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Biology of Aflatoxigenic Fungi and Biocontrol of Aflatoxins in Orchards of Nut Crops in California

Michailides, Themis
University of California - Davis
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This project will address the serious food safety problem of aflatoxin contamination in almond and pistachio nuts in California. Aflatoxin, a toxin produced as a secondary metabolite of the two filamentous fungi, Aspergillus flavus and A. parasiticus, has received greater attention and is more widely studied than any other mycotoxin due to demonstrated potent carcinogenic effects in laboratory animals and acute toxicological effects (even deaths) in people. In order to minimize potential human exposure, the aflatoxin content of food and feed is strictly regulated in most of the world. Among the cultivated nut crops in California, pistachio and almonds are the most frequently contaminated nuts with aflatoxin. Indeed, in the last few years, unusually higher levels of aflatoxin contamination were detected frequently in these two crops than in nut samples recorded in previous years, suggesting that there may be a shift towards a population of Aspergillus flavus/A. parasiticus producing unusually high levels of aflatoxins. In my laboratory, we have made major discoveries in the sources of and factors affecting aflatoxin contamination of these crops and are now working towards the discovery of methods to reduce/eliminate aflatoxin contamination in these crops. At the same time, we have been involved in research on the biological control of aflatoxin-producing fungi using atoxigenic (not producing aflatoxin) A. flavus strains. Among these strains, the atoxigenic A. flavus AF36 (a registered strain for the reduction of aflatoxin in cottonseed in Arizona and corn in Texas) has been studied in detail in an experimental pistachio and fig orchards. In January 2008 we were granted an Experimental Use Permit by the US EPA and the California Dept. of Pesticide Regulation to treat 3,000 acres of pistachio orchards. The aim of the present project is to determine factors affecting putative shifts in the soil populations of A. flavus/A. parasiticus towards an increase of the highly toxigenic populations, displace these toxigenic populations by adding a mixture of atoxigenic strains of A. flavus in commercial orchards, and reduce aflatoxin contamination in nuts. Genetic characteristics of existing atoxigenic strains will be determined and molecular markers developed to identify the atoxigenic strains. The strains most frequently encountered in commercial orchards will be selected and used as the candidate biocontrol agents. A mixture of atoxigenic strains will be applied in the soil of an experimental almond and pistachio orchard and the survival of these strains and displacement of toxigenic strains quantified using a Real-time PCR assay developed in our laboratory. The levels of the propagules produced by the applied atoxigenic strains will be monitored in the soil, air, and nuts. Subsequently, the anticipated decrease in aflatoxin contamination in nuts will be quantified. This project's research will increase nut quality, safeguard the health of the consumers, and benefit greatly at least two major California nut crop industries by supplying domestic and international markets with aflatoxin-free almonds and pistachios.
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NON-TECHNICAL SUMMARY: Aflatoxins (produced by Aspergillus flavus and A. parasiticus) are potent liver carcinogens and are widely regulated by governments who have set very low tolerances for aflatoxins in food and feed. Contamination of nuts crops with aflatoxins has become a huge issue relevant to food safety in the last several years. Recently, nut loads have been rejected frequently due to excessive aflatoxin contamination levels (far more the regulated tolerance of 10 parts per billion). Some other industries have used and registered atoxigenic (not producing aflatoxin) of A. flavus and thereby have successfully reduced aflatoxin contamination. We have used very successfully the atoxigenic strain AF36 in pistachio and fig orchards. We propose here to determine the density of A. flavus and A. parasiticus and the ratio of toxigenic to atoxigenic strains in nut crop orchards and specifically the incidence of the atoxigenic strains of A. flavus. Also we will compare isolates of A. flavus collected during 2009 to 2011 (recent populations) with isolates collected from nut crops during 1992 to 1994 (historic populations) to determine whether there was a shift of strains in recent populations towards strains highly toxigenic (producing higher amounts of aflatoxins than normal). Indeed, recent analyses of nut loads exported to Europe were found contaminated with unusually higher levels of aflatoxins than previously encountered, suggesting perhaps that there was a shift in A. flavus/parasiticus towards highly toxigenic strains. Representative aflatoxigenic strains from recent populations both from pistachio and almond orchards will be tested for amounts of aflatoxins they produce using HPLC and the levels compared with those of historic A. flavus to determine if there is a shift towards highly toxigenic strains. We also propose to apply a mixture of the most commonly encountered atoxigenic strains in a pistachio and almond orchard at Kearney Ag Center and determine establishment of these strains, survival, displacement of the aflatoxigenic toxigenic strains, and reduction of aflatoxin contamination in nuts. Samples of soils will be collected from plots of treated and non treated replicated plots before and 3 months after the application of the atoxigenic strains to determine displacement of the toxigenic strains. Similar determinations in the year following the first year of application can help determine how well the strains will survive and used as a guide whether application of the atoxigenic strains need to be done yearly or every other year. Nuts will be collected and isolations be made to determine the incidence of atoxigenic strains landed on the nuts and contamination of nuts with aflatoxins. Therefore an anticipated output of this project is that it is possible that a successful technology used to reduce aflatoxins in row crops (cotton, peanut, and corn) could be transferred and applied in nut tree crops that are also susceptible to aflatoxin contamination. Secondly, the atoxigenic strains that will be used in this project are naturally occurring in California orchards and their registration will be easier than strains introduced from other States or abroad.

APPROACH: 27 orchards in nine locations/counties will be selected where major acreage of nut crops are grown. Five composite samples will be collected from the topsoil of each orchard to determine the density of A. flavus and A. parasiticus. A. parasiticus is typically rarer than A. flavus. A semi-selective medium that has been developed and used routinely in our laboratory will be used also to isolate various strains of A. flavus and A. parasiticus. Toxigenicity of the strains will be determined using HPLC to determine the ratio of the toxigenic to atoxigenic strains and the levels of aflatoxins produced. Strain identification will be done using the VCG, Vegetative Compatibility Groups, procedures well established. We will emphasize the atoxigenic strains that are encountered more frequently, including the AF36. The frequency and the amounts aflatoxin production by the toxigenic strains of A. flavus recovered in 2009 to 2011 will be compared with those of historic strains collected in 1992 to 1994. These results should facilitate and provide useful information in preparing for the registration and application of atoxigenic strains in almond orchards. Registration of native atoxigenic strains is easier and obtained faster than that for strains brought from out of State or abroad. Mixtures of selected atoxigenic strains including the AF36 will be applied in a pistachio and almond test orchards and soil samples collected before and 3 months after application of the inoculum to determine establishment and survival of these strains in the orchard's floor. The atoxigenic strains grown on wheat seed will be applied at a rate of 10 pounds per acre. The wheat will be applied in a replicated random design with three each treated and nontreated, ground surface of 16-tree replications. After application of the wheat (atoxigenic source inoculum), the orchard will be drip-irrigated daily throughout the summer to induce sporulation of the atoxigenic strains on the wheat. Soil samples will be collected just before the application of the wheat inoculum. Soil samples and nuts will be collected during harvest (end of the first growing season). The density of A. flavus in the soils and on the nuts will be quantified. Isolates of A. flavus obtained from soils and nuts will be tested to see if they have the same VCG as the applied atoxigenic strains. Comparisons of the density of the applied strains with that of the native strains will be performed. Collected nuts will be checked for visible decay by A. flavus and A. parasiticus. All the A. flavus from infected nuts will be identified to strain to determine the incidence of the atoxigenic strains and displacement of the other A. flavus strains. Nut samples will be analyzed for aflatoxins. Sampling soil samples and nuts at harvest of the second season should provide information how well the applied atoxigenic strains do after application for a second season. We anticipate from these treatments to determine the best time for applying the atoxigenic strains (yearly or every other year), the most efficient displacement of the toxigenic A. flavus/A. parasiticus strains, and levels of reduction of aflatoxin contamination.

Funding Source
Nat'l. Inst. of Food and Agriculture
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Natural Toxins
Viruses and Prions
Bacterial Pathogens
Chemical Contaminants
Nuts, Seeds