A Focus on Aflatoxin Contamination -- Updated Version

On this page... Classification Natural Occurrence Mechanism of Infection Aflatoxicosis Foodborne Disease Outbreaks Regulatory Status Research at the USDA Agricultural Research Service References This technical fact sheet illustrates the following key points about aflatoxins: A potent natural toxin identified as a human carcinogen. Causes hepatocellular carcinoma (liver cancer) which is prevalent in developing countries. Belongs to a group of mycotoxins that are produced as secondary fungal metabolites and excreted into substrates such as plants and processed foods. Causes two types of aflatoxicosis, acute and chronic, based on dietary levels consumed. Production is enhanced by tropical weather, drought stress, insect activity, and poor harvest and storage practices. U.S. Food and Drug Administration has established action levels that specify maximum acceptable levels of aflatoxins in food or feed. Research is focused on genetic characterization, development of biocontrol technologies, and effective agricultural management for aflatoxin prevention and control. Aflatoxins are one of the most potent natural toxins and are recognized as hepatotoxic, carcinogenic, immunosuppressive, and antinutritional contaminants of many staple food commodities.9, 58 The International Agency for Research on Cancer (IARC) has classified aflatoxins as carcinogenic to humans (Group 1) based on sufficient scientific evidence.33, 44 They are predominantly implicated as a risk factor for the primary liver cancer known as hepatocellular carcinoma (HCC), a common cause of cancer morbidity and mortality in Asia, Africa, and in groups of Asian- and Hispanic-Americans.57, 53 Aflatoxins belong to a group of mycotoxins that are produced as secondary fungal metabolites and excreted into substrates including plants and processed foods intended for human consumption.14 Aflatoxins represent a worldwide threat to public health due to their frequent occurrence in food and feed.2 Aflatoxin contamination of staple diets is common in many developing countries and outbreaks have been reported in several parts of Africa and Asia including Kenya, India, and Malaysia.58, 8 The greatest recorded fatal aflatoxin outbreak occurred in Kenya in 2004 which resulted in 317 cases and 125 deaths. The implicated source was the aflatoxin contaminated homegrown maize which was stored under damp conditions.52, 36, 11 The largest reported aflatoxin outbreak occurred in western India in 1974 from the consumption of contaminated maize which resulted in 397 cases and 106 deaths.11, 40 Aflatoxin contamination in foods and agricultural commodities also has significant economic implications. The Food and Agriculture Organization (FAO) estimates that 25 percent of the world’s food crops are affected by mycotoxins, particularly aflatoxins.16 Losses incurred from rejected shipments due to aflatoxin contamination can substantially affect export markets. For instance, the Almond Board of California estimates that each rejected almond consignment can cost as much as 10 thousand dollars for demurrage, warehousing, replacement shipments, and other expenses.8, 3 This economic impact, as well as its frequent occurrence in food and feed and its deleterious effect on human health, have driven producers, manufacturers, regulatory agencies, and researchers to find measures for control and rapid detection in foods.13 Classification Aflatoxins are a group of structurally related toxic compounds produced by certain species of the fungi Aspergillus, particularly Aspergillus flavus and Aspergillus parasiticus.26 Approximately 20 aflatoxins have been isolated from various fungal species; however, only four are commonly found and have been extensively studied with respect to their biological properties and health concerns.42 These four aflatoxins are: B1 B2 G1 G2 A. parasiticus produces all four aflatoxins, while A. flavus produces only B1 and B2. The B and G designations of aflatoxins refer to the exhibition of blue and yellow-green fluorescent colors produced by these compounds on thin layer chromatography plates, respectively. The subscript numbers 1 and 2 indicate relative chromatographic mobility of aflatoxins during thin layer chromatography.31, 55 Aflatoxins display variations in the magnitude of toxicity and carcinogenicity. Aflatoxin B1 is the most toxic followed by G1, B2, and G2 as illustrated by their LD50 values for day-old ducklings.31, 41 When aflatoxins B1 and B2 are ingested by lactating cows or other ruminants mainly through the consumption of contaminated feed, a proportion of these aflatoxins undergo hydroxylation and are excreted in the milk as aflatoxin metabolites known as aflatoxins M1 and M2. Although these compounds exhibit lower toxicity than the parent molecules, they are significant because of the extensive consumption of cow’s milk by infants as well as subsequent contamination of dairy products such as cheese and yogurt through the aflatoxin contaminated milk.39, 55 Back to top Natural Occurrence Aflatoxins occur as natural contaminants in human and animal foods as a result of fungal contamination of food crops during pre and postharvest.60 They are found in a wide range of foods which include:41, 32, 60, 16 Cereals (maize, corn, sorghum, pearl millet, rice, wheat) Oilseeds (peanut, soybean, sunflower, cotton) Spices (chillies, black pepper, coriander, turmeric, ginger) Tree nuts (almonds, pistachio, walnuts, coconut) Dried fruits (sultanas, figs) Cocoa beans Milk, eggs, and meat products* * Milk, eggs, and meat products are occasionally contaminated due to the consumption of aflatoxin-contaminated feed by animals. The agricultural commodities which have the highest risk of aflatoxin contamination are:16 Corn Peanuts (groundnuts) Cottonseed Agricultural commodities grown in tropical and subtropical countries are more prone to aflatoxin contamination. The exportation of such commodities to other countries spreads the contamination and makes aflatoxin a worldwide problem.55 Some commodities which spread the contamination through international trading include:27 Peanuts Edible nuts and nut products Dried figs Spices Maize Factors Favoring Aflatoxin Development Aflatoxin development in grains depends on the growth and infestation of the fungi Aspergillus.30 Fungal invasion and contamination usually begin before harvest and can be enhanced by poor production and harvest conditions. The relative proportions and amounts of the various aflatoxins on food crops depend on several factors.58, 44 These factors include: Genotypes -- Use of crop genotypes that are susceptible to Aspergillus infection and insect or microbial infestation increases the risk of aflatoxin contamination. Seed variety should be considered and farmers should consult with the appropriate plant breeding authorities or agricultural extension services to find the best suited cultivar.15 Drought stress -- Preharvest aflatoxin contamination is a problem in semi-arid tropics where drought stress amplifies the Aspergillus infection and results in groundnut contamination. This is possibly due to loss of the phytoalexin producing capacity of the seed and altered seed composition under drought stress.34 Drought stress has also been found to increase the number of Aspergillus spores in the air which significantly increases the possibility of infection, especially during pollination.30 Soil types -- Crops grown in different soil types may have significantly different levels of aflatoxin contamination. For example, peanuts grown in light sandy soils support rapid growth of the fungi, particularly under dry conditions. Heavier soils result in less contamination of peanuts due to their high water holding capacity which helps the plant to prevent drought stress.15 Insect activity -- Insect infestations can increase the occurrence of Aspergillus and levels of aflatoxins, especially during pollination in drought-stressed corn. Insect damage to the corn ear allows easy access of Aspergillus which subsequently infects the kernels. Insects also transport Aspergillus spores to the silks and kernels thereby facilitating aflatoxin contamination.30 Growing and storage conditions -- Tropical conditions favor the development of aflatoxins, and contamination is usually higher in crops grown in tropical climates than in temperate climates. Tropical conditions include:8 High temperature High Humidity Monsoons Unseasonal rains during harvest Flash floods If high moisture and temperature conditions are sustained after harvest, aflatoxins can be produced during storage, especially at a moisture content above 12 percent and temperatures greater than 70oF.37 For example, in peanuts and corn, preharvest aflatoxin contamination is favored by high temperatures, prolonged drought conditions, and high insect activity, while postharvest aflatoxin contamination is facilitated by warm temperatures and high humidity.16 Poor harvesting practices, improper storage, and improper conditions during transport and marketing can also lead to aflatoxin development and contamination.8 Other factors -- Other crop and field factors which favor aflatoxin production include:16 Specific crop growth stages Poor fertility High crop density Weed competition Back to top Mechanism of Infection Aflatoxins are associated with both toxicity and carcinogenicity in human populations.7 A number of adverse human health effects have been associated with aflatoxins, including hepatotoxicity, liver cancer, kwashiorkor, and Reye’s syndrome. However, only a link between aflatoxin exposure and both hepatotoxicity and liver cancer are well established.20 Aflatoxin B1 (AFB1) is the most potent natural carcinogen known and is generally the main aflatoxin produced by toxigenic strains.7 AFB1 infection most commonly begins with the ingestion of contaminated food and proceeds through the following four steps: Metabolic Activation -- The cytochrome P450 (CYP450) isoenzyme system in the liver metabolizes AFB1 primarily though the process of hydroxylation. The human CYP450 isoforms, CYP3A4 and CYP1A2, catalyze the epoxidation of AFB1 to yield active forms known as AFB1-8,9-exo-epoxide and AFB1-8,9-endo-epoxide. It is the AFB1-8,9-exo-epoxide which exerts hepatocarcinogenic effects of AFB1.6, 57, 20 AFB1 metabolism also results in the formation of aflatoxins Q1, M1, and P1. These metabolites as well as other naturally occurring aflatoxins (G1, B2 and G2) are poor substrates for epoxidation and, consequently, are less mutagenic, toxigenic, and carcinogenic than AFB1.57 DNA Modification -- AFB1-8,9-exo-epoxide is highly unstable molecule which has high affinity for guanine (G) bases in the deoxyribonucleic acid (DNA).6 It binds to the N7 position of guanine through a covalent bond and forms AFB1-N7-guanine adducts in the target cells.31 These adducts induce DNA mutations primarily through a G to T (thymine) transversion in the DNA targeted to the site of the original adduct.57 Cell Deregulation -- The G to T transversion (AGG to AGT) at the third base of codon 249 of the protein 53 (p53) tumor suppressor genes in the human liver induces changes in the coding properties of the DNA . The p53 gene is a transcriptional activator which functions as a controller of the cell cycle, and has a role in the apoptosis pathway and DNA repair.43 The mutations in p53 may cause permanent transcriptional changes as well as interference with RNA synthesis.59, 31, 6 This results in DNA damage, development of DNA lesions, and consequently cell deregulation.31, 43 Development of Hepatocellular Carcinoma -- Loss of function of p53 give cells a selective growth advantage, since p53 is a tumor suppressor gene. Therefore, the deregulation of p53 leads to tumor formation in the liver known as hepatocelluar carcinoma (HCC). Aflatoxin- induced p53 mutation in liver has been found in up to 50 percent of HCC samples, while AFB1-DNA adducts at other sites within the p53 gene, which induce G to T transversions, were found to be very rare in HCC samples from areas of high aflatoxin exposure.57 Aflatoxin B1, Hepatitis B Virus, and Liver Cancer in Humans Aflatoxins and hepatitis B virus (HBV) are considered as co-carcinogens, and many studies have demonstrated a strong interaction between HBV and aflatoxin exposure for the risk of HCC.39, 53 It has been found that in HBV-infected subjects, aflatoxin is approximately 30 times more potent than in persons without the virus. The relative risk of liver cancer in HBV infection increases about 12 times when HBV infection and aflatoxin exposure are combined. The probability of people developing liver cancer is much higher in areas where both aflatoxins and HBV are prevalent, including Gambia, Taiwan, Qidong.58, 54 Hepatomas are the predominant cause of death in these areas; approximately 10 percent of male deaths in Gambia and 10 percent of adult deaths in Qiding, China were reported to be due to this cancer.58 The mutation in p53 tumor suppressor gene at the codon 249 was frequently reported in patients from regions with high dietary levels of aflatoxins and a high prevalence of HBV.19 The synergistic effects of HBV and aflatoxin in causing liver cancer is complex, but some studies have suggested that HBV infection alters the expression of aflatoxin metabolizing enzymes consequently changes the degree of aflatoxins to bind to DNA.57 Aflatoxin and Lung Cancer Aflatoxin is primarily recognized as an agent causing liver cancer; however, in some cases it has also been associated with lung cancer, particularly among workers who handle contaminated grains.58 Aflatoxin exposure in agricultural workers and farmers mainly occurs by inhalation of dust generated during the handling and processing of contaminated crops and feeds. One study reported that 7 out of 45 animal feed production plant workers in Denmark had detectable levels of AFB1 in their blood after working for four weeks in the factory or unloading raw materials from ships. In another study, aflatoxins were detected in respirable dust samples from the work place and storage areas at rice and corn processing plants in India.44 These studies indicate that aflatoxin is also a risk factor for lung cancer and appropriate measures should be taken to prevent its occupational exposure. Back to top Aflatoxicosis Aflatoxicosis is a disease that results from ingestion of aflatoxins in contaminated food or feed. The primary target organ is liver and liver damage occurs in many species, including, birds, fish, rodents, nonhuman primates, and humans.26, 58 The susceptibility to aflatoxins varies significantly between different species and members of the same species, and is dependent on the following factors:58, 7, 26 Age Sex Weight Nutritional status of diet Environmental factors Aflatoxin exposure (dose and duration) Presence of other mycotoxins and pharmacologically active substances The risk of cancers due to aflatoxin exposure is based on cumulative lifetime dose. Since all animal species are susceptible to severe toxic effects of aflatoxins, it is often assumed that human health effects are similar to that of animals. The aflatoxicosis in animals (and possibly in humans) has been categorized in two general forms:26 Acute -- This is caused from the consumption of moderate to high levels of aflatoxins. Symptoms may include: Hemorrhage Acute liver damage Edema Alteration in digestion, absorption, and nutrient metabolism Death Chronic -- This is caused from the consumption of low to moderate levels of aflatoxins. It usually results in subclinical effects which are difficult to recognize. Symptoms include: Impaired food conversion Slower rates of growth with or without production of clear symptoms. Effects of Aflatoxicosis on Human Health Humans are exposed to aflatoxins mainly by eating contaminated food. The dose and duration of aflatoxins primarily determines the toxic effects of aflatoxin, usually large doses or long-term exposure to low levels in the diet have significant adverse effects. Symptoms of acute aflatoxicosis in humans include:58, 16 Vomiting Abdominal pain Pulmonary edema Convulsions Coma Death with cerebral edema and fatty involvement of the liver, kidneys, and heart Aflatoxins and Children Many children in developing countries, including Benin, Gambia, and Togo are malnourished as well as chronically exposed to high levels of aflatoxins from the consumption of contaminated staple foods such as maize and groundnuts. Children exposed to high levels of aflatoxins throughout childhood may become stunted (an indicator of chronic malnutrition) and underweight (an indicator of acute malnutrition). They also become more susceptible to infectious diseases such as malaria, diarrhea, and respiratory infections in childhood and later in life.8, 29 In addition, it has been assumed that kwashiorkor, a severe malnutrition disease, may be a form of pediatric aflatoxicosis.7 Nursing infants may be exposed to aflatoxins through breast milk. M1 is the most common aflatoxin detected in breast milk, and in one study its concentration was found to be between 0.02 to 1.12 microgram/liter in breast milk.44 Effects of Aflatoxicosis on Animal Health The health impacts of aflatoxin exposure in animals depend on dosage and response. Low doses produce nutritional interferences and immunological suppression, while high doses lead to acute illness and death. These effects are summarized below:58, 37, 16 Nutritional Effects Covalent binding to DNA Decreased protein synthesis Altered vitamin and mineral metabolism Reduced milk and egg production Reduced feed utilization and efficiency Poor growth rates Reduced weight gain Anemia Jaundice Immunological Suppression Induced thymic aplasia Reduced T-lymphocyte function and number Suppressed phagocytic activity Reduced complement activity Impaired macrophage function Recurrent infections Liver damage and bleeding Decreased response to vaccinations Embryo toxicity* * Aflatoxins are considered to be teratogenic. In some animal species, including pigs and rats, the immunosuppressive functions of aflatoxins were reported to be transferred across the placenta harming the fetus. Acute Effects Loss of appetite Depression Hemorrhage Diarrhea Death Treatment of Aflatoxicosis Aflatoxicosis is rarely reported in humans and treatment with antibiotics or other drugs has little effect.26 Treatment options are mainly focused on animals and include the use of following substances in the feed:16, 37, 10 Chemisorbents such as hydrated calcium aluminosilicate (HSCAS) and mineral clays such as zeolite and bentonite which bind to aflatoxins in feed and prevent their absorption and bioavailability. The levels of usage and effects of these products are: Usually added to feed at the rate of 5 to 10 pounds per ton Reduce effects of aflatoxins on the liver Reduce aflatoxin residues in milk Organic acids such as propionic, benzoic, and sorbic acids as well as their salts calcium propionate, potassium sorbate, and copper sulfate inhibit mold growth in feed. Modified glucan based adsorbents, such as MycosorbTM and Alltech which may bind aflatoxins in feeds. Increased levels of high quality protein supplements and vitamins A, D, E, and K in the ration as aflatoxin affects protein synthesis and binds to vitamins. In addition, good management practices should be implemented to reduce stress and to decrease the risk of secondary infections. Prevention and Control of Aflatoxin Contamination The conventional approach to prevent aflatoxin exposure at the individual level is to avoid consumption of high risk foods such as maize. However, this approach has not been reported successful in developing countries.58, 8 Therefore, prevention strategies are mainly focused on good agricultural practices at the community level. Aflatoxin formation in crops can be reduced by several pre- and postharvest good agricultural practices. Some of these practices include:8, 30 Preharvest Practices Crop rotation Irrigation to prevent drought stress Control of weeds and insects Cultivation of mold-resistant stocks Use of biocontrols such as non-aflatoxigenic fungal strains Postharvest Practices Rapid drying after harvesting Avoid damaged kernels by sorting Appropriate moisture content during storage Detoxification and decontamination using chemical methods For additional information on the control and prevention of aflatoxins, visit the FAO -- Mycotoxin Prevention and Control in Food Grains. Aflatoxin Contamination in Foods Aflatoxins are the most frequent cause of contamination of food crops worldwide, particularly in developing countries. They contaminate a wide range of commodities, many of which include staple diets of several tropical countries.44 Foods reported to be contaminated worldwide with aflatoxins include:58 Corn, maize, sorghum, wheat Peanuts and peanut products Tree nuts Watermelon seeds Soybean Spices Milk, yogurt, and cheese In the United States (U.S.), aflatoxins have been identified in several food commodities. These include:26 Corn and corn products Peanuts and peanut products Cottonseed Milk Tree nuts such as Brazil nuts, pecans, pistachio nuts and walnuts Cereals and nuts may be contaminated with aflatoxins during production, harvest, storage, or food processing. Hot and humid climates which favor the growth of the fungus Aspergillus promote aflatoxin production.29 Once produced, aflatoxins are stable to heat, cold, and light, therefore they can not be destroyed by most food processing operations. However, they are unstable in processes which employ alkalis and oxidizing agents. Aflatoxins are also found occasionally in milk and milk products. Contaminated corn and cottonseed meal fed to dairy cows results in aflatoxin M1 contamination of milk and milk products, including non-fat dry milk, cheese, and yogurt.30, 16 Back to top Foodborne Disease Outbreaks Aflatoxin outbreaks are uncommon in developed countries where foods are effectively and strictly regulated. Regulations monitor aflatoxin levels in foods and protect human populations from acute aflatoxin exposure.26, 7 In developing countries aflatoxin exposure is more common because regulations are either non-existent or not stringent and/or a food shortage exists. Foods consumed in these countries are usually produced, stored, prepared, and consume by the local families without any consideration for the risks of aflatoxin exposure. In addition, the least contaminated food commodities are exported, and the highly contaminated products are retained enhancing the risk of aflatoxin exposure.7, 58 In the U.S., the relative frequency of aflatoxicosis in humans is unknown and no outbreaks have been reported in humans. In developing countries, very little information is available on aflatoxicosis outbreaks because many cases go unnoticed due to inadequate medical services.26 The aflatoxicosis outbreaks that have been reported in developing countries are summarized below. Selected Foodborne Outbreaks in Developing Countries The following list of outbreaks indicates that aflatoxicosis often occurs in tropical countries where people consume high risk foods, such as maize and rice, which are part of their staple diets. 2004 Kenya Outbreak36 Contaminated homegrown maize Resulted in 317 cases and 125 deaths Aflatoxin levels in 55 percent contaminated maize samples were greater than 20 parts per billion (ppb), of which 35 percent had levels greater than 100 ppb , and 7 percent had levels greater than 1,000 ppb 1988 Malaysia Outbreak12, 22 Contaminated noodles Resulted in 40 cases and 13 child deaths 1981 Kenya Outbreak38, 5 Contaminated maize Resulted in 20 cases including 12 deaths Aflatoxin levels detected in two liver samples were 39 and 89 ppb 1974 India Outbreak40, 11 Contaminated maize Resulted in 397 cases and 106 deaths Aflatoxin levels in contaminated maize were 6.25 to 15.6 parts per million 1967 Taiwan Outbreak5 Contaminated rice Resulted in 26 cases including three child deaths Aflatoxin levels in two contaminated rice samples were ~ 200 ppb For additional foodborne outbreak information, visit the Bulletin of the World Health Organization – Toxic Effects of Mycotoxins in Humans. Back to top Regulatory Status The complete elimination of aflatoxins is not possible since it is a common contaminant in many agricultural products. The U.S. Food and Drug Administration (FDA) considers aflatoxins to be an unavoidable food contaminant but recommends the amount in foods should be reduced to the lowest possible levels.58, 22 In order to minimize aflatoxin contamination in food and feed, the FDA established aflatoxin action levels which specify maximum acceptable levels at or above which FDA will take legal action to eliminate products from the market.25 However, strict limitation of aflatoxin-contaminated food cannot be realistically regulated in developing countries, where food supplies are already limited and drastic food measures may lead to food shortage and excessive prices.7 Aflatoxin action levels in food and feed are summarized below:25 FDA Action Levels for Aflatoxins in Food 20 ppb -- Foods, including grain, rice, and processed products 0.5 ppb -- Aflatoxin M1 in milk 20 ppb -- Peanuts, peanut products, Brazil nuts, and pistachio nuts FDA Action Levels for Aflatoxins in Animal Feed 20 ppb -- Corn and peanut products, other animal feeds and feed ingredients but excluding cottonseed meal, intended for immature animals 20 ppb -- Corn and other animal feeds and feed ingredients, including cottonseed meal, intended for dairy animals, or when the intended use is unknown 100 ppb -- Corn and peanut products intended for breeding beef cattle, breeding swine, or mature poultry 200 ppb -- Corn and peanut products intended for finishing swine of 100 pounds or greater 300 ppb -- Corn and peanut products intended for finishing (i.e., feedlot) beef cattle, and cottonseed meal intended for beef, cattle, swine, or poultry (regardless of age or breeding status) Worldwide Regulations for Aflatoxins in Food and Feed On a worldwide basis, approximately 100 countries have aflatoxin regulations for food and feed and most include maximum permitted or recommended levels for either AFB1 or the sum of aflatoxins B1, B2, G1, and G2 in specific commodities.35, 21 The worldwide regulatory limits for AFB1 and total aflatoxins in food, and aflatoxin M1 in dairy products are summarized below:24 Aflatoxin B1 in Food 2 microgram/kilogram (µg/kg) -- Followed by 29 countries, mostly belonging to European Union (EU), the European Free Trade Association (EFTA), and candidate EU countries 5 µg/kg -- Followed by 21 countries, spread over Africa, Asia/Oceania, Latin America and Europe U.S. and Canada do not have a single limit for AFB1 Total Aflatoxins (B1, B2, G1, and G2) in Foods 4 µg/kg -- Followed by 29 countries, mostly belonging to EU, EFTA, and candidate EU countries 20 µg/kg -- Followed by 17 countries, including U.S. , several African countries, and half of Latin American countries Aflatoxin M1 in Dairy Products Regulated by 60 countries 0.05 µg/kg -- Followed by 34 countries, mostly belonging to EU, EFTA, and candidate EU countries, and some countries belonging to Africa, Asia, and Latin America 0.5 µg/kg -- Followed by 22 countries, including U.S. , several Asian and European countries, and most Latin American countries For additional information on aflatoxin regulatory limits in food and feed, please visit FAO -- Worldwide Regulations for Mycotoxins in Food and Feed 2003. Back to top Research at the USDA Agricultural Research Service The USDA Agricultural Research Service (ARS) is actively involved in food safety research related to aflatoxins under the National Food Safety Program 108. This research program provides the means to ensure that the food supply is safe and secure for consumers and that food and feed meet foreign and domestic regulatory requirements. The following ARS research units conduct research on aflatoxins: Food and Feed Safety Research Unit U.S. Arid Land Agricultural Research Center Plant Mycotoxin Research Unit Crop Genetics and Production Research Unit Crop Protection and Management Research Unit National Peanut Research Laboratory Some of the aflatoxin research projects being conducted at these ARS units are: Aflatoxin Control in Pistachios, Almonds, and Figs: Biocontrol Using Atoxigenic Strains Location: Food and Feed Safety Research Unit Project Objectives45 Identify spatial patterns associated with aflatoxin contamination in pistachio orchards using processor library samples. Determine the incidence of AF36 among A. flavus isolates obtained from commercial pistachio orchards in 2006. Determine the incidence of atoxigenic strains among A. flavus isolates naturally occurring in almond orchards at various locations. Initiate studies on biocontrol of aflatoxin-producing fungi in a drip-irrigated almond orchard using the AF36 strain of A. flavus. Determine establishment/survival of AF36 in an almond orchard and displacement of toxigenic A. flavus and/or A. parasiticus. Follow the survival and spread of atoxigenic A. flavus strains previously applied in a research fig orchard and prepare for application of AF36 in commercial fig orchards. Accomplishments45 Collected soil samples from 28 almond orchards from three regions: southern San Joaquin Valley, northern San Joaquin Valley, and the Sacramento Valley. Determined that A. flavus was more common in orchards in the southern region and A. parasiticus in the northern region, while the incidence of both was about the same in orchards of the central region. Initiated a biocontrol study in almonds in a research almond orchard. Estimated percentages of AF36 among A. flavus isolates in pistachios grown in soil in areas treated for biocontrol for years 2005, 2006, and 2007. Indicated that treatments did not differ significantly in density of A. flavus/A. parasiticus on surfaces of the hulls of freshly harvested nuts. Aflatoxin Control Through Targeting Mechanisms Governing Aflatoxin Biosynthesis in Corn and Cottonseed Location: Food and Feed Safety Research Unit Project Objectives46 Understand the molecular basis for fungal responses to conditions encountered during invasion of crops in order to identify and modify the factors in corn and other crops that could induce or impede aflatoxin formation or fungal development. Determine, by genetic and physiological studies of diverse aflatoxin producing species, whether aflatoxin production provides an adaptive advantage for fungal survival and invasion of crops. Determine the molecular responses of aflatoxin producing fungi to stress factors, particularly with regard to developing an understanding of the ability of the fungi to adapt and produce toxins. Undertake and utilize newly available sequences of genomic DNA from Aspergillus species to develop rapid and highly sensitive polymerase chain reaction (PCR) based tests to identify aflatoxigenic fungi and their toxins in contaminated food products. Accomplishments46 Sequenced all the DNA of the fungus A. flavus and identified critical genes involved in fungal response to various environmental factors favoring toxin production. Studied the long-term survival of aflatoxin-producing A. flavus strains in comparison with non-producing strains and indicated that under temperature stress (47�C), spores of toxigenic strains survived longer than non-aflatoxin producers. Showed no difference in survival under UV light for aflatoxin-producing strains vs. non-aflatoxin-producing strains. Obtained proof of involvement of genes in aflatoxin synthesis and fungal development. Completed microarray experiments and validated candidate gene expression profiles for the genes that are potentially involved in the control of fungal development and secondary metabolism. Investigated the genetic basis for loss of aflatoxin production in toxin-deficient mutants of A. parasiticus generated by physical manipulation of toxin-producing strains using microarrays and metabolic profiling and identified specific regulatory genes causing this loss. Identified DNA probes (primer sets) for universal screening for genetic variability of Aspergillus group fungi. Elucidated details of the aflatoxin biosynthetic pathway and conducted studies to understand the roles of hypothetical genes (genes whose function is not yet known) in aflatoxin biosynthesis, as well as the importance of the protein encoded by the genes NorA, NorB and NadA in the final steps in formation of the toxic metabolites aflatoxins. Ecological Basis for Aflatoxin Reduction Through Crop Management and Biological Control Location: Food and Feed Safety Research Unit and U.S. Arid Land Agricultural Research Center Project Objectives50 Improve manufacture and formulation of atoxigenic strains (ATOX) for management of aflatoxin contamination. Characterize influences of agronomic practices (tillage, irrigation, crop rotation, application strategy) on biocontrol of aflatoxin contamination and optimize field use of ATOX on a commercial scale. Develop an epidemiological model to explain formation of aflatoxins in Texas and Arizona, with emphasis on the contamination after crop maturation. Characterize the major vegetative compatibility groups (VCGs) associated with Arizona and Texas agriculture and associated niches, and test geographical and niche specialization in order to improve understanding of the etiology of contamination and selection of ATOX with improved competitive ability in target crops/areas. Develop both a single nucleotide polymorphism (SNP) database and molecular tracking techniques useful in characterizing the composition of A. flavus communities, at both the strain and VCG level on crops and in the environment. Characterize A. flavus responses to varying environments and ecological niches (including animal and plant hosts) to assess fungal adaptations leading to niche competence and to facilitate selection of elite biocontrol strains and development of management practices. Accomplishments50 Suggested that the extent to which ATOX are genetically isolated from aflatoxin producers greatly influences the potential for area-wide and long-term beneficial impacts of biological control strategies based on ATOX of A. flavus. Identified that the S strain of A. flavus is frequently responsible for poisonous maize in Kenya. This fungus also infects maize in central Texas. Suggested that A. flavus reproduces and disperses clonally in U.S. agriculture and that biocontrol strains of A. flavus are unlikely to obtain the ability to produce aflatoxins from aflatoxin producers. Indicated that several aflatoxin-producing VCGs frequently infect cottonseed in both Texas and Arizona and suggest that these strains are adapted to success in the cotton crop environment. The A. flavus population structure detected by vegetative compatibility analyses suggested that recombination (i.e. a sexual growth phase) is uncommon or nonexistent among A. flavus associated with cotton. Biological Control of Insects and Microorganisms to Prevent Mycotoxin Contamination Location: Plant Mycotoxin Research Unit Project Objectives48 Apply the Pichia anomala yeast product to pistachio orchards early in the season prior to June 15 to be followed by ATOX technology. Measure the reduction of recoverable A. flavus spores in treated plots Pichia anomala or use some other appropriate measure of reduced colonization of natural substrates. Develop commercially viable methods for control of fungal and insect pests which contribute to pre-harvest aflatoxin contamination of tree nuts. Control mycotoxin-producing fungi using bacteria and control of A. flavus in tree nut orchards using the saprophytic yeast Pichia anomala. Development of semiochemical-based low-risk control strategies against key insect pests of tree nuts as insect feeding damage is associated with the invasion of microbial pathogens and mycotoxin contamination. Accomplishments48 Found that a major insect pest of almonds, the navel orangeworm, is a major contributor in promoting infection of almonds by the fungus that produces aflatoxin. Used other microbes as biological control agents against aflatoxin producing fungi to control aflatoxin contamination. Discovered strains of two bacteria that reduced aflatoxin producing fungi by 10 to 100 fold in soil from corn fields suggesting that environmental and nutritional conditions in this soil were not conducive to aflatoxin production. Application of a yeast resulted in significant reductions in cluster loss and significant increases in harvestable yield of pistachios. Used natural chemical lures and viruses to control insect pests of tree nuts. Molecular and Genetic Approaches to Suppressing Fungal Pathogens and Mycotoxin Contamination Location: Plant Mycotoxin Research Unit Project Objectives61 Release a promising insect resistant, premium quality almond variety, and evaluate other advanced breeding selections. Characterize almond seed coat tannins at the genetic and biochemical levels and examine associaion of specific antioxidants with seed coat ink staining. Identify and integrate multiple resistances into regionally adapted, high commercial quality breeding selections. Reduce mycotoxin contamination of agricultural commodities focusing on tree nuts (almonds, pistachios and walnuts) by identifying natural constituents or biocompetitive organisms that inhibit growth of fungi and aflatoxin production. Identify target genes in fungi that trigger mycotoxin biosynthesis focusing on stress response pathways. Accomplishments61 Found chemosensitization to be effective against a number of human pathogenic fungi. Chemosensitization enabled the use of antifungal drugs, such as itraconazole or fluconazole, against strains that had become resistant to these drugs. Conducted microarray analysis of caffeic acid-treated A. flavus and indicated that expression of almost all genes in the aflatoxin biosynthetic cluster were down-regulated. Found, in collaboration with scientists in the Food and Feed Safety Research Unit, that antioxidants induce production of peroxiredoxins, enzymes that degrade certain oxygenated compounds in fungi, with a concomitant shutdown of the aflatoxin biosynthetic pathway. Found a number of chemical compounds related to the natural compound, ferulic acid, have significant fungicidal activity. Some of these compounds show commercial promise and the structures of the compounds help to understand how they work against the fungus. Agricultural Practices, Ecological and Varietal Effects on Aflatoxins and Other Mycotoxins in Corn Location: Crop Genetics and Production Research Unit Project Objectives47 Optimize agronomic systems and environmental practices, including fertilization and rotation that minimize inoculum potential of A. flavus and other mycotoxin-producing fungi while maximizing corn yield and profits in the mid-south U.S. Gain an understanding of the role of crop management practices on the ecology of A. flavus and aflatoxin contamination in order to optimize the application of competitive exclusion products. Develop economical biologically-based strategies, including antagonists from soil fungi and bacteria, microbial competitors and antagonists, and natural compounds from biological sources, to minimize mycotoxins and their respective fungi in corn, and continue ecological studies on Aspergillus populations under various management strategies, e.g., rotations, tillage, and cover crops, and herbicide-resistant crops. Evaluate insect-resistant and susceptible maize lines for insect damage and aflatoxin and fumonisin contamination. Evaluate corn accession for aflatoxin resistance and develop isolines with stable resistance and susceptibility. Accomplishments47 Developed non-aflatoxigenic A. flavus strains. Issued a patent on a non-toxigenic A. flavus K49 that reduced aflatoxin in corn up to 93 percent under field conditions. This technology allows industry to test the use of non-toxigenic strains to develop a commercial product to control aflatoxin contamination in corn. Showed that Bt-corn hybrids are more resistance to mycotoxin than non-Bt corn hybrids and also the importance of planting dates on the value of Bt-corn hybrids in the prevention of mycotoxins. Compared herbicide treated and cultivated herbicide-resistant corn and showed that application of Round-up and Liberty herbicide to resistant corn hybrids has no adverse impact on growth, development, yield or mycotoxin incidence of those hybrids. Suggested that earlier maturing corn hybrids can avoid some of the late season heat and drought stress that facilitates A. flavus infection and aflatoxin contamination. Integrated Management of Plant-Parasitic Nematodes in Cotton and Peanut Location: Crop Protection and Management Research Unit Project Objectives51 Determine whether increased aflatoxin production in nematode-infected peanuts is due to a greater percentage of immature kernels, and the role of nematode infection of roots versus pods. Determine whether nematode-resistant peanut genotypes reduced the risk of pre-harvest aflatoxin contamination in soil infected with root-knot nematodes. Enhance native and introduced antagonists of nematodes in cotton and peanut cropping systems. Accomplishments51 Determined that three root-knot nematode species parasitize peanut in the USA--Meloidogyne arenaria (Ma), M. hapla (Mh), and M. javanica (Mj). Some commercial cultivars have resistance to Ma and Mj , but no peanut cultivar or released germplasm has resistance to Mh. Identified three breeding with high levels of resistance to Mh , with two lines also having resistance to Ma and Mj . These breeding lines will be valuable for developing peanut cultivars with resistance to multiple species of root-knot nematodes, which will improve the durability of resistant cultivars. Control Mechanisms for Mycotoxin Prevention in Peanuts and Their Rotation Crops Location: National Peanut Research Laboratory Project Objectives49 Refine aflatoxin biocontrol technology for peanuts and develop an effective system for achieving biological control of aflatoxins in corn, an important crop grown in rotation with peanuts. Determine characteristics of soil populations important for invasion of peanut seeds by aflatoxigenic fungi and evaluate the competitiveness of nontoxigenic biocontrol strains of A. flavus. Determine the chemical barriers of peanut to fungal challenge, particularly challenge by A. flavus. Investigate the basis for greater resistance to A. flavus invasion and aflatoxin contamination possessed by certain peanut genotypes for possible exploitation in breeding programs. Conduct the necessary laboratory and field trials required by the Environmental Protection Agency (EPA) to extend the use of Aflaguard to other crops susceptible to aflatoxin, such as corn. Accomplishments49 Developed a laboratory assay to evaluate nontoxigenic strains of A. flavus as potential biocontrol agents. Showed that nontoxigenic A. flavus is more competitive than nontoxigenic A. parasiticus in controlling aflatoxins and that the effectiveness of both species depends upon the density and toxigenicity of native soil populations of aflatoxin-producing fungi. Suggested that several strains may be more effective at preventing aflatoxins than the current strain comprising afla-guard�. The potential impact of this accomplishment is the development of a more effective aflatoxin biocontrol product. FSRIO Research Projects Database For additional USDA Aflatoxin Research Projects, please visit the FSRIO Research Projects Database. For additional aflatoxin research projects conducted by other U.S. government and International agencies, please search the FSRIO Research Projects Database. Back to top References 1. Abbas, H.K. (ed). 2005. Aflatoxin and Food Safety. CRC Press. 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