Location: Food and Feed Safety Research2010 Annual Report
1a. Objectives (from AD-416)
For pistachios: Apply the atoxigenic strain AF36 in commercial pistachio orchards. Complete the survival studies for atoxigenic strains previously applied in research orchards. 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. For almonds: 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. Provide data from the biocontrol for an application to be submitted to the EPA to obtain an experimental use permit (EUP) to treat major acreage of almond orchards in “aflatoxin-hot” areas to reduce aflatoxins (no-cost objective). For figs: Follow the survival and spread of atoxigenic A. flavus strains previously applied in a research fig orchard. Prepare for application of AF36 in commercial fig orchards.
1b. Approach (from AD-416)
For pistachios: Continue research on biocontrol of aflatoxin-producing fungi using atoxigenic A. flavus strains: (1) apply the atoxigenic strain AF36 (currently working with EPA in order to apply AF36 in commercial pistachio orchards); (2) determine the survival of the atoxigenic strains, including the atoxigenic strain AF36 previously applied as a wheat formulation in two research pistachio orchards; (3) use the results from the aflatoxin analyses of “library” samples to identify spatial patterns of aflatoxin contamination in California. Library samples, which consist of 20 pounds of nuts taken at the processing plant as nuts are being unloaded, represent a valuable research tool because it is easy to collect numerous nut samples from many commercial orchards representing extensive acreage. The resulting information will assist in deciding which commercial orchards would be best for applying AF36; (4) determine the incidence of AF36 naturally occurring in commercial pistachio orchards with a history of high aflatoxin contamination. For almonds: The density of A. flavus and A. parasiticus and the ratio of toxigenic to atoxigenic strains will be determined in almond orchards and specifically the incidence of the AF36 atoxigenic strain of A. flavus. AF36 will be applied in experimental plots to obtain data on its survival, displacement of the toxigenic strains, and reduction of aflatoxin contamination in almonds. Data of survival of the atoxigenic strain(s) and the displacement of toxigenic A. flavus and/or A. parasiticus will be used to expand the application of registering AF36 from pistachios to include almonds. For figs: Continue research on biocontrol of aflatoxin-producing fungi using the atoxigenic Aspergillus flavus strain AF36. AF36 has been applied in a fig orchard in California, resulting in AF36 becoming the dominant A. flavus strain where it was applied. The survival and spread of the previously applied atoxigenic strain AF36 (no AF36 will be applied in 2007) will be measured in order to determine how many years after treatment AF36 will still be the dominant strain and the extent that AF36 will move outside the treated areas. All results will be statistically analyzed and summarized in preparation for applying AF36 large scale in commercial fig orchards.
3. Progress Report
The biocontrol strain AF36 (a strain of Aspergillus (A.) flavus that does not produce the potent carcinogen, aflatoxin) was applied to a research almond orchard in 2007 and 2008. In Fall 2009, incidence of the highly toxic A. parasiticus in treated orchards had shifted from 78.4% of the isolates prior to 2.3% and the highly toxic A. flavus strain S from 13.5% to 0.0%. This was associated with great increases of the atoxigenic L strain AF36. Thus, treatments caused long-term reductions in aflatoxin-producing potential of fungal communities in the almond orchard. Tests demonstrated that: after applying AF36, A. parasiticus decreased by 97% and A. flavus strain S by 100%; the biocontrol strain AF36 increased; nuts in treated areas had no increase in hull decay; and none of the samples of nuts from the treated areas had any aflatoxins. Biocontrol of aflatoxin was also evaluated in pistachios. Commercial pistachio orchards (15) were treated with biocontrol agent AF36 in 2008 and 2009. In total, 3,000 acres were treated under an Experimental Use Permit from Environmental Protection Agency (EPA). The level of the atoxigenic strain AF36 in soil increased substantially in treated orchards. Three months after application in 2009, 89% of A. flavus isolates were AF36. This demonstrates application of the wheat-AF36 product was successful in substantially increasing AF36 levels in orchards. Nonetheless, application of the wheat-AF36 product did not result in increased levels of kernel decay. Furthermore, nuts from the orchards treated with the wheat-AF36 product were less likely to be contaminated with aflatoxin than those from nearby untreated orchards in 2008 and in 2009. For the nut samples from the 2009 main harvest, 92.5% from the treated orchards had no aflatoxins, whereas 85.9% from the untreated orchards had none. To determine the concentration of spores of Aspergillus species (especially the aflatoxin-producing species Aspergillus flavus) in the air, spore samplers were put in two commercial pistachio orchards in 2008 and 2009. One of the orchards was treated with AF36, while the other was left untreated. Spores of fungi in Aspergillus sect. Nigri (A. niger group) were much more common in the air than spores for the other Aspergillus fungi, even in the orchard that had the atoxigenic strain AF36 applied to the orchard floor. At harvest, concentrations of spores reached a high level during and right after shaking. Nonetheless, levels of Aspergillus sect. Nigri (A. niger group) were much higher during this period than levels of A. flavus/A. parasiticus, even in the orchard treated with AF36. In June 2010, AF36 was applied in the same commercial pistachio orchards as in 2008 and 2009. Conclusions in pistachio: After applying the AF36, its level in soil increased from the naturally occurring 2% to 43% in 2008 and to 89% in 2009; application of AF36 did not increase kernel decay. Research progress was monitored through telephone discussions, email exchanges, presentations at professional meetings, and exchange of materials and data.