2008 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.
Soil samples were collected in 2007 from 28 almond orchards from three regions: southern San Joaquin Valley, northern San Joaquin Valley, and the Sacramento Valley. Aspergillus 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. Densities of A. flavus/A. parasiticus in California almond orchards varied greatly from orchard to orchard, ranging from 2 to 219 cfu/g soil. The A. flavus S strain was 1.9, 1.8, and 17.0% of the Aspergillus sect. Flavi isolates for the southern, central, and northern regions, respectively. AF36 occurred naturally in almond orchards throughout California, making up 6.9% of A. flavus. However, AF36 was more common in the southern region (12.1% of the A. flavus isolates) compared to the central (5.9%) and northern regions (2.6%). Additional samples were collected in 2008. A biocontrol study in almonds was initiated in a research almond orchard. Prior to applying AF36, A. parasiticus (74.2% of the isolates) was more common in the soil than A. flavus strain L (15.0%) and A. flavus strain S (10.8%). The density of A. flavus/A. parasiticus in the soil was not significantly higher in the areas treated with AF36 than in nontreated areas. In the areas treated with AF36, 93.3% of the A. flavus isolates from soil were AF36 strain. In pistachios, percentages of AF36 among A. flavus isolates from soil in areas treated for biocontrol in 2004 and 2005 were 82.0%, 88.4%, and 50.9% for 2005, 2006, and 2007, respectively; while in the areas treated in 2003 and 2004 percentages were 84.8%, 46.5%, and 32.2% for 2005, 2006, and 2007, respectively. In addition, treatments did not differ significantly in density of A. flavus/A. parasiticus on surfaces of the hulls of freshly harvested nuts. 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 nuts. Spatial patterns of aflatoxin contamination throughout the pistachio-growing regions in California are also currently being investigated. Atoxigenic strain AF36 was applied to commercial orchards in 2008 under an Experimental Use Permit granted by EPA and CDPR. The permit requires monitoring of propagules in the air. To achieve this, monitoring stations were set up in commercial fields to be treated and in control fields. These studies provide data needed for use of fungal biological control agents to prevent aflatoxins in pistachios and almonds. Activities on the project were monitored through meetings, phone conversations and emails. The cooperators made two presentations at the Aflatoxin Elimination Workshop.