2012 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.
The AF36 product was applied in 15 commercial pistachio orchards (total treated area of 3,000 acres) in 2008, 2009, 2010, and 2011. The AF36 product was applied as a single annual application to the soil surface of the orchards in late June or early July. Soil samples were collected from 7 pairs of orchards (treated orchards and adjacent nontreated orchards), and nut samples were collected from 3 pairs of orchards. Applying the wheat-AF36 product was successful in substantially increasing the frequency of the atoxigenic strain AF36 in the soil. Prior to applying the AF36 product for the first time in 2008, only 2% of the Aspergillus (A.) flavus isolates from the soil of these orchards belonged to vegetative compatibility group (VCG) YV36 (the VCG for the isolate AF36). But this increased for soil collected in September 2011 from treated orchards. Application of AF36 did not result in increased decay of the nuts. Early split nuts (the main source of kernel decay and aflatoxin contamination in pistachio orchards) did not have significantly more decay by A. flavus in the treated orchards than in untreated orchards (in 2011 both were equal to 0.1% compared to 2.0% decay by A. niger). Nuts from the orchards treated with the AF36 product were less likely to be contaminated with aflatoxin for each of the years from 2008 to 2011. The percentage of samples contaminated with aflatoxin for nuts from treated orchards was reduced by 20.4%, 38.6%, 44.9%, and 36.7% relative to that for nontreated orchards for 2008, 2009, 2010, and 2011, respectively. The wheat-AF36 product has consistently performed well in pistachio orchards in California, and earlier in 2012 the U.S. Environmental Protection Agency approved the registration of the AF36 product for use with pistachios. In 2011 two separate studies were performed comparing a sorghum-AF36 product with the currently used wheat-AF36 product. The first study involved putting the two products on media in incubators (15°C to 35°C). After 8 days in the incubators, slightly more spores were produced on the wheat-AF36 product (mean of 17 million spores/g product for all temperatures and media) than on the sorghum-AF36 product (11 million spores/g product). Nevertheless, abundant spores (like seeds for the fungus) were produced on the sorghum-AF36 product at all of the tested temperatures. The second study was to apply the two products in a research pistachio orchard at the Kearney Agricultural Center in Parlier, California, in early June. By the end of summer, substantially more A. flavus/A. parasiticus occurred in the soil in areas treated with the sorghum-AF36 product (614 propagules/g soil) than in the areas treated with the wheat-AF36 product (11 propagules/g soil) or in the untreated areas (4 propagules/g soil). Applications of AF36 to Experimental Almond Orchards are also being investigated and show promise for use of this biological control on Almonds. Other studies included assessment of contamination and spatial variability of soil populations of A. flavus and A. parasiticus in California almonds orchards, over a three-year period. The work suggests considerable spatial variability in the populations of these fungi.