Source: UNIVERSITY OF CALIFORNIA submitted to
AFLATOXIN CONTROL IN PISTACHIOS, ALMONDS, AND FIGS: BIOCONTROL USING ATOXIGENIC STRAINS
Sponsoring Institution
Agricultural Research Service/USDA
Project Status
NEW
Funding Source
Reporting Frequency
Annual
Accession No.
0412279
Grant No.
(N/A)
Project No.
6435-42000-022-01S
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Sep 19, 2007
Project End Date
Jul 31, 2012
Grant Year
(N/A)
Project Director
COTTY P J
Recipient Organization
UNIVERSITY OF CALIFORNIA
(N/A)
PARLIER,CA 93748
Performing Department
(N/A)
Non Technical Summary
(N/A)
Animal Health Component
(N/A)
Research Effort Categories
Basic
27%
Applied
27%
Developmental
46%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7121510110216%
7121810110274%
7121219110210%
Goals / Objectives
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.
Project Methods
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.

Progress 10/01/11 to 09/30/12

Outputs
Progress Report 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. 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.

Impacts
(N/A)

Publications


    Progress 10/01/10 to 09/30/11

    Outputs
    Progress Report 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. 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. Work is carried out at the Kearney Agricultural Center of the University of California in Parlier and in commercial orchards. In early July, 2008, June, 2009, and June, 2010, wheat infected with AF36 were applied in 15 commercial pistachio orchards for a total treated area of 3,000 acres. 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. The frequency of the atoxigenic AF36 in the soil increased substantially in the treated orchards after applying the wheat- AF36 product. Prior to the first application of the wheat-AF36 product in these orchards, only 2% of the Aspergillus (A.) flavus isolates from the soil of these orchards belonged to vegetative compatibility groups (VCG) YV36 (the VCG for the isolate AF36). But this increased until 92% of the isolates were VCG YV36 in the soil collected in September 2010 from treated areas. This demonstrates that the application of the wheat- AF36 product was successful in substantially increasing AF36 in the A. flavus/A. parasiticus populations in these orchards. In addition, application of the wheat-AF36 product did not result in increased incidences of kernel decay. Early split nuts (the main source of aflatoxin contamination in pistachio orchards) did not have significantly more decay by A. flavus in the treated areas than in untreated areas (both less than 0.1% compared to 3.0% decay by A. niger). 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 2010. For the nut samples from the 2010, 97.1% of the ones from the treated orchards had no aflatoxin contamination, whereas 94.8% from the untreated orchards had none. In conclusion: after applying the AF36 starting in 2008, its level in soil increased from the naturally occurring 2% to 92% in 2010; application of AF36 did not increase kernel decay of pistachio nuts; for the 2010 harvest, 97.1% of the ones from the treated orchards had no aflatoxins, and 94.8% of those collected from untreated orchards (so there was an increase of nuts without aflatoxin contamination in the treated orchards); there are a number of atoxigenics that occur naturally in California pistachio orchards with AF36 being the most common atoxigenic. For figs: No new information to report. Progress is monitored by emails, periodic reports, phone conversations, and conference calls including, when useful, representatives of both IR-4 and industry.

    Impacts
    (N/A)

    Publications


      Progress 10/01/09 to 09/30/10

      Outputs
      Progress Report 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. 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 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.

      Impacts
      (N/A)

      Publications


        Progress 10/01/08 to 09/30/09

        Outputs
        Progress Report 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. 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. Significant Activities that Support Special Target Populations 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.

        Impacts
        (N/A)

        Publications


          Progress 10/01/07 to 09/30/08

          Outputs
          Progress Report 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. 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. Significant Activities that Support Special Target Populations 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.

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