Development of Loop-Mediated Isothermal Amplification (LAMP) Assays for the Rapid Detection of Toxigenic Aspergillus flavus and A. carbonarius in Nuts.

Aspergillus species create major postharvest problems due to the food losses caused by their mere presence and the hazardous mycotoxins they produce, such as aflatoxin B1 (AFB1) and ochratoxin A (OTA). These mycotoxins are mainly produced by A. flavus and A. carbonarius, respectively. In this study, we developed a rapid detection method for the two aforementioned species based on loop-mediated isothermal amplification (LAMP). The primers were designed to target genes belonging to the mycotoxin clusters pks and aflT for A. carbonarius and A. flavus, respectively. Result visualization was carried out in real time via the detection of fluorescent signals. The method developed showed high sensitivity and specificity, with detection limits of 0.3 and 0.03 pg/reaction of purified DNA of A. carbonarius and A. flavus, respectively. The assays were further implemented on inoculated nuts, including pistachios and almonds, after one-step crude DNA extraction. These tests revealed a detection level of 0.5 spore/g that shows the effectiveness of LAMP as a rapid method for detecting potentially toxigenic Aspergillus spp. directly in food. The validation of the assays included tests on a larger scale that further confirmed their sensitivity and specificity, as well as enabling the production of ready-to-use LAMP prototype kits. These kits are easy to use and aim to simplify the screening of food samples in order to monitor the presence of specific Aspergillus contaminations.

m6A methyltransferase AflIme4 orchestrates mycelial growth, development and aflatoxin B1 biosynthesis in Aspergillus flavus.

Aflatoxin B1 (AFB1), a highly toxic secondary metabolite produced by Aspergillus flavus, poses a severe threat to agricultural production, food safety and human health. The methylation of mRNA m6A has been identified as a regulator of both the growth and AFB1 production of A. flavus. However, its intracellular occurrence and function needs to be elucidated. Here, we identified and characterized a m6A methyltransferase, AflIme4, in A. flavus. The enzyme was localized in the cytoplasm, and knockout of AflIme4 significantly reduced the methylation modification level of mRNA. Compared with the control strains, ΔAflIme4 exhibited diminished growth, conidial formation, mycelial hydrophobicity, sclerotium yield, pathogenicity and increased sensitivity to CR, SDS, NaCl and H2O2. Notably, AFB1 production was markedly inhibited in the A. flavus ΔAflIme4 strain. RNA-Seq coupled with RT-qPCR validation showed that the transcriptional levels of genes involved in the AFB1 biosynthesis pathway including aflA, aflG, aflH, aflK, aflL, aflO, aflS, aflV and aflY were significantly upregulated. Methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) analysis demonstrated a significant increase in m6A methylation modification levels of these pathway-specific genes, concomitant with a decrease in mRNA stability. These results suggest that AflIme4 attenuates the mRNA stability of genes in AFB1 biosynthesis by enhancing their mRNA m6A methylation modification, leading to impaired AFB1 biosynthesis. Our study identifies a novel m6A methyltransferase AflIme4 and highlights it as a potential target to control A. flavus growth, development and aflatoxin pollution.

AflaILVB/G/I and AflaILVD are involved in mycelial production, aflatoxin biosynthesis, and fungal virulence in Aspergillus flavus.

Aflatoxins (AFs) are produced by fungi such as Aspergillus flavus and A. parasiticus and are one of the most toxic mycotoxins found in agricultural products and food. Aflatoxin contamination, which requires the control of A. flavus, remains problematic because of the lack of effective strategies and the exploration of new compounds that can inhibit A. flavus growth and mycotoxin production is urgently required to alleviate potential deleterious effects. Acetohydroxy acid synthase (AHAS) and dihydroxy acid dehydratase are important enzymes in the biosynthetic pathways of branched-chain amino acids (BCAAs), including isoleucine, leucine, and valine. Enzymes involved in BCAA biosynthesis are present in bacteria, plants, and fungi, but not in mammals, and are therefore, attractive targets for antimicrobial and herbicide development. In this study, we characterized AflaILVB/G/I and AflaILVD, which encode the catalytic and regulatory subunits of AHAS and dihydroxy acid dehydratase, from the pathogenic fungus Aspergillus flavus. The AflaILVB/G/I and AflaILVD deletion mutant grew slower and produced smaller colonies than the wild-type strain when grown on glucose minimal medium, potato dextrose agar, and yeast extract medium for three days at 28°C, and disruption of AflaILVB/G/I caused a significant reduction in conidia production when grown on all kinds of media. Cellular stress assays determined that all strains were sensitive to H2O2. Importantly, the pathogenicity and aflatoxin production were affected when AflaILVB/G/I and AflaILVD were knocked out, particularly AflaILVB/G/I. A series of genes that encoded enzymes involved in aflatoxin synthesis were downregulated, meaning that the knockout of AflaILVB/G/I influenced aflatoxin synthesis in A. flavus strain WT. Collectively, our results demonstrate the potential value of antifungals targeting AflaILVB/G/I in A. flavus.

Genome-Wide Characterization of the Phenylalanine Ammonia-Lyase Gene Family and Their Potential Roles in Response to Aspergillus flavus L. Infection in Cultivated Peanut (Arachis hypogaea L.).

Phenylalanine ammonia-lyase (PAL) is an essential enzyme in the phenylpropanoid pathway, in which numerous aromatic intermediate metabolites play significant roles in plant growth, adaptation, and disease resistance. Cultivated peanuts are highly susceptible to Aspergillus flavus L. infection. Although PAL genes have been characterized in various major crops, no systematic studies have been conducted in cultivated peanuts, especially in response to A. flavus infection. In the present study, a systematic genome-wide analysis was conducted to identify PAL genes in the Arachis hypogaea L. genome. Ten AhPAL genes were distributed unevenly on nine A. hypogaea chromosomes. Based on phylogenetic analysis, the AhPAL proteins were classified into three groups. Structural and conserved motif analysis of PAL genes in A. hypogaea revealed that all peanut PAL genes contained one intron and ten motifs in the conserved domains. Furthermore, synteny analysis indicated that the ten AhPAL genes could be categorized into five pairs and that each AhPAL gene had a homologous gene in the wild-type peanut. Cis-element analysis revealed that the promoter region of the AhPAL gene family was rich in stress- and hormone-related elements. Expression analysis indicated that genes from Group I (AhPAL1 and AhPAL2), which had large number of ABRE, WUN, and ARE elements in the promoter, played a strong role in response to A. flavus stress.

Biotransformation of the azo dye reactive orange 16 by Aspergillus flavus A5P1: Performance, genetic background, pathway, and mechanism.

This study reports the strain Aspergillus flavus A5P1 (A5P1), which is with the capable of degrading the azo dye reactive orange 16 (RO16). The mechanism of RO16 degradation by A5P1 was elucidated through genomic analysis, enzymatic analysis, degradation pathway analysis and oxidative stress analysis. Strain A5P1 exhibited aerobic degradation of RO16, with optimal degradation at an initial pH of 3.0. Genomic analysis indicates that strain A5P1 possesses the potential for acid tolerance and degradation of azo dye. Enzymatic analysis, combined with degradation product analysis, demonstrated that extracellular laccase, intracellular lignin peroxidase, and intracellular quinone reductase were likely key enzymes in the RO16 degradation process. Oxidative stress analysis revealed that cell stress responses may participate in the RO16 biotransformation process. The results indicated that the biotransformation of RO16 may involves biological processes such as transmembrane transport of RO16, cometabolism of the strain with RO16, and cell stress responses. These findings shed light on the biodegradation of RO16 by A5P1, indicating A5P1's potential for environmental remediation.

Liamocins overproduction via the two-pH stage fermentation and anti-Aspergillus flavus activity of Massoia lactone.

Aureobasidium melanogenum was found to be grown the best at the constant pH 7.0 and to produce the highest amount of liamocins at the constant pH 3.0. Therefore, the wild type strain A. melanogenum 9-1 and the engineered strain V33 constructed in the laboratory were grown at the constant pH 7.0 for 48 h, then, they were continued to be cultivated at the constant pH 3.0. Under such conditions, A. melanogenum 9-1 produced 36.51 ± 0.55 g L-1 of liamocin and its cell mass was 27.43 ± 0.63 and 6.00 ± 0.11 g L-1 of glucose was left in the finished medium within 168 h while the engineered strain V33 secreted 70.86 ± 2.04 g L-1 of liamocin, its cell mass was 31.63 ± 0.74 g L-1 , 0.16 ± 0.01 g L-1 of glucose was maintained in the finished medium. Then, Massoia lactone was released from the produced liamocins. The released Massoia lactone loaded in the nanoemulsions could be used to actively damage cell wall and cell membrane of both spores and mycelia of Aspergillus flavus, leading to its cell necrosis. Massoia lactone loaded in the nanoemulsions also actively inhibited cell growth of A. flavus, its conidia production and aflatoxin biosynthesis on peanuts, indicating that Massoia lactone loaded in the nanoemulsions had highly potential application in controlling cell growth of A. flavus and aflatoxin biosynthesis in foods and feedstuffs.

Aspergillus population diversity and its role in aflatoxin contamination of cashew nuts from coastal Kenya.

Cashew nuts are among the main cash crops in coastal Kenya, due in large part to their high nutritional value. Unfortunately, they also make them highly susceptible to mold contamination, resulting in biodeterioration of the nutritional value and potential contamination with toxic secondary metabolites, such as aflatoxins, that cause them to be rejected for sale at the market. We determined the population diversity of the Aspergillus species and their role in aflatoxin contamination in cashew nuts in selected coastal regions of Kenya. Fifty raw cashew nut samples were collected from post-harvest storage facilities across three counties in Kenya's coastal region and examined for moisture content and the presence of Aspergillus fungi. About 63 presumptive isolates were recovered from the cashew nuts. ITS and 28S rDNA regions were sequenced. The aflD, aflM and aflR genes were amplified to identify the potentially aflatoxigenic from the Aspergillus isolates. The Aflatoxins' presence on the isolates was screened using UV and the ammonia vapour test on coconut milk agar and validated using ELISA assay. A comparison of cashew moisture content between the three counties sampled revealed a significant difference. Sixty-three isolates were recovered and identified to section based on morphological characters and their respective ITS regions were used to obtain species identifications. Three sections from the genus were represented, Flavi and Nigri, and Terrei with isolates from the section Nigri having slightly greater abundance (n = 35). The aflD, aflM and aflR genes were amplified for all isolates to assess the presence of the aflatoxin biosynthesis pathway, indicating the potential for aflatoxin production. Less than half of the Aspergillus isolates (39.68%) contained the aflatoxin pathway genes, while 22.22% isolates were aflatoxigenic, which included only the section Flavi isolates. Section Flavi isolates identification was confirmed by calmodulin gene. The presence of species from Aspergillus section Flavi and section Nigri indicate the potential for aflatoxin or ochratoxin in the cashew nuts. The study established a foundation for future investigations of the fungi and mycotoxins contaminating cashew nuts in Kenya, which necessitates developing strategies to prevent infection by mycotoxigenic fungi, especially during the storage and processing phases.

Whole genome sequencing and annotation of Aspergillus flavus JAM-JKB-B HA-GG20.

Groundnuts are mostly contaminated with the mold Aspergillus flavus which produces a carcinogenic mycotoxin called as aflatoxin. It is very important to understand the genetic factors underlying its pathogenicity, regulation, and biosynthesis of secondary metabolites and animal toxicities, but it still lacks useful information due to certain gaps in the era of modern technology. Therefore, the present study was considered to determine the key genes and metabolites involved in the biosynthesis of aflatoxin by using a molecular approach in a virulent strain of Aspergillus. The whole genome sequence of highly toxic and virulent Aspergillus isolates JAM-JKB-B HA-GG20 revealed 3,73,54,834 bp genome size, 2, 26, 257 number of contigs with N50 value of 49,272 bp, 12,400 genes and 48.1% of GC contained respectively. The genome sequence was compared with other known aflatoxin producing and non-producing genome of Aspergillus spp. and 61 secondary metabolite (SM) gene clusters were annotated with the toxic strain JAM-JKB-BHA-GG20 which showed similarity with other Aspergillus spp. A total number of eight genes (ver-1, AflR, pksA, uvm8, omt1, nor-1, Vha and aflP) were identified related to biosynthesis of aflatoxin and ochratoxin. Also, 69 SSR with forward and reverse primers and 137 di and tri nucleotide motifs were identified in the nucleotide sequence region related to aflatoxin gene pathway. The genes and putative metabolites identified in this study are potentially involved in host invasion and pathogenicity. As such, the genomic information obtained in this study is helpful in understanding aflatoxin gene producing pathway in comparison to other Aspergillus spp. and predicted presence of other secondary metabolites clusters viz. Nrps, T1pks etc. genes associated with a biosynthesis of OTA mycotoxin.

Extracellular production of antifungal peptides from oxidative endotoxin-free E. coli and application.

Antifungal peptides (AFPs) can be used as novel preservatives, but achieving large-scale production and application remains a long-term challenge. In this study, we developed a hybrid peptide MD (metchnikowin-drosomycin fusion) secreted into Escherichia coli supernatant, demonstrating strong inhibitory activity against Aspergillus flavus and Botrytis cinerea. The fusion tag did not impact its activity. Moreover, an endotoxin-free and oxidative leaky strain was developed by knocking out the trxB, gor, and lpp genes of endotoxin-free E. coli ClearColi-BL21(DE3). This strain facilitates the proper folding of multi-disulfide bond proteins and promotes the extracellular production of recombinant bioactive AFP MD, achieving efficient production of endotoxin-free MD. In addition, temperature control replaces chemical inducers to further reduce production costs and circumvent the toxicity of inducers. This extracellularly produced MD exhibited favorable effectiveness in inhibiting fruit mold growth, and its safety was preliminarily established by gavage testing in mice, suggesting that it can be developed into a green and sustainable fruit fungicide. In conclusion, this study provides novel approaches and systematic concepts for producing extracellularly active proteins or peptides with industrial significance. KEY POINTS: • First report of extracellular production of bioactive antifungal peptide in Escherichia coli. • The hybrid antifungal peptide MD showed strong inhibitory activity against Aspergillus flavus and Botrytis cinerea, and the activity was not affected by the fusion tag. • Endotoxin-free oxidative Escherichia coli suitable for the expression of multi-disulfide bond proteins was constructed.

Revealing the formation mechanisms of key flavors in fermented broad bean paste.

Pixian Douban (PXDB) is a popular Chinese condiment for its distinctive flavor. Broad bean fermentation (Meju) is the most important process in the formation of flavor substances. Key flavors were analyzed qualitatively and quantitatively, and metagenomic technology was applied to study the microbial diversity during broad bean fermentation. In addition, the main metabolic pathways of key flavors were explored. Results indicated that Staphylococcus_gallinarum was the main microorganism in the microbial community, accounting for 39.13%, followed by Lactobacillus_agilis, accounting for 13.76%. Aspergillus_flavus was the fungus with the highest species abundance, accounting for 3.02%. The KEGG Pathway enrichment analysis showed that carbohydrate metabolism and amino acid metabolism were the main metabolic pathways. Glycoside hydrolase and glycosyltransferase genes were the most abundant, accounting for more than 70% of the total number of active enzyme genes. A total of 113 enzymes related to key flavors and 39 microorganisms corresponding to enzymes were annotated. And Staphylococcus_gallinarum, Lactobacillus_agilis, Weissella_confusa, Pediococcus_acidilactici, Staphylococcus_kloosii, Aspergillus_oryzae, and Aspergillus_flavus played a key role in the metabolic pathway. This study reveals the formation mechanism of key flavors in fermented broad bean, it is important for guiding the industrial production of PXDB and improving product quality.

Bacillus amyloliquefaciens A-1 inhibiting fungal spoilage in agricultural products is improved by metabolic engineering of enhancing surfactin yield.

Fungi and subsequent mycotoxins contamination in agricultural products have caused enormous losses and great harm to human and animal health. Biological control has attracted the attention of researchers due to its advantages, including mild conditions, low cost, high efficiency and low nutrient loss. In this study, a newly isolated strain Bacillus amyloliquefaciens A-1 (A-1), was screened for its ability to inhibit the growth and Aflatoxin B1 (AFB1) production of Aspergillus flavus NRRL 3357. Electron microscopy results revealed that mycelium and conidia of A. flavus were destroyed by A-1, affecting hyphae, cell walls, cell membranes and organelles. RNA-seq analysis indicated disturbance in gene expression profiles of A. flavus, including amino acid degradation and starch and sucrose metabolism pathways. Importantly, the biosynthesis of AFB1 was significantly inhibited by the down-regulation of key regulatory genes, aflR and aflS, and the simultaneous down-regulation of most structural genes. Genome analysis predicted six secondary metabolites biosynthetic gene clusters. Then, four surfactin synthesized by cluster C were identified as the main active substance of A-1 using HPLC-Q-TOF-MS. The addition of alanine, threonine, Fe2+ increased surfactin production. Notably, the overexpression of comX also improved surfactin production. The vivo test results indicated that A-1 could significantly inhibit the decay of pear by Aspergillus westerdijkiae, and the mildew of maize and peanuts. Especially, the overexpression of comX in A-1 could enhance the inhibitory activity. In conclusion, the inhibition mechanism of A-1 was revealed, and comX was found can improve the production of surfactin and subsequent activities, which provides the scientific basis for the development of biocontrol agents to reduce spoilage in agricultural products.

Creating large chromosomal segment deletions in Aspergillus flavus by a dual CRISPR/Cas9 system: Deletion of gene clusters for production of aflatoxin, cyclopiazonic acid, and ustiloxin B.

Aspergillus flavus produces hepatocarcinogenic aflatoxin that adversely impacts human and animal health and international trade. A promising means to manage preharvest aflatoxin contamination of crops is biological control, which employs non-aflatoxigenic A. flavus isolates possessing defective aflatoxin gene clusters to outcompete field toxigenic populations. However, these isolates often produce other toxic metabolites. The CRISPR/Cas9 technology has greatly advanced genome editing and gene functional studies. Its use in deleting large chromosomal segments of filamentous fungi is rarely reported. A system of dual CRISPR/Cas9 combined with a 60-nucleotide donor DNA that allowed removal of A. flavus gene clusters involved in production of harmful specialized metabolites was established. It efficiently deleted a 102-kb segment containing both aflatoxin and cyclopiazonic acid gene clusters from toxigenic A. flavus morphotypes, L-type and S-type. It further deleted the 27-kb ustiloxin B gene cluster of a resulting L-type mutant. Overall efficiencies of deletion ranged from 66.6 % to 85.6 % and efficiencies of deletions repaired by a single copy of donor DNA ranged from 50.5 % to 72.7 %. To determine the capacity of this technique, a pigment-screening setup based on absence of aspergillic acid gene cluster was devised. Chromosomal segments of 201 kb and 301 kb were deleted with efficiencies of 57.7 % to 69.2 %, respectively. This system used natural A. flavus isolates as recipients, eliminated a forced-recycling step to produce recipients for next round deletion, and generated maker-free deletants with sequences predefined by donor DNA. The research provides a method for creating genuine atoxigenic biocontrol strains friendly for field trial release.

Role of RNA Modifications, Especially m6A, in Aflatoxin Biosynthesis of Aspergillus flavus.

RNA modifications play key roles in eukaryotes, but the functions in Aspergillus flavus are still unknown. Temperature has been reported previously to be a critical environmental factor that regulates the aflatoxin production of A. flavus, but much remains to be learned about the molecular networks. Here, we demonstrated that 12 kinds of RNA modifications in A. flavus were significantly changed under 29 °C compared to 37 °C incubation; among them, m6A was further verified by a colorimetric method. Then, the transcriptome-wide m6A methylome and m6A-altered genes were comprehensively illuminated through methylated RNA immunoprecipitation sequencing and RNA sequencing, from which 22 differentially methylated and expressed transcripts under 29 °C were screened out. It is especially notable that AFCA_009549, an aflatoxin biosynthetic pathway gene (aflQ), and the m6A methylation of its 332nd adenine in the mRNA significantly affect aflatoxin biosynthesis in A. flavus both on media and crop kernels. The content of sterigmatocystin in both ΔaflQ and aflQA332C strains was significantly higher than that in the WT strain. Together, these findings reveal that RNA modifications are associated with secondary metabolite biosynthesis of A. flavus.

Nosocomial transmission of Aspergillus flavus in a neonatal intensive care unit: Long-term persistence in environment and interest of MALDI-ToF mass-spectrometry coupled with convolutional neural network for rapid clone recognition.

Aspergillosis of the newborn remains a rare but severe disease. We report four cases of primary cutaneous Aspergillus flavus infections in premature newborns linked to incubators contamination by putative clonal strains. Our objective was to evaluate the ability of matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) coupled to convolutional neural network (CNN) for clone recognition in a context where only a very small number of strains are available for machine learning. Clinical and environmental A. flavus isolates (n = 64) were studied, 15 were epidemiologically related to the four cases. All strains were typed using microsatellite length polymorphism. We found a common genotype for 9/15 related strains. The isolates of this common genotype were selected to obtain a training dataset (6 clonal isolates/25 non-clonal) and a test dataset (3 clonal isolates/31 non-clonal), and spectra were analysed with a simple CNN model. On the test dataset using CNN model, all 31 non-clonal isolates were correctly classified, 2/3 clonal isolates were unambiguously correctly classified, whereas the third strain was undetermined (i.e., the CNN model was unable to discriminate between GT8 and non-GT8). Clonal strains of A. flavus have persisted in the neonatal intensive care unit for several years. Indeed, two strains of A. flavus isolated from incubators in September 2007 are identical to the strain responsible for the second case that occurred 3 years later. MALDI-TOF is a promising tool for detecting clonal isolates of A. flavus using CNN even with a limited training set for limited cost and handling time.

Cutaneous aspergillosis is a rare but potentially fatal disease of the prematurely born infant. We described here several cases due to Aspergillus flavus and have linked them to environnemental strains using MLP genotyping and MALDI-TOF mass spectrometry coupled with artificial intelligence.

Simultaneous detection of mycotoxigenic Aspergillus species of sections Circumdati and Flavi using multiplex digital PCR.

Populations of ochratoxin-producing Aspergillus section Circumdati species and aflatoxin-producing Aspergillus section Flavi species frequently coexist in soil and are the main sources of mycotoxin contamination of tree nuts. Identification of mycotoxigenic Aspergillus species in these sections is difficult using traditional isolation and culture methods. We developed a multiplex digital PCR (dPCR) assay to detect and quantify Aspergillus ochraceus, Aspergillus westerdijkiae, and Aspergillus steynii (section Circumdati), as well as Aspergillus flavus and Aspergillus parasiticus (section Flavi), in environmental samples based on species-specific calmodulin gene sequences. Relative quantification of each species by dPCR of mixed-species templates correlated with corresponding DNA input ratios. Target species could be detected in soil inoculated with conidia from each species. Non-target species of sections Circumdati, Flavi, and Nigri were generally not detectable using this dPCR method. Detected non-target species (Aspergillus fresenii, Aspergillus melleus, Aspergillus sclerotiorum, and Aspergillus subramanianii) were discernible from A. ochraceus in dual-template dPCR reactions based on differential fluorescence intensity.

Understanding the mechanism of pathogenicity through interactome studies between Arachis hypogaea L. and Aspergillus flavus.

Aspergillus flavus (A. flavus) infects the peanut seeds during pre-and post-harvest stages, causing seed quality destruction for humans and livestock consumption. Even though many resistant varieties were developed, the molecular mechanism of defense interactions of peanut against A. flavus still needs further investigation. Hence, an interologous host-pathogen protein interaction (HPPI) network was constructed to understand the subcellular level interaction mechanism between peanut and A. flavus. Out of the top 10 hub proteins of both organisms, protein phosphatase 2C and cyclic nucleotide-binding/kinase domain-containing protein and different ribosomal proteins were identified as candidate proteins involved in defense. Functional annotation and subcellular localization based characterization of HPPI identified protein SGT1 homolog, calmodulin and Rac-like GTP-binding proteins to be involved in defense response against fungus. The relevance of HPPI in infectious conditions was assessed using two transcriptome data which identified the interplay of host kinase class R proteins, bHLH TFs and cell wall related proteins to impart resistance against pathogen infection. Further, the pathogenicity analysis identified glycogen phosphorylase and molecular chaperone and allergen Mod-E/Hsp90/Hsp1 as potential pathogen targets to enhance the host defense mechanism. Hence, the computationally predicted host-pathogen PPI network could provide valuable support for molecular biology experiments to understand the host-pathogen interaction. SIGNIFICANCE: Protein-protein interactions execute significant cellular interactions in an organism and are influenced majorly by stress conditions. Here we reported the host-pathogen protein-protein interaction between peanut and A. flavus, and a detailed network analysis based on function, subcellular localization, gene co-expression, and pathogenicity was performed. The network analysis identified key proteins such as host kinase class R proteins, calmodulin, SGT1 homolog, Rac-like GTP-binding proteins bHLH TFs and cell wall related to impart resistance against pathogen infection. We observed the interplay of defense related proteins and cell wall related proteins predominantly, which could be subjected to further studies. The network analysis described in this study could be applied to understand other host-pathogen systems generally.

Effect of Streptomyces roseolus Cell-Free Supernatants on the Fungal Development, Transcriptome, and Aflatoxin B1 Production of Aspergillus flavus.

Crop contamination by aflatoxin B1 (AFB1), an Aspergillus-flavus-produced toxin, is frequently observed in tropical and subtropical regions. This phenomenon is emerging in Europe, most likely as a result of climate change. Alternative methods, such as biocontrol agents (BCAs), are currently being developed to reduce the use of chemicals in the prevention of mycotoxin contamination. Actinobacteria are known to produce many bioactive compounds, and some of them can reduce in vitro AFB1 concentration. In this context, the present study aims to analyze the effect of a cell-free supernatant (CFS) from Streptomyces roseolus culture on the development of A. flavus, as well as on its transcriptome profile using microarray assay and its impact on AFB1 concentration. Results demonstrated that in vitro, the S. roseolus CFS reduced the dry weight and conidiation of A. flavus from 77% and 43%, respectively, and was therefore associated with a reduction in AFB1 concentration reduction to levels under the limit of quantification. The transcriptomic data analysis revealed that 5198 genes were differentially expressed in response to the CFS exposure and among them 5169 were downregulated including most of the genes involved in biosynthetic gene clusters. The aflatoxins' gene cluster was the most downregulated. Other gene clusters, such as the aspergillic acid, aspirochlorine, and ustiloxin B gene clusters, were also downregulated and associated with a variation in their concentration, confirmed by LC-HRMS.

Complete genome of the toxic mold Aspergillus pseudotamarii isolate NRRL 25517 reveals genomic instability of the aflatoxin biosynthesis cluster.

Fungi can synthesize a broad array of secondary metabolite chemicals. The genes underpinning their biosynthesis are typically arranged in tightly linked clusters in the genome. For example, ∼25 genes responsible for the biosynthesis of carcinogenic aflatoxins by Aspergillus section Flavi species are grouped in a ∼70 Kb cluster. Assembly fragmentation prevents assessment of the role of structural genomic variation in secondary metabolite evolution in this clade. More comprehensive analyses of secondary metabolite evolution will be possible by working with more complete and accurate genomes of taxonomically diverse Aspergillus species. Here, we combined short- and long-read DNA sequencing to generate a highly contiguous genome of the aflatoxigenic fungus, Aspergillus pseudotamarii (isolate NRRL 25517 = CBS 766.97; scaffold N50 = 5.5 Mb). The nuclear genome is 39.4 Mb, encompassing 12,639 putative protein-encoding genes and 74-97 candidate secondary metabolite biosynthesis gene clusters. The circular mitogenome is 29.7 Kb and contains 14 protein-encoding genes that are highly conserved across the genus. This highly contiguous A. pseudotamarii genome assembly enables comparisons of genomic rearrangements between Aspergillus section Flavi series Kitamyces and series Flavi. Although the aflatoxin biosynthesis gene cluster of A. pseudotamarii is conserved with Aspergillus flavus, the cluster has an inverted orientation relative to the telomere and occurs on a different chromosome.

Aneuploidy Formation in the Filamentous Fungus Aspergillus flavus in Response to Azole Stress.

Aspergillus flavus is a mycotoxigenic fungus that contaminates many important agricultural crops with aflatoxin B1, the most toxic and carcinogenic natural compound. This fungus is also the second leading cause of human invasive aspergillosis, after Aspergillus fumigatus, a disease that is particularly prevalent in immunocompromised individuals. Azole drugs are considered the most effective compounds in controlling Aspergillus infections both in clinical and agricultural settings. Emergence of azole resistance in Aspergillus spp. is typically associated with point mutations in cyp51 orthologs that encode lanosterol 14α-demethylase, a component of the ergosterol biosynthesis pathway that is also the target of azoles. We hypothesized that alternative molecular mechanisms are also responsible for acquisition of azole resistance in filamentous fungi. We found that an aflatoxin-producing A. flavus strain adapted to voriconazole exposure at levels above the MIC through whole or segmental aneuploidy of specific chromosomes. We confirm a complete duplication of chromosome 8 in two sequentially isolated clones and a segmental duplication of chromosome 3 in another clone, emphasizing the potential diversity of aneuploidy-mediated resistance mechanisms. The plasticity of aneuploidy-mediated resistance was evidenced by the ability of voriconazole-resistant clones to revert to their original level of azole susceptibility following repeated transfers on drug-free media. This study provides new insights into mechanisms of azole resistance in a filamentous fungus. IMPORTANCE Fungal pathogens cause human disease and threaten global food security by contaminating crops with toxins (mycotoxins). Aspergillus flavus is an opportunistic mycotoxigenic fungus that causes invasive and noninvasive aspergillosis, diseases with high rates of mortality in immunocompromised individuals. Additionally, this fungus contaminates most major crops with the notorious carcinogen, aflatoxin. Voriconazole is the drug of choice to treat infections caused by Aspergillus spp. Although azole resistance mechanisms have been well characterized in clinical isolates of Aspergillus fumigatus, the molecular basis of azole resistance in A. flavus remains unclear. Whole-genome sequencing of eight voriconazole-resistant isolates revealed that, among other factors, A. flavus adapts to high concentrations of voriconazole by duplication of specific chromosomes (i.e., aneuploidy). Our discovery of aneuploidy-mediated resistance in a filamentous fungus represents a paradigm shift, as this type of resistance was previously thought to occur only in yeasts. This observation provides the first experimental evidence of aneuploidy-mediated azole resistance in the filamentous fungus A. flavus.