Progress 10/01/09 to 09/30/14
Outputs
Target Audience: Almond growers and the Almond Board of California Almond growers in Australia and Spain. California Pistachio growers California Pistachio Research Board American Pistachio Growers Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Undergaduate and graduate students: Mohamed Taieb Nouri, Graduate Student Intern, Summer & Fall 2013 (5 months), Tunisia Mohamed Taieb Nouri, Graduate Student Intern, Spring 2014 (2 months), Tunisia Joel Quattrini, Graduate Student Intern, Fall 2013 & Winter 2014 (6 months). Switzerland Paulo Lichtemeberg, Graduate Student, during 2013 (6 months), Brazil Michael Luna - Undergraduate Student, Winter, Spring 2014 (part time) William Vaillancour, Undergraduate Student Intern, Summer 2014 (2.5 months), Rochester Institute of Technology, USA Postgraduate Researchers supervised: Dr. Alejandro Ortega Beltran, Post-doctorate research associate, October 2013 to present (Mexico) How have the results been disseminated to communities of interest? Industry Meetings: Statewide Pistachio Day, UC Cooperative Extension & California Pistachio Research Board, Visalia American Pistachio Growers Conference, San Diego, California Almond Board of California Conference, Sacramento, California What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objectives: 1. To evaluate new formulations of the AF36 product involving sorghum and seed coating. 2. To determine the optimal timing for the application of the AF36 product in pistachio and almond orchards. 3. To monitor the atoxigenic A. flavus strain AF36 in commercial orchards where the AF36 product has been applied. 4. To investigate the aflatoxin contamination of nuts after registration of AF36 and commercial application. 1. New formulations of AF36 product. Two changes to the AF36 product were evaluated. The first change was using sorghum as an alternative to wheat as a carrier for the AF36 fungus. The second change was using a new manufacturing process, consisting of coating the seeds with AF36 spores. These AF36 products were evaluated in a study that was initiated in 2011 and consisted of applying the sorghum- and wheat-AF36 products in a research pistachio orchard at the Kearney Agric. Research and Extension Center in late spring (7 June, 2011; 21 June, 2012; 24 June, 2013). In 2013 the AF36 products applied were made with the new seed coating process for the first time. Application of the sorghum-AF36 product resulted in high levels of the atoxigenic strain AF36 in the treated areas. In addition, application of the seed-coated products resulted in high levels of the AF36 fungus in treated areas. The exact results for the AF36 fungus in the soil varied from sampling date to sampling date with neither AF36 treatment being consistently better than the other. The results demonstrated that the sorghum-AF36 product would be an adequate alternative to the wheat-AF36 product and that the seed coated products perform well in pistachio orchards. 2. To address the optimal timing for the application of AF36, a study was initiated in 2012 in a research pistachio orchard. The AF36 product was applied every 2 weeks in 2012, starting on 11 May and continuing until 19 July. In 2013 the experiment was repeated with the AF36 product being applied every 18 days, starting on 23 May and continuing until 15 July. Soil samples were collected in May and in September of each year in order to determine how successful applying the AF36 product at the various dates was in increasing the AF36 strain within the orchard. In 2014 no AF36 product was applied, but soil samples continued to be collected. For the 2012 and the 2013 experiments, the applications resulting in the highest percentages of AF36 were the July treatments. For the 2012 experiment, the percentage of AF36 in soil samples collected in May 2014 had decreased substantially for all treatments compared to the soil samples collected in 2013. However, for the 2013 experiment, the percentage of AF36 in soil samples collected in May 2014 was approximately the same as in September 2013 for all treatments. In the soil samples collected in May 2014, the frequency of AF36 ranged substantially for the various application dates for the 2012 experiment (27% to 57% of the A. flavus isolates belonged to the AF36 strain, depending on application date) and the 2013 experiment (40% to 83%). Soil samples were collected in September 2014 and are currently being evaluated. The results so far indicate that the date that the AF36 product is applied is important for success and that application in July gives the best results. 3 and 4. To monitor the atoxigenic strain AF36 in commercial orchards, in 2012 a new study on using the AF36 product in commercial orchards was initiated. The three treatments in this study were i), applying the AF36 product annually starting in 2012 (but not prior to 2012); ii), AF36 applied annually in the period 2008-2011; and iii), the untreated control. Applying the AF36 product in 2012-2014 or in 2008-2011 did not significantly increase the density of fungi in the soil compared to the untreated control. However, the percentage of A. flavus isolates belonging to the atoxigenic strain AF36 in samples collected in 2013 was the highest for the orchards treated in 2012 and 2013 (70% belonging to AF36), followed by orchards treated in 2008-2011 (46%), and was the lowest for untreated orchards (27%). Strain identification of the A. flavus isolates obtained in 2014 is currently in progress. For the 2013 harvests, the percentage of nut samples contaminated with aflatoxin for nuts from orchards treated in 2012 and 2013 was reduced by 65% relative to that for untreated orchards. In contrast, the percentage of nut samples contaminated with aflatoxin for nuts from orchards treated in 2008-2011 was only reduced by 9%. The aflatoxin analyses of nut samples from 2014 are currently being done. The following are general conclusions from this research: 1. The changes to the formulation of the AF36 product continue to be promising. The sorghum-AF36 product is suitable for use as an alternative to the wheat-AF36 product. And the new seed-coated product performed well in pistachio orchards. 2. The date for applying the AF36 product affects the success with the best results being applications in July. 3. Probably the AF36 product needs to be applied more than one year and in large acreage in order to have a satisfactory effect on aflatoxin contamination of pistachio; however, the AF36 product might not need to be applied every year after having been applied for several years. In addition, we found out that kernels of closed shell nuts had less decay by Aspergillus fungi than kernels of open shell nuts, although some fungal decay was observed.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Doster, M.A., Cotty, P.J., and Michailides, T.J. 2014. Evaluation of the atoxigenic Aspergillus flavus strain AF36 in pistachio orchards. Plant Disease 98:948-956.
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: California pistachio growers California almond growers California walnut growers pest control advisors members of the american phytopathological society Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? n order to get practical outcomes from aflatoxin research projects one has to be persistent and analyze a very large number of samples. This is because aflatoxin is not normally distributed and the variability of aflatoxins among samples is tremendous; thus there are sampling problems. To overcome these problems we analyzed a large number of pistachio samples and determined significant reductions of aflatoxin contaminated samples in orchards where the atoxigenic strain AF36 of A. flavus was applied. The four years average aflatoxin reduction in samples from treated orchards was 40%, if one considers both the first and the second harvests, and about 55% for samples representing the second harvest. Based on these results, the AF36 biopesticide was registered in pistachio in late February 2012. This is the first such registration of the AF36 biopesticide in a nut crop worldwide. The registration is for pistachios grown in California, Arizona, New Mexico, and Texas. Following registration, 77,000 acres of pistachio were treated in 2012 and more than 150,000 acres in 2013. We also showed that the damage of pistachios and almonds by the lepidopteron pest, navel orange worm (NOW), is associated with aflatoxins contamination. The 2013 was a year with a very high damage by NOW and it is expected the nut crops to have high aflatoxin levels. In fact, our initial sample analyses showed high levels of aflatoxins. For the normal harvest, fields treated with the AF36 from 2008 to 2011 had an average of 27.4 ppb while samples from fields that were never treated had an average of 93.6 ppb aflatoxins. Samples from fields where the AF36 was applied only in 2012 and 2013 had an average of 46.9 ppb aflatoxins. Although there were some blocks with very high levels of damage by NOW and very high aflatoxin levels, the remaining fields that were treated with AF36 showed trends towards reduced aflatoxin levels in comparison with the untreated control fields. For the identification of additional atoxigenic strains, we initiated studies to evaluate the competitiveness of the selected atoxigenic strains. Almond kernels have been inoculated with each of the selected strains to confirm that they are not able to produce aflatoxins in almonds. In addition, these strains were co-inoculated with toxigenic strains of A. flavus on almond kernels to show their competitive ability in the presence of the toxigenic strains. The outcome from these experiments was that now we have a limited number of strains that have unique characteristics and from these it will be easier to select a few that can be used in mixtures as a biocontrol products. Based on the initial results of the efficacy of AF36 in almond microplots, an application has been submitted to the EPA for the approval of the AF36 in almonds. A decision has not been reached yet, but we are optimistic that our application will be approved and the almond industry will be able to use this product commercially. This project was funded by the USDA In order to get practical outcomes from aflatoxin research projects one has to be persistent and analyze a very large number of samples. This is because aflatoxin is not normally distributed and the variability of aflatoxins among samples is tremendous; thus there are sampling problems. To overcome these problems we analyzed a large number of pistachio samples and determined significant reductions of aflatoxin contaminated samples in orchards where the atoxigenic strain AF36 of A. flavus was applied. The four years average aflatoxin reduction in samples from treated orchards was 40%, if one considers both the first and the second harvests, and about 55% for samples representing the second harvest. Based on these results, the AF36 biopesticide was registered in pistachio in late February 2012. This is the first such registration of the AF36 biopesticide in a nut crop worldwide. The registration is for pistachios grown in California, Arizona, New Mexico, and Texas. Following registration, 77,000 acres of pistachio were treated in 2012 and more than 150,000 acres in 2013. We also showed that the damage of pistachios and almonds by the lepidopteron pest, navel orange worm (NOW), is associated with aflatoxins contamination. The 2013 was a year with a very high damage by NOW and it is expected the nut crops to have high aflatoxin levels. In fact, our initial sample analyses showed high levels of aflatoxins. For the normal harvest, fields treated with the AF36 from 2008 to 2011 had an average of 27.4 ppb while samples from fields that were never treated had an average of 93.6 ppb aflatoxins. Samples from fields where the AF36 was applied only in 2012 and 2013 had an average of 46.9 ppb aflatoxins. Although there were some blocks with very high levels of damage by NOW and very high aflatoxin levels, the remaining fields that were treated with AF36 showed trends towards reduced aflatoxin levels in comparison with the untreated control fields. For the identification of additional atoxigenic strains, we initiated studies to evaluate the competitiveness of the selected atoxigenic strains. Almond kernels have been inoculated with each of the selected strains to confirm that they are not able to produce aflatoxins in almonds. In addition, these strains were co-inoculated with toxigenic strains of A. flavus on almond kernels to show their competitive ability in the presence of the toxigenic strains. The outcome from these experiments was that now we have a limited number of strains that have unique characteristics and from these it will be easier to select a few that can be used in mixtures as a biocontrol products. Based on the initial results of the efficacy of AF36 in almond microplots, an application has been submitted to the EPA for the approval of the AF36 in almonds. A decision has not been reached yet, but we are optimistic that our application will be approved and the almond industry will be able to use this product commercially. This project was funded by the USDA Areawide Project on navel orangeworm (NOW) management project, the California Pistachio Research Board, the Almond Board of California, and a UC Discovery Grant. How have the results been disseminated to communities of interest? Presentations at National meetings Grower/industry meetings Statewide Pistachio Day What do you plan to do during the next reporting period to accomplish the goals? Continue and complete the work
Impacts
What was accomplished under these goals?
We have continued our efforts to reduce aflatoxin contamination in both pistachios and almonds and determined new atoxigenic strains of Aspergillus flavus. We used an atoxigenic Aspergillus flavus to displace the toxigenic A. flavus and A. parasiticus and reduce aflatoxins in nuts. We now attempted to improve the application methodology for atoxigenic strains in nut orchards and compared the sporulation of AF36 on wheat with that on the sorghum carrier. The comparison of sporulation was done in four experiments set in the experimental pistachio and almond orchards at Kearney. Although there was some variation, the sporulation of wheat started earlier in a way but by the end of the sampling period both wheat and sorghum sporulated equally well. In another experiment, a new sorghum product, seed-coated sorghum-AF36, was applied along with the wheat-AF36 product in a research orchard. Again, substantially more spores were produced on the wheat product than on the seed-coated sorghum product. Although some of the results from the research so far indicated that the sorghum-AF36 product was an adequate alternative, some results clearly demonstrated that the wheat-AF36 product was superior. However, the seed-coated sorghum-AF36 product has been shown to be very effective in cotton fields in Arizona. And the sorghum product has several advantages over the wheat product, such as easier to produce, has fewer food allergies, and is cheaper to produce. Similar sporulation patterns and quantities of spores were produced on wheat and sorghum-AF36 products when these products were applied in an experimental almond orchard at Kearney. In order to determine the optimal timing for application of the AF36-product in nut orchards in California, the wheat-AF36 product was applied in a pistachio orchard every 2 weeks, starting on 11 May and continuing until 19 July. Soil samples were collected during the harvest period in September in order to determine how successful applying the AF36 product at the various dates was in increasing the AF36 strain within the orchard. Prior to applying the AF36 product, 5% of the A. flavus isolates belonged to the AF36 strain. In September the percentage of A. flavus isolates belonging to the AF36 strain ranged from 31% to 90%, depending on treatment date. The two treatments with the highest percentages of AF36 were the two July treatments but the earliest treatment in early May performed better than most treatment dates with 63% of the A. flavus isolates belonging to AF36. Further soil samples will be collected in the late spring 2014 in order to follow the development of the AF36 strain within the A. flavus population in the treated areas. For the selection of superior atoxigenic A. flavus isolates to improve the efficacy of biocontrol of aflatoxin contamination of nut crops, we started with a collection of 300 atoxigenic isolates and from these, we have selected about 19 atoxigenics that show very promising characteristics. An ideal selection from the 19 is to choose 3 or 4 of these atoxigenic strains which show the most favorable biological and evolutionary characteristics so that they can be used in biocontrol mixtures.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Doster, M.A., Morgan, D, Cotty, P., Boeckler, Puckett, R., Luna, M., Fabier, G., and Michailides, T.J. 2013. Monitoring and improving the efficacy of the atoxigenic strain AF36 in commercial orchards and aflatoxin contamination of closed shell nuts. California Pistachio Industry report, San Diego.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Michailides, T.J. 2013. Risk factors, spatial patterns, and biocontrol of aflatoxin contamination in California almonds. Page 34 in Res. Update Almond Board of California Annual Conference. Sacramento, CA.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: We have continued our efforts to reduce aflatoxin contamination in both pistachios and almonds and determined the risk factors associated with aflatoxin contamination in these nut crops. Most importantly, we adapted the approach of using an atoxigenic Aspergillus flavus to displace the toxigenic populations of A. flavus and A. parasiticus and eventually reduce aflatoxins in the nuts. The atoxigenic isolate of A. flavus strain AF36 that was found to be the most common atoxigenic strain in soil in pistachio, almond, walnut, and fig orchards was used to treat 3,000 acres of pistachio for 3 consecutive years (2008, 2009, & 2010 under the Experimental Use Permit (EUP). The Paramount Farming Company, in cooperation with my laboratory staff, has done a tremendous job in coordinating the application of 30,000 lbs of wheat-AF36 inoculum in areas that were identified as "aflatoxin hot" in a previous study. Aflatoxin analyses of 435 to 600 library nut samples showed that the AF36 resulted in a reduction of 20% in aflatoxin-contaminated samples in 2008, 38% in 2009, 45% in 2010, and 37% in 2011. These exciting results were used to prepare and submit a 50-page report/application to EPA and the Calif. Dept. of Pesticide Regulation for the registration of AF36 in pistachio. The application was approved and the AF36 was registered in pistachio (EPA Reg. No. 71693 - 1) on 29 February 2012 and in the first year after the registration of AF36, 77,000 acres were treated in 2012. Library samples have been obtained and are being now analyzed for aflatoxins. The almond industry is now pursuing a similar path to get registration of the AF36 in almonds. Three years ago the Almond Board of California/ Safety Committee started funding a project to develop the initial data required for the EUP application. These data have been gathered, the almond industry's support letter has been sent to IR-4, and an application for a EUP to treat 5,000 acres of almonds is being pursued. Postdoc Bulent Kabak completed a study in which he showed that almond samples from northern Sacramento Valley do not become contaminated with aflatoxins while those from central and southern San Joaquin Valley consistently showed contamination for 2 years. Although this study was reviewed and published in Food Control Journal, the paper was withdrawn afterwards due to sensitivity of the issue. (The industry started receiving orders of almonds originating only from northern California.) After consultation with the marketing director of the almond industry, we agreed to withdraw the paper and avoid creating export marketing problems for our almond industry. We now have completed a study funded by the Areawide Project on navel orangeworm (NOW) management and showed that NOW not only creates wounds for infection of the crop but also acts as an active carrier of A. flavus and A. parasiticus propagules to nuts. This research is funded by the ARS/USDA Areawide NOW Project, the California Pistachio Research Board, the Almond Board of California, and a UC Discovery Grant. PARTICIPANTS: Dr. Themis J. Michailides (PI): Dr. Michailides coordinated all the activities of the other individuals who participated in this project. Dr. Mark Doster, Staff Research Associate III. Dr. Doster was involved at 50% level in each project relevant to the aflatoxin contamination and biocontrol experiments on pistachio and almond. Dr. Matthias Donner, post-doc. Dr. Donner worked on the aflatoxin contamination of almonds at a 100% level until January 2012. Marion Mimbielle, student. She worked for 3 months in the summer of 2012 and involved mainly the biocontrol of aflatoxin using the atoxigenic strain AF36 and aflatoxin contamination of closed shell nuts. Lea Marquez, student. She worked for 3 months in the summer of 2012 and involved mainly with the use of sorghum seed as inoculum of the atoxigenic Aspergillus flavus used as a biopesticide to reduce aflatoxin contamination of almonds. TARGET AUDIENCES: California Pistachio Research Board California Pistachio Marketing Order Western Pistachio Association Almond Board of California California Pistachio Processors Meetings with small groups of processors in their processing plants to deliver the findings of this project and a talk at the Pistachio Day. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
In order to get practical outcomes from aflatoxin research projects one has to be persistent and analyze for aflatoxins a very large number of samples. This is because aflatoxin is not normally distributed and the variability of aflatoxin incidence among samples is tremendous, thus there are sampling problems. To overcome these problems we analyzed a large number of pistachio samples and determined significant reductions of aflatoxin contaminated samples in orchards where the atoxigenic strain AF36 of A. flavus was applied. A total of 3,000 acres of pistachio were treated in 2008, 2009, 2010, and again in 2011. The four years average aflatoxin reduction in samples from treated orchards was 40%, if one considers both the first and the second harvests, and about 55% for samples representing the second harvest. Usually, the second harvest of pistachio, which is done about 2 to 4 weeks after the first harvest, contains nuts with higher levels of NOW infestation and aflatoxin contamination. After acceptance of the pistachio industry's application by EPA and the Calif. Department of Pesticide Regulation, the AF36 biopesticide was registered in pistachio in late February 2012. This is the first such registration of the AF36 biopesticide in a nut crop worldwide. The California (and the Arizona, New Mexico, and Texas) pistachio growers have now a unique product to reduce aflatoxins, and 77,000 acres were treated in the first year of registration. This long term research project led to a major outcome which is the registration of AF36. The impact of this registration is very significant because there are no other approaches to reduce aflatoxins in pistachio. Again after application of the AF36 in commercial orchards in 2013, we observed no negative health effects on any researcher or laborer who visited the treated orchards. The following summarizes the outcomes from these studies in 2012: 1) The first analyses of samples showed that the atoxigenic strain AF36 in the soil increased substantially in the treated orchards after applying the wheat-AF36 product. 2) There was no change in the levels of kernel decay of pistachios. 3) Most importantly, applying the wheat-AF36 product resulted in 37% (2011) and 40% average (2008-2011) reduction of aflatoxin contamination. 4) Specifically for the second harvest (reshakes), this reduction reached to 54% (3-year average). 5) The risk of exposure to A. flavus spores in treated fields does not appear to be greater than if fields were not treated. Regarding aflatoxin reduction in almonds, experiments in micro plots showed that propagules of AF36 have increased tremendously in the soil of areas treated with AF36 and displaced the toxigenic A. flavus and A. parasiticus, without resulting in any significant decay increase in almond kernels. Thus the almond industry expects to have the AF36 biopesticide registered in almond within a few years. Along these lines, because in 2012 the California Fig Industry suffered heavy aflatoxin contamination of figs, they solicited a proposal to pursue a EUP to use AF36 in fig orchards and eventually have AF36 registered in figs as well.
Publications
- Michailides, T.J. 2012. Risk factors, spatial patterns, and biocontrol of aflatoxin contamination in California almonds. Page 31 in Res. Update Almond Board of California Annual Conference. Sacramento, CA.
- Doster, M.A., Morgan, D.P., Boeckler, L., Puckett, R., Cotty, P.J., and Michailides, T.J. 2012. Monitoring and improving the efficacy of the atoxigenic strain AF36 in commercial orchards and aflatoxin contamination of closed shell nuts. Annual Report. California Pistachio Industry, San Diego, CA.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: The atoxigenic Aspergillus flavus strain AF36 (the same product that is registered for use in cotton and corn fields) was applied as a biocontrol agent in commercial pistachio orchards in California in 2008, 2009, and 2010. The AF36 strain was applied as a wheat-AF36 product as a single annual application to the soil surface of the orchards in late June or early July. Applying the wheat-AF36 product was successful in substantially increasing the level of the atoxigenic strain AF36 in the soil. Prior to applying the AF36 product, only 2% of the A. flavus isolates from the soil of these orchards belonged to the AF36 strain. But three months after the single application, 43% of the A. flavus isolates in the soil belonged to the AF36 strain. After additional applications of the AF36 product were made in these orchards in 2009 and 2010, 92% of the isolates were AF36. This also demonstrates that the application of the wheat-AF36 product was more successful at substantially increasing the level of AF36 in the orchards when applied more than one year. Nonetheless, application of the AF36 product did not result in increased kernel decay of the nuts, and no cyclopiazonic acid was detected in nuts from treated orchards. Nuts from the orchards treated with the AF36 product were less likely to be contaminated with aflatoxin than those from adjacent untreated orchards in 2008, 2009, and 2010. The percentage of samples contaminated with aflatoxin for nuts from treated orchards was reduced by 20.4%, 38.6%, and 44.9% relative to that for untreated orchards for 2008, 2009, and 2010, respectively. Furthermore, for the nut samples for 2008-2010, only 3.6% of the samples from treated orchards had greater than 15 ppb aflatoxins (the tolerance defined by Federal Marketing Order for pistachios) while 5.3% of the samples from untreated orchards had greater than 15 ppb, which represents a 32% reduction. Because of the high value of pistachio nuts and the problems that rejections cause, these reductions are significant and valuable. To determine whether applying the AF36 product would increase the number of fungal spores in the air and might pose a safety problem to humans, the concentration of spores in the air of Aspergillus species, especially A. flavus, was measured using spore samplers that were put in two commercial pistachio orchards. In one of the orchards the AF36 product was applied to the orchard floor, while the other nearby orchard was left untreated. The spores of fungi in Aspergillus niger group were at a substantially higher concentration in the air than spores for the other Aspergillus fungi, even in the treated orchard. The trend for Aspergillus spore concentrations in the air was to be low in early summer, to increase during the summer to a maximum during the period of harvest in September, and then to decrease. During the harvest period, the concentration of spores reached a high level during and right after shaking the trees to remove the nuts. Nonetheless, even during this harvest period, the levels for A. niger group (Aspergillus sect. Nigri) were much higher than the levels for A. flavus/A. parasiticus in the treated orchard. PARTICIPANTS: Dr. Themis J. Michailides (PI): Dr. Michailides coordinated all the activities of the other individuals who participated in this project. Dr. Mark Doster, Staff Research Associate III. Dr. Doster was involved at 50% level in each project relevant to the aflatoxin contamination and biocontrol experiments on pistachio and almond. Dr. Matthais Donner, post-doc. Dr. Donner worked on the aflatoxin contamination of almonds at a 100% level. Mathilde Gourguillon, student. She worked for 3 months in the summer of 2011 and involved mainly with the comparison of AF36 inocula in two substrates, wheat and sorghum. Benoit Hurth, student. He worked for 3 months in the summer of 2011 and involved mainly with inoculations of almonds with toxigenic and atoxigenic Aspergillus flavus and the effects of relative humidity on aflatoxin production. TARGET AUDIENCES: California Pistachio Research Board, California Pistachio Marketing Order, Western Pistachio Association, Almond Board of California & California Pistachio Processors. Meetings with small groups of processors in their processing plants to deliver the findings of this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Because aflatoxin is not normally distributed, the variability of aflatoxin incidence among samples is tremendous and thus there are sampling problems. Based on historical data, as the number of samples per orchard increases, the chance that aflatoxin contamination will be detected increases. Pistachios are an alternate bearing crop which means that the production level rotates between greater and lesser production. Greater production years are referred to as "on" years and years with less production are termed "off" years. In general, "off" production years have higher incidence of aflatoxin contamination than "on" production years. This could be partially explained by the fact that "on" years have greater numbers of nuts, thus there is a "dilution" factor involved, if we assume all the other factors remain the same from an "on" to an "off" year. No negative health effects were observed by any researcher who visited the treated orchards to obtain samples, even though researchers visited the treated orchard with the spore sampler frequently. Similarly, no negative health effects were reported by farm workers or growers doing all the needed cultural practices in these orchards. The following are outcomes from these studies: 1) The proportion of the atoxigenic strain AF36 in the soil increased substantially in the treated orchards after applying the wheat-AF36 product. 2) Application of the wheat-AF36 product did not result in increased levels of kernel decay of pistachios. 3) Application of the wheat-AF36 product resulted in improved reduction of the incidence of nut samples contaminated with aflatoxins from 2008 to 2010, with a 3-year statistically significant average of 40.3% reduction. 4) Specifically for the second harvest (reshakes), this reduction reached to a 2-year statistically significant average of 53.3%. 5) Furthermore, there was a 32.1% reduction in the incidence of the nut samples exceeded 15 ppb aflatoxin (the tolerance of the Federal Marketing Order and of the EU for nuts to be processed further). 6) Even during the harvest period, the levels for A. niger group were much higher than the levels for A. flavus/A. parasiticus in the treated orchard. 7) Based on the results of air trapping, exposure to A. flavus spores in treated fields does not appear to be greater than if fields were not treated. Therefore, it is unlikely that applying the AF36 product would result in any increased danger to humans. 8) AF36-treated orchards were more consistent in being able to produce pistachios without aflatoxin or cyclopiazonic acid. It is expected that the AF36 product will be registered in 2012 for the pistachio growers to apply it commercially. In a similar way, the almond industry is funding relevant research on the application of AF36 in almond orchards to reduce aflatoxins. A promising outcome from these experiments is that results are very similar with those on pistachio. It is anticipated that the registration of AF36 on almonds to reduce aflatoxins will not be delayed for long after the AF36 registration on pistachio.
Publications
- Kabak, B., Luo, Y., Reyes, H., and Michailides, T. J. 2011. Occurrence of aflatoxins in California almonds. Food Control (in press).
- Michailides, T.J. 2010. Risk factors, spatial patterns, and biocontrol of aflatoxin contamination in California almonds. Page 32 in Res. Update Almond Board of California Annual Conference. Modesto, CA.
- Doster, M.A., Morgan, D.P., Cotty, P., Boeckler, L., Puckett, R., Reyes, H., and Michailides, T.J. 2010. Biocontrol of Aflatoxin Using the Atoxigenic Strain AF36. Pages 88 to 89 in Annual Report. California Pistachio Industry, Santa Barbara, CA.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: The atoxigenic Aspergillus flavus strain AF36, a biocontrol agent that shows promise in reducing aflatoxin contamination, was applied to 3,000 acres of commercial pistachio orchards in 2010 (to the same orchards that were treated in 2008 and 2009). Soil and nut samples were collected and are in the process of being evaluated. The concentration of spores of Aspergillus species (especially the aflatoxin-producing species Aspergillus flavus) in the air was determined using spore samplers in two commercial pistachio orchards (one treated with AF36 and the other untreated) in 2008 and 2009. In 2010 we completed the strain identification of the A. flavus isolates obtained from the spore samplers. The atoxigenic AF36 was also applied in a research almond orchard at the Nickels Soil Laboratory in Arbuckle, CA in 2007 and 2008. In order to determine the spread and survival of the AF36 strain (no AF36 has been applied since July, 2008), soil and nut samples have continued to be collected during the period of commercial harvest in 2009 and 2010. The evaluation of samples collected in 2009 was completed in 2010, while the evaluation of the samples collected in 2010 are still in progress. In early 2010 we also investigated the natural occurrence of 15 atoxigenic A. flavus strains in commercial pistachio orchards. Approximately 1,250 A. flavus isolates have previously been obtained from pistachio orchards between 1990 and 2007. Currently, the frequencies of these atoxigenics among these A. flavus isolates are being completed. In addition, soil samples that were collected in 2007 and 2008 from 28 almond orchards across three major almond-growing regions from the south to the north of the San Joaquin Valley were processed in the laboratory at UC Kearney in 2009 and 2010. The density and distribution of species within the Aspergillus section Flavi isolates were determined by isolating the fungus from the soil using SI10 agar. From each of these 28 orchards, 5-10 A. flavus isolates were picked at random and analyzed for aflatoxin content. All isolates were grown in a liquid medium and the aflatoxin was extracted with acetone and methylene chloride and quantified with a HPLC. The AF36 is expected to be registered soon as a biological control agent in pistachios (and eventually in almonds) to decrease the aflatoxin contamination on these nut crops. Therefore, up to 30 A. flavus isolates from each orchard were tested to see if they belong to the atoxigenic Vegetative Compatible Group (VCG) belonging to the strain AF36. Since Aspergillus flavus strain AF36 was initially isolated in Arizona, it was important to determine if AF36 already exist natively in California. The use of native atoxigenic strains can provide a more effective biological control management because the strains are already well adapted to the environment were they will be released. Results were presented at Pistachio UC Workgroup, in Parlier in Jan 2011 and the 2010 Statewide Almond Conference in Modesto in Dec 2010. An annual report has been submitted to the IR-4 Project and the US EPA for approval of registration of this biopesticide on pistachio to reduce aflatoxin contamination. PARTICIPANTS: Dr. Themis J. Michailides (PI): Dr. Michailides coordinated all the activities for the other individuals who participated in this project. <br> Dr. Mark Doster, Staff Research Associate III. Dr. Doster was involved at 50% level in this project relevant to the aflatoxin contamination and biocontrol experiments on pistachio. <br> Dr. Matthais Donner, post-doc. Dr. Donner worked on the aflatoxin contamination of almonds at a 100% level. <br> Dr. Bulent Kabak, postdoc. He performed the aflatoxin analyses of almond and pistachio samples using a HPLC. He worked for 2.5 months in this project. <br> Mathilde Teulier, student. She worked for 3 months in the summer of 2010 and involved mainly with inoculations of pistachio with toxigenic and atoxigenic Aspergillus flavus. <br> Pauline Jiquel, student. She worked for 3 months in the summer of 2010 and involved mainly with inoculations of almonds with toxigenic and atoxigenic Aspergillus flavus. TARGET AUDIENCES: California Pistachio Research Board<br> California Pistachio Marketing Order<br> Western Pistachio Association<br> Almond Board of California<br> California Pistachio Processors <p> Meetings with small groups of processors in their processing plants to deliver the findings of this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Application of AF36 did not result in increased levels of kernel decay of pistachios. Nuts from the orchards treated with AF36 were less likely to be contaminated with aflatoxin than those from adjacent untreated orchards in 2008 and in 2009. For the main harvest nuts in 2009, 92% of the nut samples from the treated orchards had no aflatoxin contamination compared to 86% from untreated orchards. And for the reshakes in 2009, 73% of the nut samples from the treated orchards had no aflatoxin contamination compared to 65% from untreated orchards. These results suggest that the AF36 treatment contributed to the reduction of aflatoxin contamination of pistachios. It is expected that with a further increase of AF36 in the orchards there will be a further decrease in aflatoxin contamination as has been shown in other susceptible crops. In the orchard that had the atoxigenic AF36 strain applied to the orchard floor, only after tree shaking at harvest did the density of fungal spores reached high levels. Nevertheless, the levels of Aspergillus section Nigri were much higher during this period than those of A. flavus and A. parasiticus. These results suggest that applying AF36 in pistachio orchards will not increase significantly affect the density of fungal spores in the air and will not affect the health and safety of orchard workers. After applying AF36 in 2007 and 2008 in the research almond orchard at Nickels Soil Laboratory, the level of AF36 in the soil increased to 99% of all the A. flavus isolates in September, 2008. Furthermore, the level of AF36 remained high in 2009, demonstrating good persistence of AF36 in the almond orchard. However, the AF36 declined to 31%`in 2010, suggesting that maybe another application of the AF36 product would be needed for continued effectiveness. Applying AF36 did not significantly increase the incidence of hull decay of the almond nuts. The results so far demonstrate that applying the commercial product of AF36 in an almond orchard in a manner similar to that done in other crops is effective in increasing the level of this atoxigenic strain AF36. These results will help the registration of AF36 to decrease aflatoxins in almonds. Many strains of A. flavus in California pistachio orchards are atoxigenic and might be useful as biocontrol agents. Of the 15 atoxigenic strains studied, all were very rare in pistachio orchards and none occurred naturally as frequently as the AF36 strain. Analyses of A. flavus isolates from almonds though showed that the aflatoxin producing isolates of A. flavus L-strains were predominant in both years. Overall, the incidence of toxigenic L-strains increased to 80% in 2008. If this level continues to increase, the risk of further aflatoxin contamination of almonds will increase as well. Incidences of toxigenic isolates varied widely among the orchards and years. The atoxigenic AF36 occurs naturally (4.7 to 6.9%) in almond orchards throughout California. The VCG of strain AF36 is so far the largest atoxigenic VCG found in California. Therefore, the AF36 strain is well adapted to the California environment.
Publications
- Doster, M. A., Cotty, P. J., and Michailides, T. J. (2009). Description of a distinctive aflatoxin-producing strain of Aspergillus nomius that produces submerged sclerotia. Mycopathologia 168:193-201.
Luo, Y., Gao, M., Doster, M., and Michailides, T.J. (2009). Quantification of conidial density of Aspergillus flavus and A. parasiticus in soil from almond orchards using real-time PCR. Journal of Applied Microbiology 106:1649-1660. Kabak, B., Luo, Y., Reyes, H., and Michailides, T. J. (2011). Occurrence of aflatoxins in California almonds. Food Control (in press).
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