aflR Gene Research Articles

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.

Mycological detection of aflatoxin producing gene (Aflr) in Aspergillus Spp found in commercial raw groundnut and groundnut cake (Kuli kuli) in some selected market in Katsina state, Nigeria

The effect of aflatoxin intoxication from groundnut and groundnut products has lead to a large amount of deaths some of which are not yet accounted of. Kuli-kuli formally referred to as groundnut cake, is a byproduct of oil extraction from groundnut which have been further fried, sweetened or spiced. This research aimed at determining the mycological quality and molecular detection of Aflatoxin gene aflR in Aspergillus spp found in raw Groundnut and groundnut cake (Kuli-kuli). A total of 120 samples (60 groundnut samples and 60 groundnut cake samples) were randomly collected from four selected markets in Katsina state and subjected to mycological assessment based on colonial morphology on PDA and microscopy. Out of the total samples, 16 samples (8 raw groundnut samples and 8 groundnut samples) were selected for determination of total Aflatoxin concentration using ELISA technique. Isolates were also screened for aflR gene using polymerase chain reaction technique, and subsequently sequenced using Sanger method. Eleven (11) fungi isolate including Aspergillus niger, Scedosporium apiospermum, Cladophialophora bantiana, Aspergillus flavus, Aspergillus nidulans, Microsphaeropsis arundinis, Cladosporium, Cladophialophora carrionii, Rhizopus arrhizus, Aspergillus parasitus and Aspergillus fumigatus were identified to produce aflatoxin concentration ranging from 0.4–3.5 ppb from the samples of the raw groundnut and groundnut cake (Kuli-kuli). Therefore, the findings of this research has raised serious food safety concerns, especially in consumption of raw groundnut and groundnut cake (Kuli-kuli) sold in Katsina as such relevant regulatory agencies such as NAFDAC are advised to deploy drastic measure to curtail a possible aflatoxin intoxication outbreak

Potential fungicidal and antiaflatoxigenic effects of cinnamon essential oils on Aspergillus flavus inhabiting the stored wheat grains

Wheat is one of the essential crops for the human and animal nutrition, however, contamination with aflatoxigenic fungi, due to the improper storage conditions and high humidity, was the main global threats. So, preventing the growth of aflatoxigenic fungi in stored wheat grains, by using different essential oils was the main objective of this work. Aspergillus flavus EFBL-MU12 PP087400, EFBL-MU23 PP087401 and EFBL-MU36 PP087403 isolates were the most potent aflatoxins producers inhabiting wheat grains. The effect of storage conditions of wheat grains “humidity, temperature, incubation period, and pH” on growth of A. flavus, was assessed by the response surface methodology using Plackett-Burman design and FCCD. The highest yield of aflatoxins EFBL-MU12 B1 and B2 by A. flavus grown on wheat grains were 145.3 and 7.6 μg/kg, respectively, at incubation temperature 35°C, 16% moisture contents, initial pH 5.0, and incubated for 14 days. The tested oils had a powerful antifungal activity for the growth and aflatoxins production by A. flavus in a concentration-dependent manner. Among these oils, cinnamon oil had the highest fungicidal activity for A. flavus at 0.125%, with about 85-90 % reduction to the aflatoxins B1 and B2, conidial pigmentation and chitin contents on wheat grains. From the SEM analysis, cinnamon oils had the most deleterious effect on A. flavus with morphological aberrations to the conidial heads, vegetative mycelia, alteration in conidiophores identity, hyphae shrank, and winding. To emphasize the effect of the essential oils on the aflatoxins producing potency of A. flavus, the molecular expression of the aflatoxins biosynthetic genes was estimated by RT-qPCR. The molecular expression of nor-1, afLR, pKsA and afLJ genes was suppressed by 94-96%, due to cinnamon oil at 0.062% compared to the control. Conclusively, from the results, cinnamon oils followed by the peppermint oils displayed the most fungicidal activity for the growth and aflatoxins production by A. flavus grown on wheat grains.

A polyphasic characterisation of Aspergillus section Flavi isolated from Malaysian and Nigerian food grains and poultry feeds by phenotypic, chemotypic, and molecular methods

The taxonomy and toxigenicity of Aspergillus section Flavi (ASF) in various regions are still under-reported, as many potentially toxic species are continuously discovered. This study aims to determine the aflatoxigenicity and identify 74 isolates of ASF from Malaysian and Nigerian food grains and feeds using phenotypic, chemotypic, and molecular methods. Isolates were screened for sclerotium morphotype and aspergillic acid production phenotypically, while an ultra-fast reverse-phase high-performance liquid chromatography (HPLC) was optimised to quantify the cyclopiazonic acid and aflatoxins produced by the isolated ASF, based on which, they were characterised into phenotypes L-strains, S-strains, and NS-strains, and chemotypes I to VI. Chemotypic data and nucleotide sequences of β-tubulin and internal transcribed spacer (ITS) genes were utilised to identify the isolates’ species and determine their phylogenetic relationships. Aflatoxigenesis on solid media and multiplex polymerase chain reactions of four essential genes (aflR, aflM, aflD, and aflP) of aflatoxin biosynthesis were used to confirm the aflatoxigenicity of each isolate. All the six chemotypes of ASF were present among the isolated strains, with the aflatoxigenic chemotypes (55.41%) and CPA-positive isolates (68.92%) being significantly higher (p < 0.05) than the non-aflatoxigenic and CPA negative chemotypes. Aflatoxins and CPA were produced in the 0.25–19.58 ng/g range and 29.89–46.55 ng/g, respectively. Chemotypic data accurately differentiate toxigenic from atoxigenic species but only identify 90% of species names. Molecular data accurately resolved species but not toxigenicity. The results highlight the need for robust regulatory measures in the concerned regions to mitigate the risk of aflatoxicosis.

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.

Effects of Tripleurospermum caucasicum, Salvia rosmarinus and Tanacetum fruticulosum essential oils on aflatoxin B1 production and aflR gene expression in Aspergillus flavus

Aflatoxin B1 (AFB1) is one of the most hazardous mycotoxins for humans and livestock that mainly produced by members of the genus Aspergillus in a variety of food commodities. In this study, the effect of S. rosmarinus, T. fruticulosum, and T. caucasicum essential oils (EOs) was studied on fungal growth, AFB1 production and aflR gene expression in toxigenic A. flavus IPI 247. The AFB1 producer A. flavus strain was cultured in YES medium in presence of various two-fold concentrations of the plant EOs (62.5–500 μg/mL) for 4 days at 28 °C. EO composition of plants was analyzed by Gas Chromatography/Mass Spectrometry (GC/MS). The amount of fungal growth, ergosterol content of fungal mycelia and AFB1 content of EO-treated and non-treated controls were measured. The expression of aflR gene was evaluated using Real-time PCR in the fungus exposed to minimum inhibitory concentration (MIC50) of EOs. The main constituents of the oils analyzed by GC/MS analysis were elemicin (33.80 %) and 2,3-dihydro farnesol (33.19 %) in T. caucasicum, 1,8-cineole (17.87 %), trans-caryophyllene (11.14 %), α and ẞ-pinene (10.92 and 8.83 %) in S. rosmarinus, and camphor (17.65 %), bornyl acetate (15.08 %), borneol (12.48 %) and camphene (11.72 %) in T. fruticulosum. The results showed that plant EOs at the concentration of 500 μg/mL suppressed significantly the fungal growth by 35.24–71.70 %, while mycelial ergosterol content and AFB1 production were inhibited meaningfully by 36.20–65.51 % and 20.61–89.16 %. T. caucasicum was the most effective plant, while T. fruticulosum showed the lowest effectiveness on fungal growth and AFB1 production. The expression of aflR in T. caucasicum and S. rosmarinus -treated fungus was significantly down-regulated by 2.85 and 2.12 folds, respectively, while it did not change in T. fruticulosum-treated A. flavus compared to non-treated controls. Our findings on the inhibitory activity of T. caucasicum and S. rosmarinus EOs toward A. flavus growth and AFB1 production could promise these plants as good candidates to control fungal contamination of agricultural crops and food commodities and subsequent contamination by AFB1. Down-regulation of aflR as the key regulatory gene in AF biosynthesis pathway warrants the use of these plants in AF control programs. Further studies to evaluate the inhibitory activity of studied plants EOs in food model systems are recommended.

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.

Characterization of Aspergillus section Flavi associated with stored grains

Increased frequencies of Aspergillus section Flavi and aflatoxins in cereal grains have been seen in recent years due to changes in climate circumstances, such as high temperatures and drought. To assess the microbiological risks of contamination, it is critical to have a reliable and accurate means of identifying the fungi. The main goal of this study was to characterize Aspergillus species from section Flavi obtained from twenty-three samples of barley and maize grains, gathered from different markets in Qena, Egypt, using morphological and molecular techniques. Twenty-three isolates were chosen, one isolate from each sample; they were identified as A. aflatoxiformans (4 isolates), A. flavus (18), and A. parasiticus (1). The existence of four aflatoxin biosynthesis genes was also investigated in relation to the strains’ ability to produce total aflatoxins and aflatoxin B1, focusing on the regulatory gene aflR and the structural genes aflD and aflM. All strains producing aflatoxins were linked to the presence of aflR1 and/or aflR2, except two isolates that exhibited aflatoxins but from which aflR1 or aflR2 were not detected, which may be due to one or more missing or unstudied additional genes involved in aflatoxin production. AflD and aflM genes were amplified by 10 and 9 isolates, respectively. Five samples of barley and maize were contaminated by aflatoxins. Fifteen isolates were positive for producing total aflatoxins in the range of 0.1–240 ppm. Antagonistic activity of Trichoderma viride against A. flavus (F5) was assessed at 31.3%. Trichoderma reduced total aflatoxins in all treated seeds, particularly those subjected to Trichoderma formulation.

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 3,73,54,834bp genome size, 2, 26, 257 number of contigs with N50 value of 49,272bp, 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.

A New Benzaldehyde Derivative Exhibits Antiaflatoxigenic Activity against Aspergillus flavus.

Aflatoxin B1 (AFB1) is the most potent naturally occurring carcinogen for humans and animals produced by the common fungus Aspergillus flavus (A. flavus). Aflatoxin (AF) contamination in commodities is a global concern related to the safety of food and feed, and it also impacts the agricultural economy. In this study, we investigated the AFB1-inhibiting activity of a new benzaldehyde derivative, 2-[(2-methylpyridin-3-yl)oxy]benzaldehyde (MPOBA), on A. flavus. It was found that MPOBA inhibited the production of AFB1 by A. flavus, with an IC50 value of 0.55 mM. Moreover, the inhibition of conidiation was also observed at the same concentration. The addition of MPOBA resulted in decreased transcript levels of the aflR gene, which encodes a key regulatory protein for the biosynthesis of AF, and also decreased transcript levels of the global regulator genes veA and laeA. These results suggested that MPOBA has an effect on the regulatory mechanism of the development and differentiation of conidia, leading to the inhibition of AFB1 production. In addition, the cytotoxicity study showed that MPOBA had a very low cytotoxic effect on the Madin-Darby canine kidney (MDCK) cell line. Therefore, MPOBA may be a potential compound for developing practically effective agents to control AF contamination.

The biodiversity of Aspergillus flavus in stored rice grain leads to a decrease in the overall aflatoxin B1 production in these species

Aspergillus flavus is a significant fungus that poses a threat to food safety by producing mycotoxins in various crops. In this study, A. flavus isolates were obtained from storage rice collected from seven provinces in southern China, and their AFB1 production, biosynthesis genes presence, and diversity were detected. Results showed that 56 out of the 81 A. flavus isolates produced detectable levels of AFB1, and 71 isolates (87.6 %) possessed aflR gene in their AF synthesis gene cluster, while only 41 isolates (50.6 %) had the ver-1 gene present. Genetic diversity analysis using inter-simple sequence repeats (ISSR) markers revealed seven main clusters among the isolates and the genetic similarity coefficients of 81 A. flavus isolates ranged from 0.53 to 1.00. Additionally, coculture assays were conducted using two toxigenic and two atoxigenic isolates from the same grain depot to investigate the effect of intraspecific inhibition on AFB1 production and to assess the AFB1 contamination risk of storage rice. The in situ results demonstrated that the atoxigenic isolates effectively inhibited the AFB1 contamination of toxigenic isolates. These findings provide insight into the genetic diversity of A. flavus isolates populations and highlight the potential food safety hazards of them in stored rice grain in China.

Detection of Aflatoxin B1-producing Aspergillus flavus strains from pistachio orchards soil in Iran by multiplex polymerase chain reaction method.

The current study aimed to report a multiplex polymerase chain reaction (PCR) assay as a monitoring technique to differentiate aflatoxigenic from non-aflatoxigenic strains of Aspergillus flavus isolated from pistachio orchards soil. In total, 25 A. flavus strains were isolated from soil samples of pistachio orchards. To test the strains for Aflatoxin B1 (AFB1)-producing ability, thin-layer chromatography (TLC) was used and the amounts of AFB1 were measured by high-performance liquid chromatography (HPLC). Multiplex PCR was used as a genome-based method to detect genes responsible for AFB1 production by A. flavus and the results were analyzed in terms of speed and specificity of detection. A set of four primers was designed specifically for the omtA, omtB, ver-1, and aflR genes which are commonly present in aflatoxin biosynthetic pathways. The AFB1 production by the A. flavus strains ranged from 0 to 321 ρg/μl. Four-band patterns of the primer sets were observed only in AFB1-producing A. flavus strains. Moreover, 18 out of the 25 strains showed all four bands belonging to omtA, omtB, ver-1, and aflR, whereas 7 strains did not display omtA, or aflR-related bands, in non-toxigenic and low toxin-producing A. flavus. The multiplex PCR is a supplementary strategy to current conventional mycotoxin analytical techniques, such as TLC and HPLC. It could be used as an efficient method to differentiate aflatoxigenic from non-aflatoxigenic strains of A. flavus. This achievement is crucial to minimize fungal contamination of food, feed, and agricultural commodities, thereby reducing the risk of subsequent aflatoxin consumption.

Screening of Aflatoxin Production by Aspergillus flavus Isolates from Petroleum-contaminated Soil

Fungi are eukaryotic, heterogeneous, unicellular to filamentous, spore-bearing, and chemoorganotrophic organisms which lack chlorophyll. This present study was carried out to isolate and identify fungi from petroleum-contaminated soil. Several fungal genera which included Rhizopus spp., Mucor spp., Penicillium spp., Rhizoctonia spp., Aspergillus spp., Alternaria spp., and Cladosporiumspp. were isolated using potatoes dextrose agar, Czapek-Dox Agar, and Aspergillus flavus Differentiation Agar culture media that comparable with co-amoxiclav (1g) and chloramphenicol to prevent the growth of any bacteria. The direct plate and serial dilution agar plate methods were used for the isolation of fungi. Based on results, Aspergillusand Mucor spp. were the most predominant genera and had the highest number of colonies in the soil samples. In this investigation, seven out of 27 soil samples were morphologically (macroscopically and microscopically) identified, such as A. flavus. Aflatoxigenicity of A. flavus was detected using characteristics in Aspergillus differentiation agar and colony fluorescence on exposure to ultraviolet light. Moreover, molecular approaches were used for the detection of aflatoxigenic of the A. flavus isolates. Three structural (aflD, aflO, and aflP) and one regulatory (aflR) gene of the aflatoxin gene cluster of A. flavus were targeted for amplification by the polymerase chain reaction method. The aflatoxigenic of all six A. flavus isolates was detected molecularly which contained two structural (aflD, aflP)genes out of three structural genes, while there was no specific amplification of the aflO gene in the fourth, fifth, and sixth A. flavus which issimilar to the aflR gene in the first and second A. flavus.

Tracking new insights into antifungal and anti-mycotoxigenic properties of a biofilm forming Pediococcus pentosaceus strain isolated from grain silage

The present study offers detailed insights into the antifungal and anti-mycotoxigenic potential of a biofilm forming lactic acid bacterium (Pediococcus pentosaceus) against one atoxigenic (Aspergillus flavus) and two toxigenic (Aspergillus nomius and Fusarium verticillioides) fungal strains. The antifungal effect of P. pentosaceus LBM18 strain was initially investigated through comparative analysis of fungi physiology by macroscopic visual evaluations and scanning electron microscopy examinations. The effects over fungal growth rate and asexual sporulation were additionally accessed. Furthermore, analytical evaluations of mycotoxin production were carried out by HPLC-MS/MS to provide insights on the bacterial anti-mycotoxigenic activity over fungal production of the aflatoxins B1, B2, G1 and G2 as well as fumonisins B1 and B2. Finally, reverse transcription quantitative real-time PCR (RT-qPCR) analysis was employed at the most effective bacterial inoculant concentration to evaluate, at the molecular level, the down-regulation of genes aflR, aflQ and aflD, related to the biosynthesis of aflatoxins by the strain of Aspergillus nomius. The effects over mycotoxin contamination were thought to be result of a combination of several biotic and abiotic factors, such as interaction between living beings and physical-chemical aspects of the environment, respectively. Several possible mechanisms of action were addressed along with potentially deleterious effects ascribing from P. pentosaceus misuse as biopesticide, emphasizing the importance of evaluating lactic acid bacteria safety in new applications, concentrations, and exposure scenarios.

Occurrence of fungi and mycotoxins in herbal medicines and rapid detection of toxin-producing fungi

Contamination from external hazardous materials may greatly influence the safety and efficacy of herbal medicines. This paper aimed to evaluate the levels of contamination by mycotoxins and toxigenic fungi in herbal medicines and establish a rapid method for detecting toxin-producing fungi. Herein, 62.92%, 36.25%, and 64.17% of herbal medicines were contaminated by aflatoxins (AFs), ochratoxins, and fumonisins, respectively. Aspergillus (43.77%), Fusarium (5.17%), and Cladosporium (4.46%) were the three predominant genera. Spearman's correlation results showed that Aspergillus and Fusarium were significantly and positively correlated with mycotoxin content (R > 0.5, P < 0.05). In addition, 323 fungal strains were isolated from herbal medicines, and 20 species were identified, mainly belonging to Aspergillus and Penicillium. Analysis of potential mycotoxin-producing fungi showed that Aspergillus flavus can produce AFs, and Aspergillus ochraceus and Aspergillus niger can produce ochratoxin A (OTA). Multiplex real-time polymerase chain reaction showed that A. flavus harbored AF synthesis genes (aflR), and A. ochraceus and A. niger harbored OTA synthesis genes (aoksl). With these synthesis genes, 67.07% and 37.20% of 164 herbal medicines were positive for toxigenic genes. Furthermore, an excellent correlation was found between the above gene copies and mycotoxin content (R2 = 0.99). Our results confirmed the high detection rate of mycotoxins in herbal medicines and identified pivotal AF- and OTA-producing fungi. In conclusion, this paper provided the contamination status of fungi and mycotoxins in herbal medicines and established a rapid method for detecting toxigenic fungi.

Molecular characterization of aflatoxigenic and non-aflatoxigenic Aspergillus flavus strains isolated from peanut sold in northern Togo

The contamination of agricultural products and aflatoxins synthesis can be controlled and reduced by the application of a biological control product based on atoxinogenic Aspergillus flavus strains. The purpose of this work was to select atoxinogenic Aspergillus flavus strains that can be used in the biological control of aflatoxins. Thirty-four (34) strains of Aspergillus flavus were used and the Nor-1, Ver-1, Omt-A and AflR genes involved in the aflatoxin biosynthesis pathway were sought. The growth speed and ability of atoxigenic strains to inhibit the growth of toxigenic strains were also determined. The results showed that 50% of the strains have at least one of the genes sought and the percentages of inhibition (PI) of the atoxinogenic strains varied significantly from 16.98% to 62.50% at the threshold of 5% according to the Tukey test (p-value = 0.0000136). The mycelial growth speed (VCM) of the atoxinogenic strains varied from 7.5 to 10 mm/day with an average VCM of 8.4 ± 0.05 mm/day. Strains AKA-10; AGA-8; AMAN-35; ABA-28 and AGA-46 were found to be more effective in inhibiting mycelial growth of toxigenic strains.

Multiple Year Influences of the Aflatoxin Biocontrol Product AF-X1 on the A. flavus Communities Associated with Maize Production in Italy.

AF-X1 is a commercial aflatoxin biocontrol product containing the non-aflatoxigenic (AF-) strain of Aspergillus flavus MUCL54911 (VCG IT006), endemic to Italy, as an active ingredient. The present study aimed to evaluate the long-term persistence of VCG IT006 in the treated fields, and the multi-year influence of the biocontrol application on the A. flavus population. Soil samples were collected in 2020 and 2021 from 28 fields located in four provinces in north Italy. A vegetative compatibility analysis was conducted to monitor the occurrence of VCG IT006 on the total of the 399 isolates of A. flavus that were collected. IT006 was present in all the fields, mainly in the fields treated for 1 yr or 2 consecutive yrs (58% and 63%, respectively). The densities of the toxigenic isolates, detected using the aflR gene, were 45% vs. 22% in the untreated and treated fields, respectively. After displacement via the AF- deployment, a variability from 7% to 32% was noticed in the toxigenic isolates. The current findings support the long-term durability of the biocontrol application benefits without deleterious effects on each fungal population. Nevertheless, based on the current results, as well as on previous studies, the yearly applications of AF-X1 to Italian commercial maize fields should continue.

Phytochemical investigation of alkaloid fractions of Fumaria vaillantii and F. asepala and their antifungal influence on gene expression pattern in Aspergillus species

The genus Fumaria is known for its strong medicinal effects. This research aimed to assess the phytochemical profile in various alkaloid fractions from Fumaria vaillantii Loisel and Fumaria asepala Boiss. grown in the Saveh region of the central province of Iran and their antifungal activity against Aspergillus flavus and Aspergillus niger was investigated in laboratory conditions. Also, the types and amount of alkaloid compounds and the effect of compounds on the expression of key genes of aflatoxin biosynthesis were conducted in order to investigate the ability of alkaloids of two Fumaria species to prepare environmentally friendly disinfectants. The total alkaloid contents were extracted using the soxhlet apparatus and then fractioned to perform GC-MS analysis. The effect of the extract of two species of Fumaria on the activity of Aspergillus flavus and Aspergillus niger were evaluated considering time factors (24 h, 48 h, and 72 h) and two concentrations (50 and 100 mg/mL). The total alkaloids of the leaf and fruit of F. vaillantii respectively contain (3.48 and 3.32 mgg?1DW, ) and F. asupala (2.38 and 2.26 mgg?1DW, respectively). Fractions 1 and 4 of leaf and fruit showed the highest total alkaloid content, which was then mixed for GC-MS analysis; isoquinoline alkaloids, including protopine and sanguinarine; parfumine and allocryptopine accounted for the highest percentage of alkaloids in both species. F. vaillantii had a higher content of these alkaloids. The antifungal activity of fractions 1 and 4 reduced radial growth in both Aspergillus species. The alkaloid fractions at both species caused downregulation of aflatoxin B1 biosynthesis key genes (aflR, aflD, and aflM) in A. flavus and fumonisin B2, key genes(fum8 and fum6) in A. niger. The obtained data shows that alkaloid fractions extracted from Fumaria species, F. vaillantii, can be useful in the preparation of nature-friendly disinfectants for postharvest disinfection of fruits and vegetables.

Evaluation of Aflatoxin-Producing Fungi in Indoor Air of Warehouses and Houses of Farmers in Giwa, Kaduna State Nigeria

The health risks associated with ingesting food contaminated with mycotoxins, particularly aflatoxin-contaminated staple foods like maize and other cereals, have been widely studied. However, there is little knowledge about the role of inhalation of pathogenic fungi as bioaerosols in contaminated air from handling crops as an occupational health risk. This paper presents a study aimed at determining the level of airborne aflatoxin-producing fungi in the indoor air of grain stores in the Giwa community of Kaduna State. Indoor air was sampled using the settling plate technique from grain stores, warehouses and living rooms. Metrological data of the studied area were collected from the Institute of Agricultural Research, ABU, Zaria. Airborne mycofloral concentrations were determined, and colonies of Aspergillus flavus were identified. The isolates were screened for aflatoxin production on Neutral Red Desiccated Coconut Agar (NRDCA). Selected aflatoxin-producing fungal isolates were screened for the presence of aflD (nor-1), aflM (ver-1) and AflR genes by PCR. Sampling was done once every month from October to December 2020. Mycofloral concentrations were in the range of 2.77x103−4.05x103 and 1.55x103−2.17x103CFUm-3for grain stores and living rooms respectively. A total of twelve (12) strains of A. flavus were isolated from the indoor air of the grain stores and warehouses while none was obtained from the living room. Eleven (11) isolates were confirmed to be aflatoxigenic on NRDCA, presenting 30 CFUm-3 of the indoor air mycofloral composition. The aflD, aflM and aflR were amplified with aflD being the most detected gene from all the aflatoxin-producing mould isolates of Aspergillus species. The mycofloral concentrations in the grain stores were higher than those in the living room and, in all the sampling sites, exceeded the limit of the total mycofloral concentration of 500 CFUm-3 for agricultural and industrial environments. There were significant differences (p&lt;0.05) in the indoor mycofloral concentrations between the grain stores/warehouses and the living room. The presence of aflatoxigenic strains of A. flavus in the stores indicates that grain handlers and traders are at risk of occupational exposure to aflatoxigenic fungi and aflatoxins. Hence, they should wear protective materials for their safety while working in such stores.

Inhibition of Aflatoxin Production by Citrinin and Non-Enzymatic Formation of a Novel Citrinin-Kojic Acid Adduct.

Screening for microorganisms that inhibit aflatoxin production from environments showed that Penicillium citrinum inhibited aflatoxin production by Aspergillus parasiticus. The inhibitory substance in the culture medium of P. citrinum was confirmed to be citrinin (CTN). RT-PCR analyses showed that CTN did not inhibit expressions of aflatoxin biosynthetic genes (aflR, pksL1, and fas-1) of A. parasiticus, whereas feeding experiments using A. parasiticus showed that CTN inhibited the in vivo conversion of dihydrosterigmatocystin to AFB2·AFG2. These results suggest that CTN inhibits a certain post-transcriptional step in aflatoxin biosynthesis. CTN in the culture medium of A. parasiticus was found to be decreased or lost with time, suggesting that a certain metabolite produced by A. parasiticus is the cause of the CTN decrease; we then purified, characterized, and then analyzed the substance. Physico-chemical analyses confirmed that the metabolite causing a decrease in CTN fluorescence was kojic acid (KA) and the resulting product was identified as a novel substance: (1R,3S,4R)-3,4-dihydro-6,8-dihydroxy-1-(3-hydroxy-6-(hydroxymethyl)-4-oxo-4H-pyran-2-yl)-3,4,5-trimethyl-1H-isochromene-7-carboxylic acid, which was named "CTN-KA adduct". Our examination of the metabolites' toxicities revealed that unlike CTN, the CTN-KA adduct did not inhibit aflatoxin production by A. parasiticus. These results indicate that CTN's toxicity was alleviated with KA by converting CTN to the CTN-KA adduct.