Vibrio gazogenes inhibits aflatoxin production through downregulation of aflatoxin biosynthetic genes in Aspergillus flavus

Aspergillus flavus is an opportunistic pathogen of oilseed crops such as maize, peanut, cottonseed, and tree nuts and produces carcinogenic secondary metabolites known as aflatoxins during seed colonization. Aflatoxin contamination not only reduces the value of the produce but also is a health hazard to humans and animals. Previously, we observed inhibition of A. flavus aflatoxin biosynthesis upon exposure to the marine bacterium, Vibrio gazogenes (Vg). In this study, we used RNA sequencing to examine the transcriptional profiles of A. flavus treated with both live and heat-inactivated dead Vg and control samples. Fungal biomass, total accumulated aflatoxins, and expression profiles of genes constituting secondary metabolite biosynthetic gene clusters were determined at 24, 30, and 40 h after treatment. Statistically significant reductions in total aflatoxins were detected in Vg-treated samples as compared to control samples at 40 h. But no statistical difference in fungal biomass was observed upon these treatments. The Vg treatments were most effective on aflatoxin biosynthesis as was reflected in significant downregulation of majority of the genes in the aflatoxin gene cluster including the aflatoxin pathway regulator gene, aflR. Along with aflatoxin genes, we also observed significant downregulation in some other secondary metabolite gene clusters including cyclopiazonic acid and aflavarin, suggesting that the treatment may inhibit other secondary metabolites as well. Finally, a weighted gene correlation network analysis identified an upregulation of ten genes that were most strongly associated with Vg-dependent aflatoxin inhibition and provide a novel start-point in understanding the mechanisms that result in this phenomenon.

Spatial and temporal population dynamics of Aspergillus flavus in commercial pistachio orchards in Arizona

Aspergillus flavus infects a wide range of crops, including pistachios, and subsequent aflatoxin contamination results in significant economic losses. Application of biocontrol products based on non-aflatoxigenic (atoxigenic) strains of A. flavus is one of the most effective tactics for controlling aflatoxins in crops. Both risk of aflatoxin contamination and effectiveness of biocontrol are influenced by the extent to which A. flavus spores move into pistachio tree canopies during periods of nut development. Thus, the purpose of this study was to evaluate spatial and temporal population dynamics of A. flavus, including the applied biocontrol strain AF36, in canopies of pistachio orchards in Arizona. Propagule densities of A. flavus were quantified on leaf samples collected from lower, middle, and upper canopies from spring through harvest in 2018 and 2019. Aspergillus flavus propagule densities peaked during periods of high temperature and rainfall in 2018 (up to 600 CFU/g) and 2019 (up to 23 CFU/g), which coincided with nut development and maturation. The applied biocontrol strain AF36 was detected at all canopy heights, but overall propagule densities were greater in the upper and middle canopy (mean = 70 CFU/g) compared to the lower canopy (mean = 47 CFU/g). Results suggest June to August is the period during which A. flavus inoculum increases in Arizona pistachio orchards, and to most effectively displace aflatoxin-producing fungi in tree canopies, biocontrol applications should precede this period. In addition, this study demonstrates that soil-applied biocontrol strains can successfully disperse throughout the canopies of commercial tree nut orchards.

Microbial Quality and Aflatoxin Levels of Bread and Flour Products Vended in Akure Metropolis, Ondo State, Nigeria

Aim: This study evaluated the microbial quality characteristics of bread and flour-made products vended for human consummation in Akure metropolis. Methods: The sample products including bread, buns, puff puff, meat pie and cake collected from different locations were analysed using standard microbiological methods to enumerate the bacterial and fungal consortia. Macro and micro-morphological identification of the implicated fungi in the food samples were done via standard techniques. The presence and quantity of some aflatoxin types were also investigated using standard techniques. Results: The fungal organisms enumerated include species of Fusarium, Aspergillus, Cladosporium, Mucor, Sacharomyces cerevisiae, Rhizopus and Penicillium. Bacteria consortium implicated in sample products include; Staphylococcus aureus, Bacillus sp., Escherichia coli, Clostridium sp., Pseudomonas aeruginosa and the likes. The levels of aflatoxin B1 and B2 produced were predominantly associated with Aspergillus flavus enumerated from bread products which serve as a rider to the aflatoxin contamination in vended flour products. Conclusion: The toxicity and potency of aflatoxins make them a primary health hazard and as well accountable for losses associated with contamination of processed foods and ready-to-eat foods. It is recommended that bakers should implement the use of heat-treated flour in the production process of ready-to-eat products for human safety.

Genetic Variation and Aflatoxin Accumulation Resistance among 36 Maize Genotypes Evaluated in Ghana

Aflatoxins are carcinogenic secondary metabolites produced predominantly by the fungi Aspergillus flavus and parasiticus. The toxin contaminate maize grains and threatens human food safety. Survey in Ghana revealed aflatoxin contamination of maize in excess of 941 ppb which is way beyond WHO and USA approved limits of 15 ppb and 20 ppb respectively. Host plant resistance is considered as the best strategy for reducing aflatoxins. This study was designed to (1) identify and select suitable maize lines that combine aflatoxin accumulation resistance and good agronomic traits under tropical conditions and (2) assess the genetic diversity among the exotic and locally adapted maize genotypes using significant morphological traits. Thirty-six maize genotypes, 19 from Mississippi State University, USA and 17 locally adapted genotypes in Ghana were evaluated for aflatoxin accumulation resistance and good agronomic characteristics across six contrasting environments using a 6x6 lattice design with three replicates. Five plants each per genotype were inoculated with a local strain of Aspergillus flavus inoculum at a concentration of 9 x 107/3.4 ml, two weeks after 50% mid silking. Total aflatoxin in the kernels were determined at harvest using HPLC method. Statistical analysis for agronomic traits and aflatoxin levels were performed using PROC GLM procedure implemented in SAS. The result indicated that genotype by environment interaction was significant (p < 0.05) for aflatoxin accumulation resistance and many other agronomic traits. Five genotypes (MP715, NC298, MP705, MP719, CML287 and TZEEI- 24) consistently displayed stable resistance across the environments and may serve as suitable candidates for developing aflatoxin resistant hybrids. Cluster analysis showed two distinct groups (locally adapted and exotic genotypes), an indication of re-cycled alleles per region. Broad sense heritability estimates for grain yield and aflatoxin accumulation resistance were moderately high, which could permit transfer of traits during hybrid development.

Enzymatic degradation is an effective means to reduce aflatoxin contamination in maize

Background Aflatoxins are carcinogenic compounds produced by certain species of Aspergillus fungi. The consumption of crops contaminated with this toxin cause serious detrimental health effects, including death, in both livestock and humans. As a consequence, both the detection and quantification of this toxin in food/feed items is tightly regulated with crops exceeding the allowed limits eliminated from food chains. Globally, this toxin causes massive agricultural and economic losses each year. Results In this paper we investigate the feasibility of using an aflatoxin-degrading enzyme strategy to reduce/eliminate aflatoxin loads in developing maize kernels. We used an endoplasmic reticulum (ER) targeted sub-cellular compartmentalization stabilizing strategy to accumulate an aflatoxin-degrading enzyme isolated from the edible Honey mushroom Armillariella tabescens and expressed it in embryo tissue in developing maize kernels. Three transgenic maize lines that were determined to be expressing the aflatoxin-degrading enzyme both at the RNA and protein level, were challenged with the aflatoxin-producing strain Aspergillus flavus AF13 and shown to accumulate non-detectable levels of aflatoxin at 14-days post-infection and significantly reduced levels of aflatoxin at 30-days post-infection compared to nontransgenic control Aspergillus-challenged samples. Conclusions The expression of an aflatoxin-degrading enzyme in developing maize kernels was shown to be an effective means to control aflatoxin in maize in pre-harvest conditions. This aflatoxin-degradation strategy could play a significant role in the enhancement of both US and global food security and sustainability.

Flavonoids Modulate the Accumulation of Toxins From Aspergillus flavus in Maize Kernels

Aspergillus flavus is an opportunistic fungal pathogen capable of producing aflatoxins, potent carcinogenic toxins that accumulate in maize kernels after infection. To better understand the molecular mechanisms of maize resistance to A. flavus growth and aflatoxin accumulation, we performed a high-throughput transcriptomic study in situ using maize kernels infected with A. flavus strain 3357. Three maize lines were evaluated: aflatoxin-contamination resistant line TZAR102, semi-resistant MI82, and susceptible line Va35. A modified genotype-environment association method (GEA) used to detect loci under selection via redundancy analysis (RDA) was used with the transcriptomic data to detect genes significantly influenced by maize line, fungal treatment, and duration of infection. Gene ontology enrichment analysis of genes highly expressed in infected kernels identified molecular pathways associated with defense responses to fungi and other microbes such as production of pathogenesis-related (PR) proteins and lipid bilayer formation. To further identify novel genes of interest, we incorporated genomic and phenotypic field data from a genome wide association analysis with gene expression data, allowing us to detect significantly expressed quantitative trait loci (eQTL). These results identified significant association between flavonoid biosynthetic pathway genes and infection by A. flavus. In planta fungal infections showed that the resistant line, TZAR102, has a higher fold increase of the metabolites naringenin and luteolin than the susceptible line, Va35, when comparing untreated and fungal infected plants. These results suggest flavonoids contribute to plant resistance mechanisms against aflatoxin contamination through modulation of toxin accumulation in maize kernels.

Mitigation of Aflatoxin Contamination in Groundnuts using Trichoderma viride

Groundnuts are often prone to contamination by Microorganisms during pre-harvest or post-harvest storage. One such contaminant is Aspergillus flavus which is abundantly found in soil and air. Several strains of A. flavus are known to produce mycotoxins named as aflatoxins. These aflatoxins are potent carcinogenic agents whose destruction has become a challenging task in the present scenario. Various physical and chemical methods are available to eliminate the growth of Aspergillus flavus but these methods have several demerits. The present study is based on biological control of Aspergillus flavus using Trichoderma viride strain TV 10. Antagonistic studies of Tv 10 against A.flavus were carried out by performing dual culture technique.

Development and scale-up of bioprotectants to keep staple foods safe from aflatoxin contamination in Africa

Aflatoxins pose a significant public health risk, decrease productivity and profitability and hamper trade. To minimize aflatoxin contamination a biocontrol technology based on atoxigenic strains of Aspergillus flavus that do not produce aflatoxin is used widely in the United States. The technology, with the generic name Aflasafe, has been improved and adapted for use in Africa. Aflasafe products have been developed or are currently being developed in 20 African countries. Aflatoxin biocontrol is being scaled up for use in several African countries through a mix of public, private, and public-private interventions. Farmers in several countries have commercially treated nearly 400,000 ha of maize and groundnut achieving >90% reduction in aflatoxin contamination. This chapter summarizes the biology of aflatoxin-producing fungi and various factors affecting their occurence, including climate change. Various management practices for aflatoxin mitigation are then discussed. These include biological control, which is increasingly being adopted by farmers in several countries. We discuss biocontrol product development and commercialization in various African countries. Subsequently, we highlight some barriers to adoption and other challenges.

Peanut Seed Coat Acts as a Physical and Biochemical Barrier against Aspergillus flavus Infection

Aflatoxin contamination is a global menace that adversely affects food crops and human health. Peanut seed coat is the outer layer protecting the cotyledon both at pre- and post-harvest stages from biotic and abiotic stresses. The aim of the present study is to investigate the role of seed coat against A. flavus infection. In-vitro seed colonization (IVSC) with and without seed coat showed that the seed coat acts as a physical barrier, and the developmental series of peanut seed coat showed the formation of a robust multilayered protective seed coat. Radial growth bioassay revealed that both insoluble and soluble seed coat extracts from 55-437 line (resistant) showed higher A. flavus inhibition compared to TMV-2 line (susceptible). Further analysis of seed coat biochemicals showed that hydroxycinnamic and hydroxybenzoic acid derivatives are the predominant phenolic compounds, and addition of these compounds to the media inhibited A. flavus growth. Gene expression analysis showed that genes involved in lignin monomer, proanthocyanidin, and flavonoid biosynthesis are highly abundant in 55-437 compared to TMV-2 seed coats. Overall, the present study showed that the seed coat acts as a physical and biochemical barrier against A. flavus infection and its potential use in mitigating the aflatoxin contamination.

An in Vitro Antifungal and Antiaflatoxigenic Properties of Commiphora myrrha and Prunus mahaleb

Aflatoxins and especially aflatoxin B, are the devastating contaminant of food and feed products with hazardous effects to mankind and his domestic animals. These investigations were set to evaluate the effect of various levels of Commiphora myrrha resin (1.0, 1.25, 2.25, and 3.25 g/100 ml) and Prunus mahaleb seed extract (0.75, 1.5, 2.5, and 3.5 g/100 ml) on the growth and aflatoxin secretion by two aflatoxigenic strains of Aspergillus flavus and A. parasiticus. The two plant extracts significantly (p&lt;0.05) decreased aflatoxin secretion, and inhibited the fungal growth. Resin of C. myrrha displayed 51.9-95.7% reduction in total aflatoxin secretion by A. flavus, and 46.9-92% for A. parasiticus, and Seed extract of P. mahaleb decreased aflatoxin up to 53.7-95.8% and 40-94.7%, respectively. The inhibition of aflatoxin B (B1 and B2) by myrrh resin and seed extract of mahaleb ranged between 51.7-93.5, 50-93.6% (A. flavus) and 39.5-89.7%, 37.9-93% (A. parasiticus). The mycelial dry weight of A. flavus and A. parasiticus ws decreased up to 46.1-58.7%, 28.9-51.3% (Myrrh resin), and between 45-56.9%, 33.3-55.9% (Mahaleb seed extract). Nonetheless, the two plant extracts did not detoxify aflatoxin B1. Therefore, it apparent that the resin of C. myrrha and seed extract of P. mahaleb affected the biosynthesis pathway of aflatoxins. Thus, they can be recommended as effective natural plant biopreservative against aflatoxin contamination of food and feed products.