New perspectives for the application of bioplastic materials in the biocontrol of Aspergillus flavus in corn

Mycotoxins are secondary metabolites produced by certain filamentous fungi that can contaminate a large variety of agricultural commodities before and after harvest. Among different mycotoxins, aflatoxins and especially aflatoxin B1 are of particular concern because they are potent natural carcinogens. Aflatoxin-producing fungi, mainly Aspergillus flavus and A. parasiticus, are ubiquitous, being commonly isolated from agricultural soil and crop debris. Although many aspects of the ecology of aflatoxin-producing fungi have been elucidated, control of aflatoxin contamination of agricultural crops remains a difficult task. Agronomical practices promoting general plant health have shown variable and more frequently limited success in preharvest control of aflatoxin contamination. Competitive replacement of indigenous toxigenic soil isolates is considered a more promising and effective approach. This biocontrol strategy is based on field application of a large number of propagules of nontoxigenic strains of A. flavus. Biocontrol strains are typically formulated as inoculated or spore-coated grain seeds. More recently, efforts to explore new approaches and technologies have resulted in the development of other practical solutions, including a bioplastic-based formulation. This formulation originally developed in 2008, consists of bioplastic granules entrapping spores of the nontoxigenic biocontrol strain, A. flavus NRRL 30797. Laboratory and field studies that have been conducted until now have clearly shown that granules of the starch-based bioplastic Mater-Bi^® are effective in delivering this biocontrol strain. In addition to having a satisfactory shelf life, the granules are easy to prepare, handle, and apply to agricultural fields. More importantly, this novel bioplastic formulation is capable of efficiently reducing aflatoxin contamination of corn. The bioplastic Mater-Bi^® can also have other applications. For instance, rods or granules prepared using a slightly modified Mater-Bi^® bioplastic matrix can be used to selectively isolate A. flavus from soil and corn kernels.     

Current Research on Reducing Pre‐ and Post‐harvest Aflatoxin Contamination of U.S. Almond,Pistachio, and Walnut

Aflatoxins are considered to be potent carcinogens and teratogens to humans and farm animals. A variety of species of the fungal genus Aspergillus (mainly A. flavus and A. parasiticus) synthesize aflatoxins. Spores of these fungi are common in air and soil of agricultural areas of temperate and tropical environments. Because aflatoxigenic fungi are ubiquitous and opportunistic, aflatoxin contamination has become a food safety concern. The chief U.S. crops affected by the threat of contamination with aflatoxin include corn, peanuts, cottonseed, and certain tree nuts. Additionally, aflatoxin contamination has also become an international trade issue. Major trading partners of U.S. agricultural products have set total aflatoxin action threshold levels at four ng/g (ppb). This action level is far below the 20 ppb level recommended by the U.S. Food and Drug administration for domestic foods. Almonds, pistachios and walnuts are one of the major food commodities affected by food safety and trade issues associated with aflatoxin contamination. Commercial domestic production of these tree nuts in the U.S. is entirely in California. Moreover, 50 to 75% of domestically produced tree nuts are exported, chiefly to countries of the European Union (EU), which adhere to the four ppb action threshold level. Scientists at the USDA's Western Regional Research Center and the University of California, Davis' Department of Pomology and Kearney Agricultural Center have developed products and methods to reduce aflatoxin contamination of tree nuts. Control of insect pests in tree nut orchards is a major strategy to curtail aflatoxin contamination. Insect feeding damage can lead to fungal infection and concomitant aflatoxin contamination. This is especially the case with navel orangeworm on pistachio and almond. A new and potent lure has been developed to control codling moth, a major insect pest of walnuts whose feeding damage potentially leads to fungal infection. Through breeding and genetic engineering, new varieties of almonds and walnuts have been developed which are resistant to insect attack. New orchard management strategies have been prescribed to reduce reservoirs of A. flavus in tree nut orchards. A number of saprophytic yeasts, natural to tree nut orchards, have been discovered which show promise as biological control agents of A. flavus, in vitro, and are awaiting field testing. New and improved risk assessment models have been developed for sampling and measuring aflatoxin contamination through the processing stream and in bulk shipping lots of tree nuts. An automated sorter that detects and removes aflatoxin contaminated nuts from a processing stream in real time was developed. It was also concluded that methods currently used for hand‐cracking of closed shell pistachios result in a higher risk of aflatoxin contamination. Perhaps the foremost breakthrough to date, however, is that constituents of walnut seed coat, especially from the cultivar ‘Tulare’, are potent inhibitors of aflatoxin biosynthesis, capable of rendering aflatoxigenic A. flavus virtually atoxigenic.     

Ecology and Population Biology of Aflatoxigenic Fungi in Soil

Soil serves as a reservoir for Aspergillus flavus and A. parasiticus, fungi that produce carcinogenic aflatoxins in agricultural commodities. Populations in soil are genetically diverse and individual genotypes show a clustered distribution pattern within fields. Surveys over large geographic regions suggest that climate and crop composition influence species density and aflatoxin‐producing potential. Aflatoxigenic fungi reside in soil as conidia, sclerotia and hyphae, which act as primary inocula for directly infecting peanuts or for infecting aerial crops (corn, cottonseed, tree nuts) through wind and insect dispersal. Infected crops periodically replenish soil populations during drought years.     

Antifungal and anti-aflatoxigenic effect of probiotics against Aspergillus flavus and Aspergillus parasiticus

Aflatoxins are carcinogenic mycotoxin, produced by Aspergillus species. These molds infect food crops in warm humid conditions causing economic losses and affecting the consumers' health adversely. In this study, antifungal activity and aflatoxin inhibiting ability of four probiotic strains against Aspergillus flavus and Aspergillus parasiticus were studied. The aflatoxin secreted was analyzed and quantified by both UV spectrophotometer and HPLC. It was found that Lactobacillus delbrueckii subsp. lactis showed maximal antifungal (67.43% reduction) and anti-aflatoxigenic (94.33% reduction) activity against A. flavus whereas A. parasiticus was inhibited by Lactobacillus brevis with the antifungal reduction of 69.38% and anti-aflatoxigenic reduction of 96.12%.     

Aflatoxin Contamination,Its Impact and Management Strategies: An Updated Review

Aflatoxin, a type of mycotoxin, is mostly produced by Aspergillus flavus and Aspergillus parasiticus. It is responsible for the loss of billions of dollars to the world economy, by contaminating different crops such as cotton, groundnut, maize, and chilies, and causing immense effects on the health of humans and animals. More than eighteen different types of aflatoxins have been reported to date, and among them, aflatoxins B1, B2, G1, and G2 are the most prevalent and lethal. Early detection of fungal infection plays a key role in the control of aflatoxin contamination. Therefore, different methods, including culture, chromatographic techniques, and molecular assays, are used to determine aflatoxin contamination in crops and food products. Many countries have set a maximum limit of aflatoxin contamination (2–20 ppb) in their food and agriculture commodities for human or animal consumption, and the use of different methods to combat this menace is essential. Fungal infection mostly takes place during the pre- and post-harvest stage of crops, and most of the methods to control aflatoxin are employed for the latter phase. Studies have shown that if correct measures are adopted during the crop development phase, aflatoxin contamination can be reduced by a significant level. Currently, the use of bio-pesticides is the intervention employed in many countries, whereby atoxigenic strains competitively reduce the burden of toxigenic strains in the field, thereby helping to mitigate this problem. This updated review on aflatoxins sheds light on the sources of contamination, and the on occurrence, impact, detection techniques, and management strategies, with a special emphasis on bio-pesticides to control aflatoxins.     

An industry perspective on the use of “atoxigenic” strains of Aspergillus flavus as biological control agents and the significance of cyclopiazonic acid

Several nonaflatoxigenic strains of Aspergillus flavus have been registered in the United States to reduce aflatoxin accumulation in maize and other crops, but there may be unintended negative consequences if these strains produce cyclopiazonic acid (CPA). AF36, a nonaflatoxigenic, CPA-producing strain has been shown to produce CPA in treated maize and peanuts. Alternative strains, including Afla-Guard^® brand biocontrol agent and K49, do not produce CPA and can reduce both aflatoxin and CPA in treated crops. Chronic toxicity of CPA has not been studied, and recent animal studies show significant harmful effects from short-term exposure to CPA at low doses. Grower and industry confidence in this approach must be preserved through transparency.     

Characterization of Ugandan Endemic Aspergillus Species and Identification of Non-Aflatoxigenic Isolates for Potential Biocontrol of Aflatoxins

Acute stunting in children, liver cancer, and death often occur due to human exposure to aflatoxins in food. The severity of aflatoxin contamination depends on the type of Aspergillus fungus infecting the crops. In this study, Aspergillus species were isolated from households’ staple foods and were characterized for different aflatoxin chemotypes. The non-aflatoxigenic chemotypes were evaluated for their ability to reduce aflatoxin levels produced by aflatoxigenic A. flavus strains on maize grains. Aspergillus flavus (63%), A. tamarii (14%), and A. niger (23%) were the main species present. The A. flavus species included isolates that predominantly produced aflatoxins B1 and B2, with most isolates producing a high amount (>20 ug/µL) of aflatoxin B1 (AFB1), and a marginal proportion of them also producing G aflatoxins with a higher level of aflatoxin G1 (AFG1) than AFB1. Some non-aflatoxigenic A. tamarii demonstrated a strong ability to reduce the level of AFB1 by more than 95% when co-inoculated with aflatoxigenic A. flavus. Therefore, field evaluation of both non-aflatoxigenic A. flavus and A. tamarii would be an important step toward developing biocontrol agents for mitigating field contamination of crops with aflatoxins in Uganda.     

Identification and Quantification of a Toxigenic and Non-Toxigenic Aspergillus flavus Strain in Contaminated Maize Using Quantitative Real-Time PCR

Aflatoxins, which are produced by Aspergillus flavus, are toxic to humans, livestock, and pets. The value of maize (Zea mays) grain is markedly reduced when contaminated with aflatoxin. Plant resistance and biological control using non-toxin producing strains are considered effective strategies for reducing aflatoxin accumulation in maize grain. Distinguishing between the toxin and non-toxin producing strains is important in determining the effectiveness of bio-control strategies and understanding inter-strain interactions. Using polymorphisms found in the fungal rRNA intergenic spacer region (IGS) between a toxigenic strain of A. flavus (NRRL 3357) and the non-toxigenic strain used in the biological control agent Afla-Guard^® (NRRL 21882), we developed a set of primers that allows for the identification and quantification of the two strains using quantitative PCR. This primer set has been used to screen maize grain that was inoculated with the two strains individually and co-inoculated with both strains, and it has been shown to be effective in both the identification and quantification of both strains. Screening of co-inoculated ears from multiple resistant and susceptible genotypic crosses revealed no significant differences in fungal biomass accumulation of either strain in the field tests from 2010 and 2011 when compared across the means of all genotypes. Only one genotype/year combination showed significant differences in strain accumulation. Aflatoxin accumulation analysis showed that, as expected, genotypes inoculated with the toxigenic strain accumulated more aflatoxin than when co-inoculated with both strains or inoculated with only the non-toxigenic strain. Furthermore, accumulation of toxigenic fungal mass was significantly correlated with aflatoxin accumulation while non-toxigenic fungal accumulation was not. This primer set will allow researchers to better determine how the two fungal strains compete on the maize ear and investigate the interaction between different maize lines and these A. flavus strains.     

Cyclopiazonic acid biosynthesis by Aspergillus flavus

Cyclopiazonic acid (CPA) is an indole-tetramic acid mycotoxin produced by some strains of Aspergillus flavus. Characterization of the CPA biosynthesis gene cluster confirmed that formation of CPA is via a three-enzyme pathway. This review examines the structure and organization of the CPA genes, elucidates the specific roles of functional domains of each enzyme in carrying out catalytic conversions leading to CPA production, and delineates the molecular basis for the lack of CPA production by A. flavus strains currently being used in biocontrol of aflatoxin contamination of crops.     

Selection of Aspergillus flavus isolates for biological control of aflatoxins in corn

The fungus Aspergillus flavus is responsible for producing carcinogenic mycotoxins, the aflatoxins, on corn (maize) and other crops. An additional harmful toxin, cyclopiazonic acid, is produced by some isolates of A. flavus. Several A. flavus strains that do not produce one or both of these mycotoxins are being used in biological control to competitively exclude the toxin-producing strains from the agroecosystem, particularly from seeds, grain and other marketable commodities. Three well-studied non-aflatoxigenic strains, including two that are commercially available, have been compared in side-by-side field trials. The results of that study, together with a growing understanding of A. flavus ecology and new genetic insights, are guiding the selection of biocontrol strains and influencing crop management decisions for safe and sustainable production.     

Aflatoxins in Maize: A Mexican Perspective

Aflatoxins are carcinogenic metabolites produced by Aspergillus flavus, a fungal pathogen that infects maize both in the field and during storage. Mexico is the center of origin of maize and its production in most parts in the country is characterized by the employment of a wide diversity of open‐pollinated genotypes adapted to certain environments. In most regions, maize is produced under rain fed conditions with low fertilizer and pesticide input and consequent low yields, probably fostering A. flavus infection in drought‐stressed plants. In addition, poor pest control increases insect damage, facilitating fungal infection and aflatoxin contamination. Ideally, management of aflatoxin contamination should begin with the employment of resistant genotypes as has been demonstrated by several U.S. breeding programs. However, in Mexico the wide genetic diversity of maize has not been fully exploited to identify resistance to aflatoxin contamination in breeding programs, thus impeding the reduction of aflatoxin levels in the field. Additional complications come from the fact that transgenic maize expressing insecticidal protein or any other trait to reduce aflatoxin is not viable in Mexico due to a government prohibition on the use of genetically modified maize. Maize is a staple crop in Mexico with high consumption in forms such as tortillas; thus, aflatoxin contamination is a significant threat to human health. Although aflatoxins are partially destroyed during the alkaline cooking procedure (called nixtamalization) to prepare tortillas, residual levels of aflatoxins might be considerable. Although important research has been conducted in several aspects of aflatoxin contamination of maize by universities, agricultural centers, and some government agencies, a full mycotoxin research program is needed in Mexico to ascertain the extents of aflatoxin contamination in different parts of the country and to develop economically viable technology to reduce aflatoxin exposure.     

Enhancing Maize Germplasm with Resistance to Aflatoxin Contamination

Preharvest kernel infection by Aspergillus flavus and the subsequent accumulation of aflatoxin in maize grain are chronic problems in the southeastern United States. Aflatoxin is a natural carcinogen, and its presence markedly reduces the value of grain. Losses to aflatoxin contamination reach devastating levels some years. Development and deployment of maize hybrids with resistance to aflatoxin contamination is generally considered the most feasible method of reducing or eliminating the problem. Research to address the aflatoxin problem was initiated by USDA–ARS at Mississippi State, MS, in the late 1970s. The goals of the research were to identify and develop aflatoxin‐resistant maize germplasm. First, reliable techniques for screening germplasm were developed. Then, germplasm from numerous sources was screened. The release of Mp313E in 1988 was the first release of maize germplasm with resistance to aflatoxin contamination. Two other germplasm lines, Mp420 and Mp715, were released in 1991 and 1999, respectively. Additional germplasm lines have been developed, but not yet released. Efforts are currently underway to identify other sources of resistance.When used in crosses with other lines, the aflatoxin‐resistant lines markedly reduce the level of aflatoxin contamination in the resulting hybrids. Analysis of a diallel cross indicated that general combining ability was a significant source of variation in the inheritance of resistance to aflatoxin contamination. Efforts to combine resistance to aflatoxin combination and agronomic qualities using both conventional breeding methods and molecular marker assisted selection have been initiated.     

Controlling Aflatoxin and Fumonisin in Maize by Crop Management

Maize is a vital food and feed grain worldwide. Aflatoxin and fumonisin, mycotoxins produced primarily by the fungi Aspergillus flavus and Aspergillus parasiticus Speare, and Fusarium moniliforme J. Sheld, respectively, are very potent carcinogens in both humans and livestock and can readily contaminate maize grain in the field and in storage. Stress on developing maize, particularly during reproductive growth, facilitates infection by the fungi, production of mycotoxins and contamination of the grain. Drought, excessive heat, inadequate plant nutrition, insect feeding on developing kernels, weeds, excessive plant populations, and other plant diseases can produce plant stress and facilitate the infection of maize grain by mycotoxin producing fungi. Timely planting of adapted hybrids, proper plant nutrition, irrigation, and insect control either by insecticides or the use of transgenic hybrids all assist in curbing mycotoxin contamination. Production practices that produce high yields are basically the same ones that help control mycotoxins. Care must also be exercised in harvesting and handling grain in transport and storage to reduce kernel breakage and prevent contamination. Harvesting early and artificial drying helps reduce the incidence of mycotoxins as well as preventing kernel breakage and stored‐grain insect infestations.     

Inoculation Techniques Used to Quantify Aflatoxin Resistance in Corn

The development of Aspergillus flavus inoculation techniques has played an important part in developing corn (Zea mays L.) germplasm resistant to aflatoxin contamination. Corn genotypes evaluated for aflatoxin resistance in field studies must be artificially inoculated due to the sporadic nature of aflatoxin contamination from year to year. A number of different inoculation techniques are used by researchers in the South and Midwest. Field inoculation techniques either wound developing kernels or leave the kernels intact. Non‐wounding techniques apply A. flavus conidia to exposed silks or silks inside the husks without damaging kernels. Wounding techniques deliver A. flavus conidia onto kernels that have been mechanically damaged. Inoculation techniques utilizing ear feeding insects to vector conidia have also been used in field studies. Environmental conditions such as ambient temperature and drought stress appear to have a significant impact on artificial inoculations. Laboratory evaluation techniques have been developed to confirm aflatoxin resistance identified in corn genotypes in the field. Color mutants and transformants of Aspergillus spp. have been used in field and laboratory studies to identify resistant genotypes. More efficient, less labor intensive, and less costly inoculation techniques need to be developed to aid in the production of aflatoxin resistant corn hybrids.     

Toxigenic Species Aspergillus parasiticus Originating from Maize Kernels Grown in Serbia

In Serbia, aspergillus ear rot caused by the disease pathogen Aspergillus parasiticus (A. parasiticus) was first detected in 2012 under both field and storage conditions. Global climate shifts, primarily warming, favour the contamination of maize with aflatoxins in temperate climates, including Serbia. A five-year study (2012–2016) comprising of 46 A. parasiticus strains isolated from maize kernels was performed to observe the morphological, molecular, pathogenic, and toxigenic traits of this pathogen. The HPLC method was applied to evaluate mycotoxin concentrations in this causal agent. The A. parasiticus isolates synthesised mainly aflatoxin AFB1 (84.78%). The percentage of isolates synthesising aflatoxin AFG1 (15.22%) was considerably lower. Furthermore, the concentration of AFG1 was higher than that of AFB1 in eight isolates. The polyphase approach, used to characterise isolates, showed that they were A. parasiticus species. This identification was verified by the multiplex RLFP-PCR detection method with the use of restriction enzymes. These results form an excellent baseline for further studies with the aim of application in the production, processing, and storage of cereal grains and seeds, and in technological processes to ensure the safe production of food and feed.     

Development of PCR,LAMP and qPCR Assays for the Detection of Aflatoxigenic Strains of Aspergillus flavus and A. parasiticus in Hazelnut

Aspergillus flavus and A. parasiticus are two species able to produce aflatoxins in foodstuffs, and in particular in hazelnuts, at harvest and during postharvest phase. As not all the strains of these species are aflatoxin producers, it is necessary to develop techniques that can detect aflatoxigenic from not aflatoxigenic strains. Two assays, a LAMP (loop-mediated isothermal amplification) and a real time PCR with TaqMan^® probe were designed and validated in terms of specificity, sensitivity, reproducibility, and repeatability. The capability of the strains to produce aflatoxins was measured in vitro and both assays showed to be specific for the aflatoxigenic strains of A. flavus and A. parasiticus. The limit of detection of the LAMP assay was 100–999 picograms of DNA, while the qPCR detected 160 femtograms of DNA in hazelnuts. Both techniques were validated using artificially inoculated hazelnuts and naturally infected hazelnuts. The qPCR was able to detect as few as eight cells of aflatoxigenic Aspergillus in naturally infected hazelnut. The combination of the LAMP assay, which can be performed in less than an hour, as screening method, with the high sensitivity of the qPCR, as confirmation assay, is able to detect aflatoxigenic strains already in field, helping to preserve the food safety of hazelnuts.     

Comparison of Expression of Secondary Metabolite Biosynthesis Cluster Genes in Aspergillus flavus,A. parasiticus,and A. oryzae

Fifty six secondary metabolite biosynthesis gene clusters are predicted to be in the Aspergillus flavus genome. In spite of this, the biosyntheses of only seven metabolites, including the aflatoxins, kojic acid, cyclopiazonic acid and aflatrem, have been assigned to a particular gene cluster. We used RNA-seq to compare expression of secondary metabolite genes in gene clusters for the closely related fungi A. parasiticus, A. oryzae, and A. flavus S and L sclerotial morphotypes. The data help to refine the identification of probable functional gene clusters within these species. Our results suggest that A. flavus, a prevalent contaminant of maize, cottonseed, peanuts and tree nuts, is capable of producing metabolites which, besides aflatoxin, could be an underappreciated contributor to its toxicity.     

Developing resistance to aflatoxin in maize and cottonseed

At this time, no "magic bullet" for solving the aflatoxin contamination problem in maize and cottonseed has been identified, so several strategies must be utilized simultaneously to ensure a healthy crop, free of aflatoxins. The most widely explored strategy for the control of aflatoxin contamination is the development of preharvest host resistance. This is because A. flavus infects and produces aflatoxins in susceptible crops prior to harvest. In maize production, the host resistance strategy has gained prominence because of advances in the identification of natural resistance traits. However, native resistance in maize to aflatoxin contamination is polygenic and complex and, therefore, markers need to be identified to facilitate the transfer of resistance traits into agronomically viable genetic backgrounds while limiting the transfer of undesirable traits. Unlike maize, there are no known cotton varieties that demonstrate enhanced resistance to A. flavus infection and aflatoxin contamination. For this reason, transgenic approaches are being undertaken in cotton that utilize genes encoding antifungal/anti-aflatoxin factors from maize and other sources to counter fungal infection and toxin production. This review will present information on preharvest control strategies that utilize both breeding and native resistance identification approaches in maize as well as transgenic approaches in cotton.     

Control of Aflatoxin Production of Aspergillus flavus and Aspergillus parasiticus Using RNA Silencing Technology by Targeting aflD (nor-1) Gene

Aspergillus flavus and Aspergillus parasiticus are important pathogens of cotton, corn, peanuts and other oil-seed crops, producing toxins both in the field and during storage. We have designed three siRNA sequences (Nor-Ia, Nor-Ib, Nor-Ic) to target the mRNA sequence of the aflD gene to examine the potential for using RNA silencing technology to control aflatoxin production. Thus, the effect of siRNAs targeting of two key genes in the aflatoxin biosynthetic pathway, aflD (structural) and aflR (regulatory gene) and on aflatoxin B₁ (AFB₁), and aflatoxin G₁ (AFG₁) production was examined. The study showed that Nor-Ib gave a significant decrease in aflD mRNA, aflR mRNA abundance, and AFB₁ production (98, 97 and 97% when compared to the controls) in A. flavus NRRL3357, respectively. Reduction in aflD and aflR mRNA abundance and AFB₁ production increased with concentration of siRNA tested. There was a significant inhibition in aflD and AFB₁ production by A. flavus EGP9 and AFG₁ production by A. parasiticus NRRL 13005. However, there was no significant decrease in AFG₁ production by A. parasiticus SSWT 2999. Changes in AFB₁ production in relation to mRNA levels of aflD showed a good correlation (R = 0.88; P = 0.00001); changes in aflR mRNA level in relation to mRNA level of aflD also showed good correlation (R = 0.82; P = 0.0001). The correlations between changes in aflR and aflD gene expression suggests a strong relationship between these structural and regulatory genes, and that aflD could be used as a target gene to develop efficient means for aflatoxin control using RNA silencing technology.     

Aflatoxins and growth impairment: A review

Aflatoxins, fungal toxins produced by Aspergillus flavus and Aspergillus parasiticus in a variety of food crops, are well known as potent human hepatocarcinogens. Relatively less highlighted in the literature is the association between aflatoxin and growth impairment in children. Foodborne aflatoxin exposure, especially through maize and groundnuts, is common in much of Africa and Asia—areas where childhood stunting and underweight are also common, due to a variety of possibly interacting factors such as enteric diseases, socioeconomic status, and suboptimal nutrition. The effects of aflatoxin on growth impairment in animals and human children are reviewed, including studies that assess aflatoxin exposure in utero and through breastfeeding. Childhood weaning diets in various regions of the world are briefly discussed. This review suggests that aflatoxin exposure and its association with growth impairment in children could contribute a significant public health burden in less developed countries.