2009 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.
Activities on the project were monitored through annual meetings, bimonthly phone calls, exchanges of data, and written reports. In California, treenuts are contaminated with aflatoxins. Biological control using an atoxigenic strain of the fungus, Aspergillus flavus, is considered to be an effective strategy to minimize toxin contamination. In early July 2008, wheat infected with AF36 was applied in 15 commercial pistachio orchards. The 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 wheat with the atoxigenic A. flavus strain AF36 applied to the orchard floor. Spores of the fungal species A. flavus/A. parasiticus and of Aspergillus sect. Circumdati (A. ochraceus group) only occurred at relatively low levels in both orchards. For the aflatoxin-producing fungi, spores of A. flavus L were much more common in the air than those of A. flavus S type strain or another species, A. parasiticus. The trend for the spore concentrations in the air was to be low in early summer, to increase during the summer and reach a maximum during harvest, and then decrease. Percentage of A. flavus isolates from the air belonging to the strain AF36 increased from 14.4 to 26.3% in November in treated orchard, while for untreated orchard AF36 increased from 7.8% to 32.1% in October (then decreasing to 7.1% in November). Conidia of AF36 are moving from the treated areas to the untreated areas and vice-versa. Treatments did not differ significantly in the density of A. flavus/A. parasiticus on the surface of the hulls of freshly harvested pistachio nuts. The density of the fungus Aspergillus sect. Nigri in the soil and on the surface of nuts in treated areas was substantially more than that of the density of toxin producing fungi A. flavus/A. parasiticus. Only one kernel out of 4,242 early split nuts was decayed by A. flavus, suggesting that applying AF36 does not significantly increase decay of the nuts. In 2008/2009, another emphasis was creation of maps of pistachio contamination. In order to create the maps, the samples were separated in 5 categories based on the aflatoxin contamination levels as 0, 0.1-2.9, 3.0-11.9, 12.0-19.9, and >20 parts per billon (ppb). For each year (2001 to 2005), the total number of orchards in each category of aflatoxin contamination level was calculated. Over 75% of the pistachio orchards in Kern County had no aflatoxin in 2001 through 2004, while about 67% of them in 2005 were free of any aflatoxin contamination. Depending on the year, 0 to 21% of the orchards had < 4.0 ppb, 2-11% had 4.0 to 11.9 ppb, 2-11% had 4.0 to 19.9 ppb, and 1-6% orchards had > 20 ppb aflatoxins.