistent with a facultative mutualism that has broad implications for pest management efforts and basic understanding of Lepidoptera-plant interactions.