Reduced Aflatoxin Production Research Articles

Effect of Standard and Nano-Urea on the Inhibition of the Fungus Aspergillus flavus Growth and Reduction of Aflatoxin B1 Production in the Laboratory

Hussein, A.H. and H.Z. Hussein. 2024. Effect of Standard and Nano-Urea on the Inhibition of the Fungus Aspergillus flavus Growth and Reduction of Aflatoxin B1 Production in the Laboratory. Arab Journal of Plant Protection, 42(2): 184-188. https://doi.org/10.22268/AJPP-001235 The results obtained from this study showed that the standard and nano-urea at concentrations 1, 2, and 3% inhibited the growth of Aspergillus flavus on PDA medium by 54.90, 72.55 and 100% for normal urea, and 60.78, 100 and 100% for nano-urea, respectively, compared with normal growth in the control treatment. The results obtained also showed that adding the most effective concentrations of normal and nano urea to corn kernels with A. flavus and stored for 30 days led to the inhibition of Aspergillus flavus growth and reduced aflatoxin production by 73.22 and 78.30%, respectively, as compared to the control treatment. In addition, results showed that adding regular urea at a concentration of 3% and nano-urea at a concentration of 2% to maize grains contaminated with AFB1 and stored for 30 days led to the reduction of aflatoxin B1 production by A. flavus by 78.74 and 83.36%, respectively, compared to the control treatment. Keywords: Maize, Reduction ratio, Inhibition ratio, Nanotechnology, HPLC.

Cocoa-associated filamentous fungi for the biocontrol of aflatoxigenic Aspergillus flavus.

Aflatoxin and other mycotoxin contamination are major threats to global food security and present an urgent need to secure the global food crop against spoilage by mycotoxigenic fungi. Cocoa material is noted for naturally low aflatoxin contamination. This study was designed to assess the potential for harnessing cocoa-associated filamentous fungi for the biocontrol of aflatoxigenic Aspergillus flavus. The candidate fungi were isolated from fermented cocoa beans collected from four cocoa-growing areas in Ghana. Molecular characterization included Internal Transcribed Spacer (ITS)-sequencing for identification and polymer chain reaction (PCR) to determine mating type. Effects of the candidate isolates on growth and aflatoxin-production by an aflatoxigenic A. flavus isolate (BANGA1) were assessed. Aflatoxin production was monitored by UV fluorescence and quantified by enzyme-linked immunosorbent assay (ELISA). Thirty-six filamentous fungi were cultured and identified as Aspergillus, Cladosporium, Lichtheimia, or Trichoderma spp. isolates. The isolates generally interacted negatively with BANGA1 growth and aflatoxin production. The Aspergillus niger and Aspergillus aculeatus biocontrol candidates showed the strongest colony antagonism (54%-94%) and reduction in aflatoxin production (12%-50%) on agar. In broth, the A. niger isolates reduced aflatoxin production by up to 97%. Metabolites from the A. niger isolates showed the strongest inhibition of growth by BANGA1 and inhibited aflatoxin production. Four of the candidate isolates belonged to the MAT1-1 mating type and 12 identified as MAT1-2. This may be indicative of the potential for genetic recombination events between fungi in the field, a finding which is particularly relevant to the risk posed by A. flavus biocontrol measures that rely on atoxigenic A. flavus strains.

Targeting the Aspergillus flavus p2c gene through host-induced gene silencing reduces A. flavus infection and aflatoxin contamination in transgenic maize.

Aspergillus flavus is an opportunistic fungal pathogen that infects maize and produces aflatoxins. Using biocontrol or developing resistant cultivars to reduce aflatoxin contamination has only achieved limited success. Here, the A. flavus polygalacturonase gene (p2c) was targeted for suppression through host-induced gene silencing (HIGS) to reduce aflatoxin contamination in maize. An RNAi vector carrying a portion of the p2c gene was constructed and transformed into maize B104. Thirteen out of fifteen independent transformation events were confirmed to contain p2c. The T2 generation kernels containing the p2c transgene had less aflatoxin than those without the transgene in six out of eleven events we examined. Homozygous T3 transgenic kernels from four events produced significantly less aflatoxins (P ≤ 0.02) than the kernels from the null or B104 controls under field inoculation conditions. The F1 kernels from the crosses between six elite inbred lines with P2c5 and P2c13 also supported significantly less aflatoxins (P ≤ 0.02) than those from the crosses with null plants. The reduction in aflatoxin ranged from 93.7% to 30.3%. Transgenic leaf (T0 and T3) and kernel tissues (T4) were also found to have significantly higher levels of p2c gene-specific small RNAs. Further, homozygous transgenic maize kernels had significantly less fungal growth (27~40 fold) than the null control kernels 10 days after fungal inoculation in the field. The calculated suppression of p2c gene expression based on RNAseq data was 57.6% and 83.0% in P2c5 and P2c13 events, respectively. These results indicate clearly that the reduced aflatoxin production in the transgenic kernels is due to RNAi-based suppression of p2c expression, which results in reduced fungal growth and toxin production.

Prevalence of Aflatoxins in Camel Milk from the Arabian Peninsula and North Africa: A Reduction Approach Using Probiotic Strains.

Camel milk is known as a source of nutritional and health supplements. It is known to be rich in peptides and functional proteins. One main issue facing it is related to its contamination, mainly with aflatoxins. The present study aimed to evaluate camel milk samples from different regions while trying to reduce its toxicity using safe approaches based on probiotic bacteria. Collected samples of camel milk were sourced from two main regions: the Arabic peninsula and North Africa. Samples were tested for their contents of aflatoxins (B1 and M1) using two techniques to ensure desired contamination levels. Additionally, feed materials used in camel foods were evaluated. Applied techniques were also tested for their validation. The antioxidant activity of camel milk samples was determined through total phenolic content and antioxidant activity assays. Two strains of probiotic bacteria (Lactobacillus acidophilus NRC06 and Lactobacillus plantarum NRC21) were investigated for their activity against toxigenic fungi. The result revealed high contamination of aflatoxin M1 for all samples investigated. Furthermore, cross-contamination with aflatoxin B1 was recorded. Investigated bacteria were recorded according to their significant inhibition zones against fungal growth (11 to 40 mm). The antagonistic impacts were between 40% and 70% against toxigenic fungi. Anti-aflatoxigenic properties of bacterial strains in liquid media were recorded according to mycelia inhibition levels between 41 to 52.83% against Aspergillus parasiticus ITEM11 with an ability to reduce aflatoxin production between 84.39% ± 2.59 and 90.4% ± 1.32 from media. Bacteria removed aflatoxins from the spiked camel milk in cases involving individual toxin contamination.

Lysine 2‐hydroxyisobutyrylation orchestrates cell development and aflatoxin biosynthesis in Aspergillus flavus

Lysine 2-hydroxyisobutyrylation (Khib ) is a recently identified post-translational modification (PTM) that regulates numerous cellular metabolic processes. In pathogenic microorganism, although glycolysis and fungal virulence are regulated by Khib , its potential roles in fungi remain to be elusive. Our preliminary results showed that levels of Khib fluctuate over time in Aspergillus flavus, which frequently contaminates crops and produces carcinogenic aflatoxins. However, the perception of Khib function in A. flavus is limited, especially in mycotoxin-producing strains. Here, we performed a comprehensive analysis of Khib in A. flavus, and 7156 Khib sites were identified in 1473 proteins. Notably, we demonstrated that Khib of AflM, a key enzyme in aflatoxin biosynthesis, affected conidia production and sclerotia formation. Furthermore, aflM deletion impaired aflatoxin biosynthesis, and more importantly, strains in which Khib was mimicked by K to T mutation at K49, K179 and K180 sites showed reduced aflatoxin production compared with wild type and ΔaflM complementation strains. These results indicate that Khib at these sites of AflM negatively regulates aflatoxin biosynthesis in A. flavus. In summary, our study revealed the potential roles of Khib in A. flavus, and particularly shed light on a new way to regulate aflatoxin production via Khib .

Down-regulation of aflatoxin biosynthetic genes in Aspergillus parasiticus by Heliopsis longipes roots and affinin for reduction of aflatoxin production

Affinin present in Heliopsis longipes roots has been identified as an anti-aflatoxin molecule. However, its mechanism of action has yet to be clarified. Aflatoxins biosynthesis involves not less than 27 enzymatic reactions. In this work, the genes aflG, aflH, aflI, aflK, aflL, aflM, aflO, aflP, and aflQ of the aflatoxins cluster and the aflS gene encoding an internal regulatory factor involved in aflatoxins biosynthesis in Aspergillus parasiticus, were studied by qRT-PCR. Results demonstrated that ethanolic extract of H. longipes roots and affinin inhibit aflatoxin biosynthesis and fungal growth in a dose-dependent manner. At 300 µg/mL, ethanolic extract and affinin presented the highest inhibition of radial growth (86% and 94%) and aflatoxin production (68% and 80%). The qRT-PCR analysis demonstrated that nine tested genes were down-regulated by affinin and ethanolic extract. The most down-regulated was the aflK, a gene that encodes an enzyme cyclase with double function during the aflatoxin biosynthesis. While no significant down-regulation was obtaining for aflH gene. Exposure to affinin also resulted in decreased transcript levels of the internal regulator factor aflS. Based on our results, a model showing the regulatory mechanism in aflatoxin biosynthesis and its role in gene expression was proposed. In conclusion, affinin modulates the expression of several aflatoxin biosynthetic genes, leading to mycotoxin biosynthesis inhibition. Therefore, H. longipes roots is a suitable candidate to developed control strategies via lowering gene expressions as a future perspective in reducing aflatoxin contamination.

Aflatoxin, microbial contamination, sensory attributes, and morphological analysis of pistachio nut coated with methylcellulose.

Pistachio is a nut with high consumption that could be affected by aflatoxin contamination, which affects the consumption market; therefore, broad studies seem to be necessary for this area. In the current study, pistachio nuts (Abbasali variety) were coated with different concentrations (0.1%, 0.5%, 1%, and 2%) of methylcellulose (MC) by immersion method and then stored in the incubator (25°C) for four months. The inhibitory effect of hydrocolloid coating on microbial (mold, yeast, and total count) and aflatoxin (B1, B2, G1, G2, and total aflatoxin) contamination, as well as sensory attributes (flavor, color, crispiness, aroma, and total acceptability), was investigated during storage periods. Results showed that the storage period had a significant effect on yeast, mold, and total count. HPLC analysis results showed that coating with MC had a significant inhibitory effect on aflatoxin contamination, and the highest amount of aflatoxin contamination was related to the control sample (3.5%). All samples except sample coated with MC 0.5% had appropriate total acceptability. Regarding the inhibitory effect of MC edible coating on aflatoxin contamination, its application on pistachio nut could be a promising approach to control the fungus infection and reduce aflatoxin production in coated pistachio.

Anti-fungal activity of lactic acid bacterial isolates against aflatoxigenic fungi inoculated on peanut kernels

Aflatoxigenic fungi infect various agricultural commodities and produce a class I carcinogen aflatoxin B1 (AFB1). Sixteen bacterial isolates were screened from idli batter and other fermented foods, for antagonistic effect against Aspergillus spp. Based on the preliminary observation, three bacterial isolates from idli batter namely, IB1, IB4, IB7, and one from mango wine namely, MW2 were selected and further characterized. IB1 was found to be closely related to Lactobacillus plantarum argentoratensis while IB4, IB7, and MW2 were closely related to Enterococcus faecium according to their 16sRNA sequencing. Invitro and invivo antifungal tests in peanut revealed that cell free supernatant (CFS) obtained from these isolates inhibited fungal growth and significantly suppressed AFB1 production, and expression of aflD, aflP, OrdB and aflR genes. Further, microscopic examination showed that all isolates could reduce the conidial development and inhibits spore formation. The highest antifungal activity was observed in CFS of L. plantarum MN880325 and E. faecium MN880376, with an inhibition zone of ≥28 mm in PDA and ≥97% reduction in aflatoxin production on peanut kernels. ESI-MS and NMR spectroscopy revealed the active compounds from L. plantarum MN880325 and E. faecium MN880376 to be 3-phenyllactic acid and 4-hydroxyphenyllactic acid respectively.

Transcriptome Analysis Identified Coordinated Control of Key Pathways Regulating Cellular Physiology and Metabolism upon Aspergillus flavus Infection Resulting in Reduced Aflatoxin Production in Groundnut.

Aflatoxin-affected groundnut or peanut presents a major global health issue to both commercial and subsistence farming. Therefore, understanding the genetic and molecular mechanisms associated with resistance to aflatoxin production during host–pathogen interactions is crucial for breeding groundnut cultivars with minimal level of aflatoxin contamination. Here, we performed gene expression profiling to better understand the mechanisms involved in reduction and prevention of aflatoxin contamination resulting from Aspergillus flavus infection in groundnut seeds. RNA sequencing (RNA-Seq) of 16 samples from different time points during infection (24 h, 48 h, 72 h and the 7th day after inoculation) in U 4-7-5 (resistant) and JL 24 (susceptible) genotypes yielded 840.5 million raw reads with an average of 52.5 million reads per sample. A total of 1779 unique differentially expressed genes (DEGs) were identified. Furthermore, comprehensive analysis revealed several pathways, such as disease resistance, hormone biosynthetic signaling, flavonoid biosynthesis, reactive oxygen species (ROS) detoxifying, cell wall metabolism and catabolizing and seed germination. We also detected several highly upregulated transcription factors, such as ARF, DBB, MYB, NAC and C2H2 in the resistant genotype in comparison to the susceptible genotype after inoculation. Moreover, RNA-Seq analysis suggested the occurrence of coordinated control of key pathways controlling cellular physiology and metabolism upon A. flavus infection, resulting in reduced aflatoxin production.

Host Induced Gene Silencing Targeting Aspergillus flavus aflM Reduced Aflatoxin Contamination in Transgenic Maize Under Field Conditions.

Maize (Zea mays L.) is one of the major crops susceptible to Aspergillus flavus infection and subsequent contamination with aflatoxins, the most potent naturally produced carcinogenic secondary metabolites. This pathogen can pose serious health concerns and cause severe economic losses due to the Food and Drug Administration (FDA) regulations on permissible levels of aflatoxins in food and feed. Although biocontrol has yielded some successes in managing aflatoxin contamination, enhancing crop resistance is still the preferred choice of management for long-term sustainability. Hence, host induced gene silencing (HIGS) strategy was explored in this study. The A. flavus gene aflM encoding versicolorin dehydrogenase, a key enzyme involved in the aflatoxin biosynthetic pathway, was selected as a possible target for suppression through HIGS. An RNAi vector containing a portion of the aflM gene was constructed and introduced into immature B104 maize zygotic embryos through Agrobacterium transformation. PCR analysis of the genomic DNA from T0 leaf tissue confirmed the presence of the transgene in six out of the seven events. The seeds from the lines that showed reduced aflatoxin production in laboratory aflatoxin kernel screening assay (KSA) have been increased from T1 to T4 generation in the past four years. Changes in aflatoxin resistance in these transgenic kernels have been evaluated under both field and laboratory conditions. The T2 generation kernels containing the transgene from two events out of four examined had less aflatoxin (P ≤ 0.01 and P ≤ 0.08) than those without the transgene. Field-inoculated homozygous T3 and T4 transgenic kernels also revealed lower levels of aflatoxins (P ≤ 0.04) than kernels from the null (segregated non-transgenic samples) or B104 controls. A similar result was observed when the harvested T3 and T4 homozygous transgenic kernels were evaluated under KSA conditions without inoculation (P ≤ 0.003–0.05). These two events were crossed with LH195, LH197, LH210, and PHW79 elite breeding lines and the resulting crosses supported less aflatoxin (P ≤ 0.02) than the crosses made with non-transgenic lines. In addition, significantly higher levels of aflM gene-specific small RNAs were detected in the transgenic leaf and kernel tissues, indicating that the enhanced aflatoxin resistance in the homozygous transgenic kernels is likely due to suppression of aflM expression through HIGS.

Assessment of the Effect of Satureja montana and Origanum virens Essential Oils on Aspergillus flavus Growth and Aflatoxin Production at Different Water Activities.

Aflatoxin contamination of foodstuffs poses a serious risk to food security, and it is essential to search for new control methods to prevent these toxins entering the food chain. Several essential oils are able to reduce the growth and mycotoxin biosynthesis of toxigenic species, although their efficiency is strongly influenced by the environmental conditions. In this work, the effectiveness of Satureja montana and Origanum virens essential oils to control Aspergillus flavus growth was evaluated under three water activity levels (0.94, 0.96 and 0.98 aw) using a Bioscreen C, a rapid in vitro spectrophotometric technique. The aflatoxin concentrations at all conditions tested were determined by HPLC-FLD. Aspergillus flavus growth was delayed by both essential oil treatments. However, only S. montana essential oil was able to significantly affect aflatoxin production, although the inhibition percentages widely differed among water activities. The most significant reduction was observed at 0.96 aw, which is coincident with the conditions in which A. flavus reached the highest levels of aflatoxin production. On the contrary, the treatment with S. montana essential oil was not effective in significantly reducing aflatoxin production at 0.94 aw. Therefore, it is important to study the interaction of the new control compounds with environmental factors before their application in food matrices, and in vitro ecophysiological studies are a good option since they provide accurate and rapid results.

Effects of zinc chelating nutrients on Aflatoxin production in Aspergillus flavus

Aflatoxins are carcinogenic metabolites produced by Aspergillus and Penicillium spp. Aflatoxin contamination of food is a serious health hazard. Some metal ions (such as Zn2+) affect Aspergillus growth and aflatoxin biosynthesis. Presence of zinc in the growth medium incites aflatoxin production. This study investigates the effect of zinc binding amino acids and peptides on aflatoxin synthesis in indigenous toxigenic Aspergillus species isolated from agro-ecological zones in Northern Iran. Zinc (II) chelating nutrients (such as Histidine (His), Cysteine (Cys), Histidine-Cysteine (His-Cys), and triple peptide (Asn-Cys-Ser) were added to the growth medium of toxigenic Aspergillus isolates and incubated at temperature range of 25–40 °C. Aflatoxin production on different culture media was tested using ELISA. Addition of cysteine to Sabouraud dextrose broth (SDB) medium significantly reduced aflatoxin production, which could be related to its zinc chelating property. Aflatoxin production was drastically restrained at high concentration of His, especially in combination with Cys, at high pH values and incubation temperature (pH = 7.5, temperature = 40 °C). Aflatoxin production was low in presence of triple peptide (Asn-Cys-Ser) at concentration of 500 mg/L. From the application perspective, natural zinc chelators can be used as harmless aflatoxin-production inhibitors.

Biological control of aflatoxin-producing Aspergillus flavus by volatile organic compound-producing antagonistic yeasts

Aspergillus spp. are the most common phytopathogenic fungi able to produce various types of aflatoxins. Yeasts can produce volatile organic compounds (VOCs) that may be used as biocontrol agents against mycotoxigenic fungi. In this study, we aimed to evaluate antagonistic yeasts that are potentially capable of producing active VOCs against the aflatoxin-producing fungus, Aspergillus flavus A39. In total, 366 epiphytic and endophytic yeast strains isolated from leaves of rice, sugarcane, and corn in Thailand were screened for their potential. Only 49 yeast strains were able to produce antifungal volatile organic compounds (VOCs). Candida nivariensis DMKU-CE18 was the most effective yeast strain to inhibit the mycelium growth (64.9 ± 7.0% inhibition) and conidial germination (49.3 ± 3.3% inhibition) of A. flavus A39, and to reduce aflatoxin production (74.8 ± 6.5% reduction) in corn grains. The analysis results of headspace gas chromatography/mass spectrometry (GC/MS) revealed that the major VOC produced by this yeast strain was closest to 1-pentanol.

Comparison of the Antifungal and Antiaflatoxigenic Potential of Liquid and Vapor Phase of Thymus vulgaris Essential Oil Against Aspergillus flavus

The antifungal and antiaflatoxigenic activity of Thymus vulgaris essential oil (EO) against Aspergillus flavus was evaluated over a range of concentrations in vapor- and liquid-phase contact tests. Total reduction in mycelial growth in the vapor- and liquid-phase tests was detected at EO concentrations of 20 and 400 μg/mL, respectively. Treatment with 10 μg/mL EO reduced aflatoxin production by 97.0 and 56.4% in the vapor- and liquid-phase tests, respectively. Greater inhibition of the expression of both fungal development-related genes (brlA, abaA, and wetA) and aflatoxin biosynthesis-related genes (aflR, aflD, and aflK) was also observed in the vapor-phase test. A substantial reduction in aflatoxin production was also observed in brown rice (72.7%) and white rice (18.0%). Our results indicate that the way this EO contacts fungal cells significantly affects its antifungal activity and that T. vulgaris EO in vapor phase might be a good strategy to control fungal contamination.

Function of crzA in Fungal Development and Aflatoxin Production in Aspergillus flavus.

The calcineurin pathway is an important signaling cascade for growth, sexual development, stress response, and pathogenicity in fungi. In this study, we investigated the function of CrzA, a key transcription factor of the calcineurin pathway, in an aflatoxin-producing fungus Aspergillus flavus (A. flavus). To examine the role of the crzA gene, crzA deletion mutant strains in A. flavus were constructed and their phenotypes, including fungal growth, spore formation, and sclerotial formation, were examined. Absence of crzA results in decreased colony growth, the number of conidia, and sclerocia production. The crzA-deficient mutant strains were more susceptible to osmotic pressure and cell wall stress than control or complemented strains. Moreover, deletion of crzA results in a reduction in aflatoxin production. Taken together, these results demonstrate that CrzA is important for differentiation and mycotoxin production in A. flavus.

Synergistic Inhibition of Mycotoxigenic Fungi and Mycotoxin Production by Combination of Pomegranate Peel Extract and Azole Fungicide.

Fungal plant pathogens cause considerable losses in yield and quality of field crops worldwide. In addition, under specific environmental conditions, many fungi, including such as some Fusarium and Aspergillus spp., are further able to produce mycotoxins while colonizing their host, which accumulate in human and animal tissues, posing a serious threat to consumer health. Extensive use of azole fungicides in crop protection stimulated the emergence of acquired azole resistance in some plant and human fungal pathogens. Combination treatments, which become popular in clinical practice, offer an alternative strategy for managing potentially resistant toxigenic fungi and reducing the required dosage of specific drugs. In the current study we tested the effect of pomegranate peel extract (PPE) on the growth and toxin production of the mycotoxigenic fungi Aspergillus flavus and Fusarium proliferatum, both alone and in combination with the azole fungicide prochloraz (PRZ). Using time-lapse microscopy and quantitative image analysis we demonstrate significant delay of conidial germination and hyphal elongation rate in both fungi following PPE treatment in combination with PRZ. Moreover, PPE treatment reduced aflatoxin production by A. flavus up to 97%, while a combined treatment with sub-inhibitory doses of PPE and PRZ resulted in complete inhibition of toxin production over a 72 h treatment. These findings were supported by qRT-PCR analysis, showing down-regulation of key genes involved in the aflatoxin biosynthetic pathway under combined PPE/PRZ treatment al low concentrations. Our results provide first evidence for synergistic effects between the commercial drug PRZ and natural compound PPE. Future application of these findings may allow to reduce the required dosage of PRZ, and possibly additional azole drugs, to inhibit mycotoxigenic fungi, ultimately reducing potential concerns over exposure to high doses of these potentially harmful fungicides.

Biocontrol Activity of Volatile-Producing Bacillus megaterium and Pseudomonas protegens against Aspergillus flavus and Aflatoxin Production on Stored Rice Grains

In our previous study, three bacterial strains, Bacillus megaterium KU143, Microbacterium testaceum KU313, and Pseudomonas protegens AS15, were selected as effective biocontrol agents against Aspergillus flavus on stored rice grains. In this study, we evaluated the inhibitory effects of the volatiles produced by the strains on A. flavus growth and aflatoxin production on stored rice grains. The three strains significantly reduced mycelial growth of A. flavus in dual-culture assays compared with the negative control strain, Sphingomonas aquatilis KU408, and an untreated control. Of these tested strains, volatiles produced by B. megaterium KU143 and P. protegens AS15 markedly inhibited mycelial growth, sporulation, and conidial germination of A. flavus on agar medium and suppressed the fungal populations in rice grains. Moreover, volatiles produced by these two strains significantly reduced aflatoxin production in the rice grains by A. flavus. To our knowledge, this is the first report of the suppression of A. flavus aflatoxin production in rice grains using B. megaterium and P. protegens volatiles.

A Novel Method for Preventing and Reducing Aflatoxin Production from Toxigenic Fungi by Using Calcium Bentonite Clay Dip Treatment in Smoked Fish

A Novel Method for Preventing and Reducing Aflatoxin Production from Toxigenic Fungi by Using Calcium Bentonite Clay Dip Treatment in Smoked Fish

Genotypic Regulation of Aflatoxin Accumulation but Not Aspergillus Fungal Growth upon Post-Harvest Infection of Peanut (Arachis hypogaea L.) Seeds.

Aflatoxin contamination is a major economic and food safety concern for the peanut industry that largely could be mitigated by genetic resistance. To screen peanut for aflatoxin resistance, ten genotypes were infected with a green fluorescent protein (GFP)—expressing Aspergillus flavus strain. Percentages of fungal infected area and fungal GFP signal intensity were documented by visual ratings every 8 h for 72 h after inoculation. Significant genotypic differences in fungal growth rates were documented by repeated measures and area under the disease progress curve (AUDPC) analyses. SICIA (Seed Infection Coverage and Intensity Analyzer), an image processing software, was developed to digitize fungal GFP signals. Data from SICIA image analysis confirmed visual rating results validating its utility for quantifying fungal growth. Among the tested peanut genotypes, NC 3033 and GT-C20 supported the lowest and highest fungal growth on the surface of peanut seeds, respectively. Although differential fungal growth was observed on the surface of peanut seeds, total fungal growth in the seeds was not significantly different across genotypes based on a fluorometric GFP assay. Significant differences in aflatoxin B levels were detected across peanut genotypes. ICG 1471 had the lowest aflatoxin level whereas Florida-07 had the highest. Two-year aflatoxin tests under simulated late-season drought also showed that ICG 1471 had reduced aflatoxin production under pre-harvest field conditions. These results suggest that all peanut genotypes support A. flavus fungal growth yet differentially influence aflatoxin production.

Antifungal Activity and Aflatoxin Degradation of Bifidobacterium Bifidum and Lactobacillus Fermentum Against Toxigenic Aspergillus Parasiticus.

Food and feedstuff contamination with aflatoxins (AFTs) is a serious health problem for humans and animals, especially in developing countries. The present study evaluated antifungal activities of two lactic acid bacteria (LAB) against growth and aflatoxin production of toxigenic Aspergillus parasiticus. The mycelial growth inhibition rate of A. parasiticus PTCC 5286 was investigated in the presence of Bifidobacterium bifidum PTCC 1644 and Lactobacillus fermentum PTCC 1744 by the pour plate method. After seven days incubation in yeast extract sucrose broth at 30°C, the mycelial mass was weighed after drying. The inhibitory activity of LAB metabolites against aflatoxin production by A. parasiticus was evaluated using HPLC method. B. bifidum and L. fermentum significantly reduced aflatoxin production and growth rate of A. parasiticus in comparison with the controls (p≤0.05). LAB reduced total aflatoxins and B1, B2, G1 and G2 fractions by more than 99%. Moreover, LAB metabolites reduced the level of standard AFB1, B2, G1 and G2 from 88.8% to 99.8% (p≤0.05). Based on these findings, B. bifidum and L. fermentum are recommended as suitable biocontrol agents against the growth and aflatoxin production by aflatoxigenic Aspergillus species.