Flavus Isolates Research Articles

Physicochemical characteristics, mycoflora and aflatoxins in corn grown and stored in Northern Tamaulipas, Mexico

In northern Tamaulipas, México, the contamination of corn by toxigenic fungi reduces grain production and quality. Corn contaminated by mycotoxins puts humans and livestock at risk. Continuous monitoring of the sanitary quality of grain at harvest and in storage will define preventive and corrective strategies for contamination by mycotoxigenic fungi. In this work, we identified toxigenic fungi associated with corn grown and stored in northern Tamaulipas, identified and quantified aflatoxins and their relationships with the physicochemical characteristics of the grain, and identified the main genes responsible for aflatoxin production in A. flavus. Fungal incidence was evaluated in vitro, aflatoxin production was evaluated via HPLC, and physicochemical traits were evaluated via spectrophotometry. Three genera were identified: Fusarium, Aspergillus, and Penicillium; the latter had the highest incidence in both 2011 and 2012. The incidence was higher in 2012 (82.3%) than in 2011 (4.5%), and storage did not affect the incidence. Associations among fungal incidences and physicochemical traits were significant and intermediate in both years. AFB1 production was negatively associated with hectoliter weight, and total fungal incidence was positively related to the incidence of Penicillium, Fusarium, and Aspergillus and negatively related to the flotation index. AFB1 was detected in 13.18% of the samples, with values ranging from 3.4881.33 ppb upon receipt and from 4.3245.92 ppb after storage. Two samples exceeded the allowed limits for Mexico (20 ppb). The aflD and aflQ genes were detected in 52.1 and 56.3%, respectively, of the A. flavus isolates.

Enhancing Ras cheese safety: antifungal effects of nisin and its nanoparticles against Aspergillus flavus

BackgroundDue to the adverse effects of industrial chemicals and their carcinogenicity and toxicity for humans, the debates have increased on using natural preservatives. This study was conducted to investigate the inhibitory effect of pure nisin and nisin nanoparticles (nisin NPs) against Aspergillus flavus in vivo by inoculation in laboratory-manufactured Ras cheese. A novel, safe, and natural approach of nanoprecipitation using acetic acid was employed to prepare nisin nanoparticles. The prepared NPs were characterized using zeta-sizer, FTIR, and transmission electron microscopy (TEM). Furthermore, the cytotoxicity of nisin NPs on Vero cells was assessed. The minimum inhibitory concentrations (MICs) of nisin and its nanoparticles were determined in vitro against A. flavus isolates using the agar well-diffusion method. The sensory evaluation of manufactured Ras cheese was conducted over a 60-day storage period.ResultsThe obtained results showed a strong antifungal activity of nisin NPs (0.0625 mg/mL) against A. flavus strain in comparison with pure nisin (0.5 mg/mL). Notably, the count decreased gradually by time from 2 × 108 at zero time and could not be detected at the 7th week. The count with pure nisin decreased from 2 × 108 at zero time and could not be detected at the 10th week where it’s enough time to produce aflatoxins in cheese. The MICs of nisin and nisin NPs were 0.25 and 0.0313 mg/mL, respectively. Nisin NPs used in our experiment had good biocompatibility and safety for food preservation. Additionally, the sensory parameters of the manufactured Ras cheese inoculated with nisin and nisin NPs were of high overall acceptability (OAA).ConclusionsOverall, the results of this study suggested that adding more concentration (˃0.0625 mg/mL) from nisin nanoparticles during the production of Ras cheese may be a helpful strategy for food preservation against A. flavus in the dairy industry.

Prevalence of azole-resistant Aspergillus fumigatus and other aspergilli in the environment from Argentina.

Azole resistance has emerged as a new therapeutic challenge in patients with aspergillosis. Various resistance mutations are attributed to the widespread use of triazole-based fungicides in agriculture. This study explored the prevalence of azole-resistant Aspergillus fumigatus (ARAF) and other aspergilli in the Argentine environment. A collection of A. fumigatus and other aspergilli strains isolated from soil of growing crops, compost, corn, different animal feedstuffs, and soybean and chickpea seeds were screened for azole resistance. No ARAF was detected in any of the environmental samples studied. However, five A. flavus, one A. ostianus, one A. niger and one A. tamarii recovered from soybean and chickpea seeds showed reduced susceptibility to medical azole antifungals (MAA). The susceptibility profiles of five A. flavus isolates, showing reduced susceptibility to demethylase inhibitors (DMIs), were compared with those of 10 isolates that exhibited susceptibility to MAA. Aspergillus flavus isolates that showed reduced MAA susceptibility exhibited different susceptibility profiles to DMIs. Prothioconazole and tebuconazole were the only DMIs significantly less active against isolates with reduced susceptibility to MAA. Although no ARAF isolates were found in the samples analysed, other aspergilli with reduced susceptibility profile to MAA being also important human pathogens causing allergic, chronic and invasive aspergillosis, are present in the environment in Argentina. Although a definitive link between triazole-based fungicide use and isolation of azole-resistant human pathogenic aspergilli from agricultural fields in Argentina remains elusive, this study unequivocally highlights the magnitude of the environmental spread of azole resistance among other Aspergillus species.

Impact of storage conditions on the shelf life of aflatoxin biocontrol products containing atoxigenic isolates of Aspergillus flavus as active ingredient applied in various countries in Africa

Aflatoxin contamination significantly threatens food safety and security, particularly in tropical and sub-tropical regions where staple crops such as maize, groundnut, and sorghum become frequently affected. This contamination is primarily caused by the fungus Aspergillus flavus. The contamination causes adverse health effects, reduced income, and trade restrictions. In response to this challenge, various technologies have been developed to mitigate the impacts of aflatoxin. Among these, biocontrol products containing atoxigenic isolates of A. flavus as the active ingredient can effectively reduce aflatoxin levels both at pre- and post-harvest. A notable example of such products is Aflasafe, which contains four atoxigenic isolates native to specific target regions. These products have undergone rigorous testing, have received regulatory approval, and are commercially available in multiple African countries. However, their manufacturing processes have evolved, and comprehensive shelf life studies for current formulations are lacking. Evaluations of the spore production ability of atoxigenic A. flavus isolates in Aflasafe products over 4 years, under various storage conditions, revealed a significant linear decrease in sporulation with storage months (P < 0.001; R2 = 0.203), with no significant differences observed between treatments. However, this marginal decline (P = 0.398) is unlikely to be sufficient to prevent the effectiveness in limiting aflatoxin. In addition, storing the products for 2 weeks at 54 °C did not affect (P > 0.05) the ability of the coated fungi to produce spores compared to when the products were stored at 24 °C. The findings contribute valuable insights for manufacturers and users of atoxigenic-based aflatoxin biocontrol products, informing best practices for product storage and utilization to ensure prolonged effectivenes in aflatoxin mitigation efforts.

Pathogenicity is associated with population structure in a fungal pathogen of humans.

Aspergillus flavus is a clinically and agriculturally important saprotrophic fungus responsible for severe human infections and extensive crop losses. We analyzed genomic data from 250 (95 clinical and 155 environmental) A. flavus isolates from 9 countries, including 70 newly sequenced clinical isolates, to examine population and pan-genome structure and their relationship to pathogenicity. We identified five A. flavus populations, including a new population, D, corresponding to distinct clades in the genome-wide phylogeny. Strikingly, > 75% of clinical isolates were from population D. Accessory genes, including genes within biosynthetic gene clusters, were significantly more common in some populations but rare in others. Population D was enriched for genes associated with zinc ion binding, lipid metabolism, and certain types of hydrolase activity. In contrast to the major human pathogen Aspergillus fumigatus, A. flavus pathogenicity in humans is strongly associated with population structure, making it a great system for investigating how population-specific genes contribute to pathogenicity.

Reliability of the chemical, metabolic, and molecular methods in discriminating aflatoxigenic from non-aflatoxigenic Aspergillus isolates

Aflatoxins, potent carcinogens produced by Aspergillus species (A. flavus and A. parasiticus), are primarily consumed through dietary sources. Aflatoxin management using non-aflatoxigenic Aspergillus to outcompete their aflatoxigenic congeneric to achieve low residual toxin levels is a promising biological control (BC) field intervention. A prerequisite for the BC program is the accurate delineation of aflatoxigenic fungi from their non-aflatoxigenic relatives. In this study, we use three methods: Chemical (Dichlorvos-ammonia), Metabolic (ELISA coupled live cultures), and Molecular (10 aflatoxin biosynthetic genes) to determine the aflatoxigenicity of Aspergillus spp. previously collected from Eastern Kenya, an aflatoxin hotspot. Thirty-six different Aspergillus isolates were identified and studied microscopically from a total of 314 fungal cultures. The chemical method reported 21 aflatoxigenic and 15 non-aflatoxigenic isolates, while the metabolic technique distinguished 20 aflatoxigenic and 16 non-aflatoxigenic isolates. A large proportion (97.22 %) of the isolates harboured the aflR, aflO, and aflM genes, while 91.67 % of the isolates possessed the aflS gene. All isolates (100 %) harboured the aflD, aflQ, aflP, and aflK genes. ELISA-coupled live cultures positively correlated with Dichlorvos-ammonia (p > 0.01), only differing in one sample, indicating a strong concurrence between the two methods at segregating toxigenic A. flavus isolates. Cluster analysis based on Multiple Correspondence revealed that the non-aflatoxigenic group exhibited similar characteristics correlating with the absence of aflR, aflS, aflI, and aflO genes. These findings indicate the reliability of the Chemical and Metabolic methods and can function as confirmatory assays to complement the molecular detection approach in determining aflatoxigenicity among Aspergillus spp.

Molecular and aflatoxigenicity analyses of Aspergillus flavus isolates indigenous to grain corn in Malaysia; potentials for biological control.

The present work aimed to distinguish the indigenous Aspergillus flavus isolates obtained from the first (pioneer) grain corn farms in Terengganu, Malaysia, into aflatoxigenic and non-aflatoxigenic by molecular and aflatoxigenicity analyses, and determine the antagonistic capability of the non-aflatoxigenic isolates against aflatoxigenic counterparts and their aflatoxin production in vitro. Seven A. flavus isolates previously obtained from the farms were characterized molecularly and chemically. All isolates were examined for the presence of seven aflatoxin biosynthesis genes, and their aflatoxigenicity were confirmed using high performance liquid chromatography with fluorescence detector (HPLC-FLD). Phylogenetic relationships of all isolates were tested using ITS and β-tubulin genes. Of the seven isolates, two were non-aflatoxigenic, while the remaining were aflatoxigenic based on the presence of all aflatoxin biosynthesis genes tested, and the productions of aflatoxins B1 and B2. All isolates were also confirmed as A. flavus following phylogenetic analysis. The indigenous non-aflatoxigenic isolates were further examined for their antagonistic potential against aflatoxigenic isolates on 3% grain corn agar (GCA). Both non-aflatoxigenic isolates significantly reduced AFB1 production of the aflatoxigenic isolates. The indigenous non-aflatoxigenic A. flavus strains identified in the present work were effective in controlling the aflatoxin production by the aflatoxigenic A. flavus isolates in vitro and can be utilized for in situ testing.

Application of Cystoseira myrica phycosynthesized selenium nanoparticles incorporated with nano-chitosan to control aflatoxigenic fungi in fish feed.

The recurrent contaminations of feed materials with mycotoxigenic fungi can endanger both farmed animals and humans. Biosynthesized nanomaterials are assumingly the ideal agents to overcome fungal invasion in feed/foodstuffs, especially when utilizing sustainable sources for synthesis. Herein, the phycosynthesis of selenium nanoparticles (SeNPs) was targeted using Cystoseira myrica algal extract (CE), and the conjugation of CE/SeNPs with chitosan nanoparticles (NCt) to produce potential antifungal nanocomposites for controlling Aspergillus flavus isolates in fish feed. The phycosynthesis of SeNPs with CE was effectually carried out and validated using visible/UV analysis, X-ray diffraction and transmission microscopy; CE/SeNPs had diameters of 8.7 nm and spherical shapes. NCt/CE/SeNPs nanocomposite (173.3 nm mean diameter) was achieved and the component interactions were validated via infrared spectroscopic analysis. The antifungal assessment of screened nanomaterials against three Aspergillus flavus strains indicated that NCt/CE/SeNPs exceeded the fluconazole action using qualitative/quantitative assays. Severe alteration/distortions in A. flavus mycelial structure and morphology were microscopically observed within 48 h of NCt/CE/SeNPs treatment. The treatment of feed ingredients (crushed corn and feed powder) by blending with nanomaterials (NCt, CE/SeNPs and NCt/CE/SeNPs) led to significant reduction in A. flavus count/growth after storage for 7 days; NCt/CE/SeNPs could completely inhibit any fungal growth in feed material. The pioneering phycosynthesis of CE/SeNPs and their nanoconjugation with NCt generated bioactive antifungal agents to control A. flavus strains. The innovatively constructed NCt/CE/SeNPs nanocomposite is advised for application as an effectual, biosafe and natural fungicidal conjugate for the protection of fish feed from mycotoxigenic fungi. © 2024 Society of Chemical Industry.

Molecular Characterization and Tolerance Potential of Culturable Crude Oil-Degrading Microbes in Santa-Barbara River, Bayelsa State, Nigeria

There has been chronic contamination of the surrounding aquatic and terrestrial environments in the Niger Delta region of Nigeria due to the enormous activities of crude oil exploration. This study aimed to characterize and assess the crude oil tolerance potential of indigenous microbes from crude oil spills in Santa Barbara River, Bayelsa State, Nigeria, that could serve as potential consortia for bioremediation of the crude oil spills. Total crude oil hydrocarbon-utilizing bacteria count (THUBC) and total crude oil hydrocarbon-utilizing fungi count (THUFC) in the samples were determined with a culture-dependent spread plate technique. Bacterial and fungal isolates were characterised using standard phenotypic and 16S/Internal Transcribed Spacer region rRNA gene sequencing techniques, respectively. The tolerance of autochthonous bacterial isolates to different concentrations of crude oil was subsequently determined. THUBC and THUFC in crude oil-polluted water samples ranged from 1.88 log10 CFU/ml to 2.74 log10 CFU/ml and from 0.00 log10 CFU/ml to 1.70 log10 CFU/ml, respectively. Representative strains of Proteus mirabilis, Bacillus subtilis, Pseudomonas aeruginosa, Micrococcus luteus and Aspergillus flavus isolates obtained from crude oil-polluted water samples were deposited in the GenBank (NCBI) under accession numbers OQ969924, OQ969951, OQ969987, OQ970009 and OQ975908. Proteus mirabilis, Pseudomonas aeruginosa and Aspergillus flavus demonstrated the most significant tolerance to crude oil pollutants (minimum crude oil inhibitory concentrations (MIC) = 80%) followed by Bacillus subtilis and Micrococcus luteus (MIC= 40%). The findings from this study are pointers to the potential role of the microbial isolates as bioremediation consortia to remediate the polluted Santa Barbara River.

Investigation of Azole Resistance Involving cyp51A and cyp51B Genes in Clinical Aspergillus flavus Isolates.

This study aimed to investigate azole resistance mechanisms in Aspergillus flavus, which involve cyp51A and cyp51B genes. Real-time Reverse Transcriptase qPCR method was applied to determine the overexpression of cyp51A and cyp51B genes for 34 A. flavus isolates. PCR sequencing of these two genes was used to detect the presence of gene mutations. Susceptibility test found sensitivity to voriconazole (VOR) in all strains. 14.7% and 8.8% of isolates were resistant to itraconazole (IT) and posaconazole (POS), respectively, with a cross-resistance in 5.8%. For the double resistant isolates (IT/POS), the expression of cyp51A was up to 17-fold higher. PCR sequencing showed the presence of 2 mutations in cyp51A: a synonymous point mutation (P61P) in eight isolates, which did not affect the structure of CYP51A protein, and another non synonymous mutation (G206L) for only the TN-33 strain (cross IT/POS resistance) causing an amino acid change in the protein sequence. However, we noted in cyp51B the presence of the only non-synonymous mutation (L177G) causing a change in amino acids in the protein sequence for the TN-31 strain, which exhibits IT/POS cross-resistance. A short single intron of 67 bp was identified in the cyp51A gene, whereas three short introns of 54, 53, and 160 bp were identified in the cyp51B gene. According to the models provided by PatchDock software, the presence of non-synonymous mutations did not affect the interaction of CYP51A and CYP51B proteins with antifungals. In our study, the overexpression of the cyp51A and cyp51B genes is the primary mechanism responsible for resistance in A. flavus collection. Nevertheless, other resistance mechanisms can be involved.

Aspergillus flavus pangenome (AflaPan) uncovers novel aflatoxin and secondary metabolite associated gene clusters

BackgroundAspergillus flavus is an important agricultural and food safety threat due to its production of carcinogenic aflatoxins. It has high level of genetic diversity that is adapted to various environments. Recently, we reported two reference genomes of A. flavus isolates, AF13 (MAT1-2 and highly aflatoxigenic isolate) and NRRL3357 (MAT1-1 and moderate aflatoxin producer). Where, an insertion of 310 kb in AF13 included an aflatoxin producing gene bZIP transcription factor, named atfC. Observations of significant genomic variants between these isolates of contrasting phenotypes prompted an investigation into variation among other agricultural isolates of A. flavus with the goal of discovering novel genes potentially associated with aflatoxin production regulation. Present study was designed with three main objectives: (1) collection of large number of A. flavus isolates from diverse sources including maize plants and field soils; (2) whole genome sequencing of collected isolates and development of a pangenome; and (3) pangenome-wide association study (Pan-GWAS) to identify novel secondary metabolite cluster genes.ResultsPangenome analysis of 346 A. flavus isolates identified a total of 17,855 unique orthologous gene clusters, with mere 41% (7,315) core genes and 59% (10,540) accessory genes indicating accumulation of high genomic diversity during domestication. 5,994 orthologous gene clusters in accessory genome not annotated in either the A. flavus AF13 or NRRL3357 reference genomes. Pan-genome wide association analysis of the genomic variations identified 391 significant associated pan-genes associated with aflatoxin production. Interestingly, most of the significantly associated pan-genes (94%; 369 associations) belonged to accessory genome indicating that genome expansion has resulted in the incorporation of new genes associated with aflatoxin and other secondary metabolites.ConclusionIn summary, this study provides complete pangenome framework for the species of Aspergillus flavus along with associated genes for pathogen survival and aflatoxin production. The large accessory genome indicated large genome diversity in the species A. flavus, however AflaPan is a closed pangenome represents optimum diversity of species A. flavus. Most importantly, the newly identified aflatoxin producing gene clusters will be a new source for seeking aflatoxin mitigation strategies and needs new attention in research.

First report of Aspergillus species in green pistachio of Bronte

Aspergillus contamination of pistachios causes significant product losses and potential presence of mycotoxins, particularly aflatoxin B1 (AFB1), and ochratoxin A (OTA). These toxins, which threaten human health, are strictly monitored by most nations. Italian pistachios produced in Bronte, Sicily, have high nutritional value and unique organoleptic properties, but the extent to which they contain these contaminants is unknown. Aspergillus spp. isolated from Bronte pistachios (cultivar Napoletana) were assessed for their ability to synthesize OTA or AFB1. Aspergillus occurrence in pistachio samples was measured at 1137 cfu g-1 for in shell pistachios and 770 cfu g-1 for kernels. The predominant isolated Aspergillus species was A. niger representing 74% of section Nigri (black isolates) and 47% of all Aspergillus isolates. Within section Flavi, A. flavus comprised 83% of green isolates. Only one black isolate (identified as A. carbonarius) had high OTA production, but all the A. flavus isolates had potential to produce AFG1 and AFB1, with AFB1 produced amount ranging from 0.1 to 8498 ng mL-1 of culture filtrate.

Melanin depletion affects Aspergillus flavus conidial surface proteins, architecture, and virulence.

Melanin is an Aspergillus flavus cell wall component that provides chemical and physical protection to the organism. However, the molecular and biological mechanisms modulating melanin-mediated host-pathogen interaction in A. flavus keratitis are not well understood. This work aimed to compare the morphology, surface proteome profile, and virulence of melanized conidia (MC) and non-melanized conidia (NMC) of A. flavus. Kojic acid treatment inhibited melanin synthesis in A. flavus, and the conidial surface protein profile was significantly different in kojic acid-treated non-melanized conidia. Several cell wall-associated proteins and proteins responsible for oxidative stress, carbohydrate, and chitin metabolic pathways were found only in the formic acid extracts of NMC. Scanning electron microscopy (SEM) analysis showed the conidial surface morphology difference between the NMC and MC, indicating the role of melanin in the structural integrity of the conidial cell wall. The levels of calcofluor white staining efficiency were different, but there was no microscopic morphology difference in lactophenol cotton blue staining between MC and NMC. Evaluation of the virulence of MC and NMC in the Galleria mellonella model showed NMC was less virulent compared to MC. Our findings showed that the integrity of the conidial surface is controlled by the melanin layer. The alteration in the surface protein profile indicated that many surface proteins are masked by the melanin layer, and hence, melanin can modulate the host response by preventing the exposure of fungal proteins to the host immune defense system. The G. mellonella virulence assay also confirmed that the NMC were susceptible to host defense as in other Aspergillus pathogens. KEY POINTS: • l-DOPA melanin production was inhibited in A. flavus isolates by kojic acid, and for the first time, scanning electron microscopy (SEM) analysis revealed morphological differences between MC and NMC of A. flavus strains • Proteome profile of non-melanized conidia showed more conidial surface proteins and these proteins were mainly involved in the virulence, oxidative stress, and metabolism pathways • Non-melanized conidia of A. flavus strains were shown to be less virulent than melanised conidia in an in vivo virulence experiment with the G. melonella model.

Aflatoxin profiles of Aspergillus flavus isolates in Sudanese fungal rhinosinusitis.

Aspergillus flavus is a commonly encountered pathogen responsible for fungal rhinosinusitis (FRS) in arid regions. The species is known to produce aflatoxins, posing a significant risk to human health. This study aimed to investigate the aflatoxin profiles of A. flavus isolates causing FRS in Sudan. A total of 93 clinical and 34 environmental A. flavus isolates were studied. Aflatoxin profiles were evaluated by phenotypic (thin-layer and high-performance chromatography) and genotypic methods at various temperatures and substrates. Gene expression of aflD and aflR was also analyzed. A total of 42/93 (45%) isolates were positive for aflatoxin B1 and AFB2 by HPLC. When the incubation temperature changed from 28°C to 36°C, the number of positive isolates decreased to 41% (38/93). Genetic analysis revealed that 85% (79/93) of clinical isolates possessed all seven aflatoxin biosynthesis-associated genes, while 27% (14/51) of non-producing isolates lacked specific genes (aflD/aflR/aflS). Mutations were observed in aflS and aflR genes across both aflatoxin-producers and non-producers. Gene expression of aflD and aflR showed the highest expression between the 4th and 6th days of incubation on the Sabouraud medium and on the 9th day of incubation on the RPMI (Roswell Park Memorial Institute) medium. Aspergillus flavus clinical isolates demonstrated aflatoxigenic capabilities, influenced by incubation temperature and substrate. Dynamic aflD and aflR gene expression patterns over time enriched our understanding of aflatoxin production regulation. The overall findings underscored the health risks of Sudanese patients infected by this species, emphasizing the importance of monitoring aflatoxin exposure.

Efficacy of atoxigenic Aspergillus flavus from southern China as biocontrol agents against aflatoxin contamination in corn and peanuts

Aspergillus flavus is a ubiquitous facultative pathogen that routinely infects important crops leading to formation of aflatoxins during crop development and after harvest. Corn and peanuts in warm and/or drought-prone regions are highly susceptible to aflatoxin contamination. Controlling aflatoxin using atoxigenic A. flavus is a widely adopted strategy. However, no A. flavus genotypes are currently approved for use in China. The current study aimed to select atoxigenic A. flavus endemic to Guangxi Zhuang Autonomous Region with potential as active ingredients of aflatoxin biocontrol products. A total of 204 A. flavus isolates from corn, peanuts, and field soil were evaluated for ability to produce the targeted mycotoxins. Overall, 57.3% could not produce aflatoxins while 17.15% were incapable of producing both aflatoxins and CPA. Atoxigenic germplasm endemic to Guangxi was highly diverse, yielding 8 different gene deletion patterns in the aflatoxin and CPA biosynthesis gene clusters ranging from no deletion to deletion of both clusters. Inoculation of corn and peanuts with both an aflatoxin producer and selected atoxigenic genotypes showed significant reduction (74 to 99%) in aflatoxin B1 (AFB1) formation compared with inoculation with the aflatoxin producer alone. Atoxigenic genotypes also efficiently degraded AFB1 (61%). Furthermore, atoxigenic isolates were also highly efficient at reducing aflatoxin concentrations even when present at lower concentrations than aflatoxin producers. The use of multiple atoxigenics was not always as effective as the use of a single atoxigenic. Effective atoxigenic genotypes of A. flavus with known mechanisms of atoxigenicity are demonstrated to be endemic to Southern China. These A. flavus may be utilized as active ingredients of biocontrol products without concern for detrimental impacts that may result from introduction of exotic fungi. Field efficacy trials in the agroecosystems of Southern China are needed to determine the extent to which such products may allow the production of safer food and feed.

Preliminary study for evaluation of some fungicides against Aspergillus flavus isolated from historical illuminated paper manuscript dated back to the Mamluk period

Various valuable illuminated paper manuscripts in museums and libraries suffer from fungal deterioration. The treatment and protection of illuminated paper manuscripts from fungal damage is an important process. Therefore, the current study aimed to evaluate different fungicides against fungi isolated from historical illuminated paper manuscript dated back to the Mamluk period (1250–1516 AD). Fungi isolated from infected historical illuminated paper manuscripts were identified and their cellulolytic enzyme activity was performed. The identified fungi were A. fumigatus, A. niger, A. flavus, Alternaria alternate, and Trichoderma sp. Isolate (4-1), identified as Aspergillus flavus, possessed the highest cellulase activity. This isolate was tested against the following fungicides: azoxystrobin, boscalid, difenoconazole, dimethomorph, propiconazole, pyraclostrobin, and thiophanate-methyl at concentrations from 1 to 200 ppm, nanoparticles of TiO2 and SiO2 at concentrations of 0.5%, and 1% and mixtures of fungicides [difenoconazole + propiconazole (1:1)] at 100 and 200 ppm and [boscalid + pyraclostrobin (2:1)] at 150 and 300 ppm. The disc diffusion method (DDM) was used to evaluate the efficiency of the prepared individual and mixed fungicides against Aspergillus flavus (4-1) isolate. The results of this study revealed that treatments with individual difenoconazole at 200 ppm exhibited the best antifungal activity, followed by the propiconazole fungicide. Further research is needed to evaluate the in vitro practical application of these fungicides with infected illuminated paper samples with Aspergillus flavus agents in the field.

Molecular identification and management of mycotoxigenic fungi in stored corn Grains

Mycotoxin-producing molds which considered as common maize grains contaminants are the genera Fusarium, Aspergillus and Penicillium. There are natural and safe ways to protect grains from mold contamination as the use of essential oils and chemical treatments. A total number of 25 samples were used to study the natural frequency in five governorates in Egypt, Molecular identification indicated that the most frequent fungi were Fusarium verticillioides, Aspergillus niger, Talaromyces verruculosus, Aspergillus flavus and Aspergillus terreus. The in vitro studies have been done to determine mycelial growth and spore germination inhibition of the two A. flavus; isolated and reference isolates. Thyme and acetic acid were tested in direct contact assay to study their effects on mycelial growth. Treatments showed significant impact on mycelial growth and spore germination inhibition of both A. flavus isolates. In the postharvest application treatments: as vapour and carrier contact assay, Thyme and Acetic acid were tested to determine their influence on growth and aflatoxin production in A. flavus isolates by liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS). Results indicated that both treatments were effective in inhibition of aflatoxin production in both vapour and carrier assays as they succeeded in reducing AFB1 while they inhibited completely the production of AFB2. The extent of the inhibition of aflatoxin production was dependent on the concentration and storage duration of treatments applied.

Evaluation of Mustard oil and temperature in the toxicity reduction of Aspergillus flavus in Walnut fruits

The study aimed to isolate and identify fungi that produce mycotoxins from walnut fruits and the possibility of limiting their presence in the fruits and reducing their toxic effects. The molecular diagnosis in this study of the fungus Aspergillus flavus was carried out using the internal transcribed spacer (ITS) genetic marker. As for the results of testing the ability of some A. flavus isolates to produce aflatoxins B1 including the ammonia test, the results of this test showed the ability of four out (of six) isolates of A. flavus on the production of aflatoxin B1 by changing the color of the base of the coconut medium on which the fungus isolates are grown, with a percentage of 66.66 %. The results of the chemical analysis using TLC technology for Aspergillus flavus showed the ability of three isolates to produce aflatoxin B1 out of six isolates of A. flavus with a percentage of 50 %. As for the most important results of the effectiveness of mustard oil in inhibiting the growth of the fungus A. flavus in walnut fruits, the highest percentage of inhibition was at 15% concentration, which amounted to 57.33%. The most important results of the storage experiment after three months of storage, included the test of the effect of mustard oil and temperature on the growth and density of A. flavus. The test was the effect of mustard oil on the reduction ratio of aflatoxin B1 as it reached the highest percentage of toxin reduction when adding mustard oil only, which amounted to 98.213%, compared to other treatments, the test was the effect of mustard oil and temperature on the amount of aflatoxin B1 toxin, which gave the highest rate when adding A. flavus to walnuts, which amounted to 13.425 micrograms/ g,. At a temperature of 5 °C, it gave the lowest rate of aflatoxin B1 toxin, which was 0.880 µg.gm-1. The highest rate was 8.620 µg.gm-1, at 25°C.

Evaluation of the efficacy of cinnamon oil on Aspergillus flavus and Fusarium proliferatum growth and mycotoxin production on paddy and polished rice: Towards a mitigation strategy

In the present investigation, the effect of cinnamon oil (CO) (10, 30, 50 and 70 %) on the growth rate (mm/day) and aflatoxin B1 (AFB1) and fumonisin B1 (FB1) production of Aspergillus flavus (AF01) and Fusarium proliferatum (FP01) isolates, respectively was determined at optimum water activities (0.95 and 0.99 aw) and temperatures (25, 30 and 35 °C) on paddy and polished rice grains. The results showed that the growth rate, AFB1 and FB1 production of all the fungal isolates decreased with an increase in CO concentrations on both matrices. AF01 and FP01 failed to grow under all conditions on paddy at 50 % of CO concentration whereas both fungi were completely inhibited (No Growth-NG) at 70 % of CO on polished rice. Regarding mycotoxin production, 30 % of CO concentrations could inhibit AFB1 and FB1 production in both matrices (No Detection-ND). In this study, the production of mycotoxins was significantly influenced by cinnamon oil compared to the growth of both fungi. These results indicated the promising potential of CO in improving the quality of rice preservation in post-harvest; however, further investigations should be evaluated on the effects on the qualitative characteristics of grains. Especially, the prospective application of CO in rice storage in industry scales to mitigate mycotoxin contamination need also to be further researched. Moreover, collaboration between researchers, agricultural experts, and food industry should be set up to achieve effective and sustainable strategies for preserving rice.

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.