Source: UNIVERSITY OF CALIFORNIA, DAVIS submitted to
BIOLOGICAL CONTROL OF AFLATOXINS CONTAMINATING PISTACHIO, ALMOND, AND FIGS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1005544
Grant No.
(N/A)
Project No.
CA-D-PPA-2025-H
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Dec 12, 2014
Project End Date
Sep 30, 2019
Grant Year
(N/A)
Project Director
Michailides, T.
Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671
Performing Department
Plant Pathology
Non Technical Summary
Aflatoxins are toxins produced by certain molds (Aspergillus flavus and Aspergillus parasiticus) while these molds grow in various crops. Even though aflatoxin contamination occurs less frequently in pistachio and almond nuts compared to other crops such as corn and peanuts, some pistachio and almond nuts do become aflatoxin-contaminated in commercial orchards. Aflatoxins are widely regulated by governments, who have set very low tolerances for aflatoxins in food and feed. Strains of A. flavus that are not able to produce aflatoxin ("atoxigenic" strains) are able to successfully reduce aflatoxin contamination by displacing or excluding the aflatoxin-producing strains of A. flavus and A. parasiticus. Although several atoxigenic strains have been registered and are being applied commercially for aflatoxin reduction, the main atoxigenic strain used in the western United States is the atoxigenic A. flavus strain AF36. The use of the atoxigenic A. flavus strain AF36 as a biocontrol agent was developed first in Arizona. We have found that the atoxigenic strain AF36 is naturally widespread in California, occurring in all of the major pistachio- and almond-growing counties. After doing research for 10 years and obtaining efficacy data in commercial pistachio orchards, the atoxigenic strain AF36 was registered for pistachio in the states of California, Arizona, New Mexico, and Texas. In 2013, about 150,000 acres of commercial pistachios were treated with AF36. In this new project, we plan to evaluate new formulations of the atoxigenic AF36 product involving sorghum and seed coating (possibility for the sorghum inoculum to replace the newly produced sorghum AF36-inoculum. and determine the optimal time of application of AF36 strain for one time application within a year. Along the same lines, it is essential to determine the population dynamics and survival of AF36 in almond and pistachio orchards under different commercial settings and evaluate aflatoxin contamination levels of nuts as affected by commercial application of AF36 after registration. Furthermore, we will continue searching for more atoxigenic strains in pistachio and almond orchards so that to adapt mixtures for applying them in the field. It is expected these mixtures to have a better fit in the environment and colonize more diverse habitats. The methodology used to investigate these objectives has been commonly used in our laboratory for years now with a lot of success. We expect some major outcomes after the completion of this project. First, the growers will have a putative new product (AF36 sorghum inoculum) to use; second, information on the optimal time for application of the AF36; third, an answer on whether the atoxigenic strain would be needed to be applied once yearly or once every other year; forth, information about fitness and survival of mixtures of atoxigenic strains in comparison with the single registered strain AF36. It is expected that a mixture of atoxigenic strains will occupy a more diverse habitat in almond and pistachio orchard, thus have a better fit in the environment, and expected to survive more diverse soil environmental conditions.
Animal Health Component
0%
Research Effort Categories
Basic
30%
Applied
50%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2124020106030%
2155010107020%
2165240110220%
2164020117030%
Knowledge Area
215 - Biological Control of Pests Affecting Plants; 212 - Pathogens and Nematodes Affecting Plants; 216 - Integrated Pest Management Systems;

Subject Of Investigation
4020 - Fungi; 5010 - Food; 5240 - Seeds and other plant propagules;

Field Of Science
1070 - Ecology; 1170 - Epidemiology; 1060 - Biology (whole systems); 1102 - Mycology;
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.5. Select new atoxigenic Aspergillus flavus strains better suited to pistachio and almond orchards in California.
Project Methods
Objective 1. Two changes have been developed to the original formulation of the AF36 product. First, sorghum has been used as the carrier for the AF36 fungus instead of wheat. And second, the AF36 fungus has been attached to the seeds of wheat and sorghum by applying a coating of fungal spores instead of allowing the fungus to grow into the seeds as was done originally. In 2011, we initiated a replicated experiment in a research pistachio orchard at the Kearney Agricultural Center. The treatments are 1, sorghum-AF36 product; 2, wheat-AF36 product; and 3, untreated control. Samples will continue to be collected from this experiment in 2015 and in subsequent years to determine the long-term survival of AF36 after application. In addition, we made an additional application of the seed-coated sorghum and wheat products in June 2014. Soil samples were collected before application and again during the harvest period in order to determine how successful the application of the products are in increasing the levels of AF36. The density of A. flavus/A. parasiticus in soil samples will be determined by plating soil on the surface of a selective isolation (SI) medium containing chloramphenicol and dichloran and then incubating at 30°C for 7 days. Any colony of A. flavus and A. parasiticus will be isolated into pure culture to verify the species identification and to identify the strain. In another experiment, we will compare the production of spores on the seed-coated sorghum-AF36 product with that on the seed-coated wheat-AF36 product after applying both products to the soil surface in late spring/early summer in a research orchard at the Kearney Agricultural Research and Extension Center. Sporulation will be quantified in several ways routinely used in the Kearney plant path laboratory. Objective 2. Because the optimal timing for application of AF36 has not been determined for pistachio orchards in California, we will initiate a study to investigate the application of AF36 at different times in detail. The AF36 product will be applied in a research pistachio orchard on different dates in 2015 and again in 2016. Starting on early May and continuing until the middle July and using different plots, the AF36 will be applied at four different dates, 15 May, 5 and 25 June, or 15 July. Soil samples will be collected during the harvest period in September 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. The frequency of the AF36 fungus in the soil is quantified using the methods described earlier. The date that the AF36 product is applied is very important for successfully increasing the AF36 fungus in the orchard. Following the September sampling, the soil samples will be collected again in the late spring and during the harvest period in 2016 and 2017 in order to follow the development of the AF36 strain within the A. flavus population in the treated areas. The results from this study will help in designing a larger experiment in commercial orchards by using hundreds of acres of commercial pistachios. Objective 3. A study investigating AF36 in commercial orchards was initiated in 2012, the first year when AF36 was registered (EPA Reg. No. 71693-1, 29 February 2012). The three treatments in this study are 1, treated with AF36 annually in 2008-2011 (part of the Experimental Use Permit study) but not after 2011--- long-term survival of AF36; 2, treated with AF36 in 2012 for the first time and again in 2013, 2014, and every other year (2016, 2018, and 2020) until the end of the project in 2010 --- identify if application every other year will be sufficient; and 3, untreated control (never treated and will not be treated with AF36 --- to compare with the other two treatments). This study consists of five groups (replications) of three orchards that are adjacent or nearby each other (with each of the three orchards having one of the three treatments). The AF36 product was applied and will applied in treated areas at the label rate (10 lbs/acre) in June 2016, 2018, and 2020. Soil samples will be collected during the harvest period (September) in order to determine how successful the application of the AF36 product is in increasing the level of AF36 in the soil. The density of A. flavus/A. parasiticus in soil samples will be determined by plating soil on the surface of a selective isolation (SI) medium containing chloramphenicol and dichloran and then incubating at 30°C for 7 days as described earlier. Any colony of A. flavus and A. parasiticus will be isolated into pure culture to verify the species identification and to identify the strain. Identification of the AF36 strain will be done by vegetative compatibility group assays. Results of levels of AF36 in soil from subsequent applications (2013 and 2014) will be compared with results of levels of AF36 in soil from the every-other-year application of AF3. Objective 4. Aflatoxin contamination in the nuts from the above three treatments will be determined by obtaining the relevant library samples from a processor. Library samples consist of 20 pounds of fresh nuts taken at the processing plant as nuts are being unloaded and are used by the processor to determine how much growers will be paid. The library samples for the orchards involved in this study will be brought from the processor to the Kearney Agricultural Center, where we will combine library samples representing the same orchard and with the same harvest date (± 24 hours) to make a composite sample of dry nuts. These samples will be analyzed for aflatoxins with our HPLC at Kearney. Objective 5. The next generation of biocontrol formulations will carry at least three atoxigenic A. flavus strains from different VCGs to ensure additive effect. For this research, a fungal collection of 378 atoxigenic strains of A. flavus collected across California will be used. This collection has been characterized using molecular markers (SSR) and VCGs that have allowed identifying the dominant genetic/VCG group, considered the best adapted. Ten of atoxigenic A. flavus strains will be evaluated for competitiveness against one high AF-producing strain of A. parasiticus, which is easily distinguished from A. flavus. The competitive ability will be tested by co-inoculating pistachio and almond kernels in three ways: i) quantifying the proportion of spores produced by the two co-inoculants; ii) quantifying the proportions of respective DNA by a real-time PCR method developed at Kearney Ag Research & Extension center; and iii) measuring the reduction in AFs produced in the co-inoculated pistachio and almond kernels using the AflaTest® column-HPLC protocol. The treatments will be compared with kernels inoculated only with A. flavus as control. To ensure the safety of the promising strains, the mechanism by which AF production is inhibited will be determined. We will focus on the five most promising strains. Portions of genes from the AF biosynthetic pathway, including aflR, will be amplified by PCR and the sequences examined for defects that might result in atoxigenicity. For that, oligonucleotide primers sets will be used, which will be derived from the known pathway of aflatoxin biosynthetic genes. Primers pairs of the protein-coding regions will be designed for 13 genes (C1, C3, norB-cypA, aflT, pksA, aflR, norA, ver1, verA, omtA, ordA, hypA, and glcA), which are located at the telomeric region toward the adjacent sugar cluster of the AF gene cluster. Those isolates that yield amplicons will be sequenced to analyze for synonymous (silent) and non-synonymous (amino acid-altering) nucleotide substitutions in the AF biosynthesis pathway and sugar utilization cluster. This analysis will verify that the selected strains will be stable and safe to apply in the orchards and allow us selecting new BCAs from USA for their patents.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:California pistachio growers California pistachio processors Arizona pistachio growers Arizona pistachio processors Califonria Almond growers California Almond Processor Califonria fig growers California fig processors Changes/Problems:1. We will empasize ways to make the inoculum sporulate better than in previous studies in order to increase efficacy of the biological control agent. Apparently, in 2017 we noticed that if the inoculum was not applied properly, a major portion fell onto dry soil and it never had a chance to produce spores of the atoxigenic strain. Limited sporulation of the inoculum on the orchard floor leads to lower efficacy of the biological control agent in reducing aflatoxin contamination. 2. We added a study to determine how much inoculum reaches the tree canopy by trapping spores of the biological control agent at different heights within the tree canopy. 3. One more study was added in 2017 to determine spread of inoculum from the source to different distances from the source. All these changes (additions) in the original objectives of the project contribute in our better understanding of the spread of inoculum from the source. What opportunities for training and professional development has the project provided?Presented 3 talks to growers and pest control advisers to demonstrate how to apply the biocontrol agent in the field and pointed to them the link for a video exaplaining the various steps during application, adjustments of inoculum spreaders, and the challenges in the field for making the application more efficient. How have the results been disseminated to communities of interest?Presentations at University of California Cooperative Extension organized meetings. Presentations at metings organized by agricultural organizations. One to one meetings in my officeta with growers and farm managers responsible for applying the biopesticide in pistachio orchards. Provided power point presentations to industry administrators to develop prochures for the proper dissemination of the procedure's steps to almond growers. What do you plan to do during the next reporting period to accomplish the goals?Organize additional field meetings to demonstrate the proper dissemination of the biological control inoculum in pistachio, almond, and fig orchards to achieve an increase efficacy.

Impacts
What was accomplished under these goals? Aflatoxins are toxic metabolites produced by Aspergillus flavus and A. parasiticus in several crops including pistachio. Pistachio is occasionally contaminated with aflatoxin, but this poses a high risk to international trade due to strict regulations of aflatoxin contamination in food and feeds. Aflatoxin control in crops is difficult and the only reliable method of control is using atoxigenic (do not produce toxins) strains of A. flavus to displace or exclude the aflatoxin-producing strains of both A. flavus and A. parasiticus. Currently the only atoxigenic strain registered for use in pistachios in California is A. flavus AF36. Experimental applications of the atoxigenic biocontrol agent A. flavus AF36 to reduce aflatoxin contamination in pistachios in California started in 2002. The application of the AF36 product was successful in substantially increasing the proportion of the atoxigenic strain AF36 within the population of A. flavus/A. parasiticus fungi (Doster et al., 2014). In addition, the nuts from the orchards treated with the AF36 product did not have a higher incidence of kernel decay by A. flavus than nuts from untreated areas, suggesting that applying AF36 will not increase the number of moldy nuts. Nut samples from the orchards treated with the AF36 and the soil water content were periodically evaluated. Results indicate that sporulation was optimal where soil moisture (dry soil based) was between 13 and 18%. Conversely, AF36 sporulation was practically nonexistent in soil where there was excess (> 24% water content) or limited amount (<6% water content) of soil water content. Knowledge of the capacity of the biocontrol to move and disperse throughout the orchard is also critical to devise the best management practices. We study both the vertical and horizontal mobility of the fungus. Vertical dispersal was measured by spore density of the applied AF36 fungus at different heights above the sporulating product. Results indicate that A. flavus spores decreased exponentially as a function of height, while the density of A. niger spores increased. It is important to consider that A. niger does not produce aflatoxins, but some isolates can produce ochratoxins, another important concern for the nut industries in California. To determine the distance that the atoxigenic biocontrol fungus A. flavus AF36 is able to disperse from the source of inoculum in nut orchards, we applied the AF36 product on the soil around one tree in the center of an orchard. Preliminary results indicate that the fungus is able to move all directions and that the spore density of total Aspergillus flavus decreased exponentially with increased distance from the source. The impact of different arthropods as a potential cause of atoxigenic biocontrol product loss was evaluated under field conditions. Results indicate that in non-tilled soils, Oniscidea species (roly poly or pill bugs) and different ant species can feed on the applied sorghum grain in the soil. Conversely, the impact of these arthropods is minimal in frequently tilled soil. Additionally, we will continue to analyze pistachio library samples for aflatoxins and evaluate the effect of commercial application of AF36 Prevail. These samples originate from commercial blocks that have been part of AF36 biocontrol research efforts since 2008. Some of the blocks have been left untreated since the start of the project. Other blocks have been treated with 5 or 10 lbs. per acre of the product annually. Following harvest, nuts samples are obtained and analyzed for aflatoxin contamination. Currently the aflatoxin analysis for the 2017 season is underway and results are not still available. Also soil samples from these orchards were analyzed to determine the percentage of isolates of Aspergillus that belong to the applied biocontrol strain A. flavus AF36. Results for the 2017 season indicate that the soils treated with 10 lbs. per acre had significantly higher percentage (77.8%) compared to 5 lbs. per acre (58.4%) and the not treated control (23.7%).

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Picot, A., Doster, M., Islam, Md-S., Callicott, K., Ortega-Beltran, A., Cotty, P., and Michailides, T. J. Diistribution and incidence of atoxigenic Aspergillus flavus VCG in tree crop orchards in California: A strategy for identifying potential antagonists, the example of almonds. Interl. J. of Food Microbiol. 265:55-64.


Progress 10/01/15 to 09/30/16

Outputs
Target Audience:The target audience reached by our research efforts of this project include the California and Arizona Pistachio Industries, the California Almond industry, and the California Fig Industry. These industries include large and small acreage individuals of different background and ethnicity, and all these individuals were part of this audience. For instance, when presentations are made to various groups, our meetings to present this research are open to all. The specific audiences include: Almond growers and the Almond Board of California (ABC) Almond growers in Australia and Spain California pistachio growers The American Pistachio Growers Association (inlcuding pistachio growers in California, Arizona, and New Mexico) California Pistachio Research Board California Pistachio Processors Californ9ia fig grower California Fig Institute members Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A training demonstration of applying the AF36 in the field was done for PCAs and some growers. application. How have the results been disseminated to communities of interest?Presentation to UC ANR officials and invited growers from the central valley. Seminar at the Pistachio Day and the Annual Almond conference. Seminar at the UC/ and California Fig Institute Fig Day Seminars for organized meetings sponsored by various chemical companies. What do you plan to do during the next reporting period to accomplish the goals?Complete the registration of AF36 for use in almond and fig orchards and initiate experiments on determining its efficacy in reducing aflatoxin on a large scale under commercial application and conditions. Intensify studies to select other atoxigenic strains that can be used in mixtures of atoxigenics to be eventually registered and used in pistachio, almond and fig orchards in California. Another plan is to replace our ancient HPLC that gives us problems with frequent repairs and secure funding by the industries to get a new HPLC.

Impacts
What was accomplished under these goals? The use of the atoxigenic Aspergillus flavus strain AF36 (a strain not able to produce aflatoxins) as a biopesticide has been successful in reducing aflatoxin contamination of pistachio nuts in commercial pistachio orchards in California in addition to reducing contamination of cottonseed and corn. The AF36 product was registered by the US Environmental Protection Agency (EPA) in 2012 for application in commercial pistachio orchards in California. We are attempting to obtain registration with the EPA of the AF36 product for application in almond orchards. To accomplish this, we compared the AF36 product in an almond orchard with that in a pistachio orchard. In our study, we examined spore production on the AF product in both types of orchard, since the spores produced would spread the biocontrol agent throughout the orchard contributing to its effectiveness. We found that the spore production on the AF36 product was similar in almond and pistachio orchards. The manufacturer who produces the AF36 product has now started using sorghum instead of wheat as a carrier of the AF36 inoculum. Furthermore, a new process is being used to produce the AF36 product. This new process coats the seeds with spores of the AF36 fungus (instead of inoculating and incubating the seeds), resulting cheaper and faster production. In our study, the new seed-coated sorghum-AF36 product performed similarly to the wheat-AF36 product in the almond orchard, suggesting that the new AF36 product is an adequate replacement for the original product. General information. After applying the AF36 product in a research almond orchard, 98% of the A. flavus isolates from the soil in treated areas were the AF36 fungus (2.5% A. flavus naturally belonged to AF36 in this orchard prior to the AF36 application). Before applying the AF36 product, 92% of the isolates in the soil of a research almond orchard belonged to A. parasiticus or A. flavus S strain, both of which consistently produce high levels of aflatoxins. However, after applying the AF36 product, the frequency of A. parasiticus and A. flavus S strain in the fungal population decreased substantially and only 2% of the isolates were A. parasiticus or A. flavus strain S. The percentages of hulls decayed by A. flavus/A. parasiticus in treated areas in a research almond orchard were never significantly different from those for hulls from untreated areas. For comparison, the incidence of hull decay caused by A. niger was always substantially higher. Inoculation studies. AF36's was co-inoculated independently with highly toxigenic isolates of A. flavus and A. parasiticus on living almond kernels. Conducive conditions for aflatoxin formation were provided. Accumulation of aflatoxin B1 was reduced well over 95% in comparison to those levels accumulated on almonds inoculated only with the highly toxigenic isolates. Several other atoxigenic isolates were evaluated in these experiments. In all cases, AF36 was amongst the most effective isolates in reducing aflatoxin accumulation regardless of the toxigenic isolate used in the co-inoculations. This demonstrates AF36's superior ability in inhibiting aflatoxin formation on almond living tissues. Sorghum experiments. AF36's ability to reproduce was examined on the original wheat carrier and different sorghum sources varying in polyphenol content. A sorghum cultivar containing low phenol, no tannins allowed significantly greater sporulation in comparison to all the other cultivars tested, including the original wheat formulation. As polyphenol and tannin content increased on sorghum cultivars, less sporulation was produced by AF36. Selecting the appropriate sorghum cultivar for increasing the reproduction rates of AF36 is critical in order to provide the nutritive source for optimal fungal reproduction. However, it should be further investigated the effect of selecting a low phenol, no tannin containing sorghum cultivar on kernel decay after harvest. No health problems have been observed associated with applying AF36 in commercial pistachio orchards after 7 years of commercial application (including the EUP years 2008-2011), many of which are adjacent to almond orchards. In addition, we are not aware of any health problems associated with applying AF36 in research almond orchards. A package of this information has been submitted on time to the EPA and registration of Aspergillus flavus AF36 Prevail was approved on March 22, 2017 (EPA Reg. No. 71693-2) for almond and fig by expanding the pistachio label. We are expecting now approval by the California Department of Pesticide Regulations before applying it in commercial almond and fig orchards.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Picot A., A. Ortega-Beltran,R. D. Puckett, J. P. Siegel, and T. J. Michailides. Period of susceptibility of almonds to aflatoxin contamination. DOI 10.1007/s10658-016-1108-2


Progress 12/12/14 to 09/30/15

Outputs
Target Audience:The 6,800 California almond growers and the Almond Board of California. Almond growers in Australia and Spain Western Pistachio Association California pistachio growers & California Pistachio Research Board Pistachio growers in Mediterranean countries Pistachio growers worlwide California fig growers and California fig growers California fig growers of Mediterranean countries and California Dried Fig Association Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A major accomplishment is the finding that the application of AF36 in almond orchards acts in a manner similar to that found in pistachio orchards, information that can be used to get the AF36 strain registered on almond. Another positive achievement was that the sorghum-AF36 inoculum produces the same amounts of spores as the wheat-AF36 inoculum. Therefore the wheat inoculum can be replaced by the sorghum inoculum. How have the results been disseminated to communities of interest?a) Publications (popular and technical). b) Power point presentations in pistachio meetings (University of California & California Pistachio Research Board). c) Power point presentations in almond workgroup meetings (University of California & Almond Board of California) d) Power point presentation in the Fig Day (California Fig Institute) Day with referee What do you plan to do during the next reporting period to accomplish the goals?After the AF36 strain is registered on almond, we plan to a) monitor almonds from fields where the biocontrol strain is applied commercially to determine the effects on aflatoxin contamination of nuts; and b) determine when is the best time for application of the AF36 inoculum in the field. Also, we have collected a large number of atoxigenic strains; we plan to screen and select a few that perform well so that they can be used in mixtures of atoxigenic strains.

Impacts
What was accomplished under these goals? The atoxigenic strain AF36 of Aspergillus flavus works that id registered to reduce aflatoxin contamination in pistachio works in almond orchards in a manner similar to how it works in pistachio orchards. Almond crops are very similar to pistachio crops in many aspects, including production, management practices, environmental (climatic) requirements, and the aflatoxin contamination process. Most of the information concerning the application of atoxigenic A. flavus to limit aflatoxin contamination in pistachios is applicable to almond crops. Irrigation (amount, timing, and type) in almond orchards is similar to that in pistachio orchards. Spacings and canopy sizes of pistachio and almond trees are somewhat similar with almond orchards sometimes with trees slightly further apart and slightly larger. Herbicides used to control weeds, and cover crops are similar for both nut crops. Almond orchards are frequently adjacent to pistachio orchards, in some instances separated by just farm road, and the regions in California where almond trees are grown are also where pistachio trees are grown. The majority of fungicides utilized in pistachio crop to control fungal diseases are used also in almond crop. Extensive use of fungicides does not affect efficacy of AF36 in limiting aflatoxin accumulation in pistachio. Similar results have been seen with AF36 experimental treatments on almond. Sporulation on the AF36 product is similar in almond and pistachio orchards. The amount of visible sporulation by the atoxigenic strain on the AF36 product was very similar over time in a research almond orchard and a nearby research pistachio orchard. Applications of AF36 effectively increased the frequency of the atoxigenic strain AF36 in a research almond orchard. After applying the AF36 product in 2007 and 2008 in a research almond orchard, 98% of the A. flavus isolates from the soil in treated areas were the AF36 fungus (2.5% A. flavus naturally belonged to the vegetative group YV36 in this orchard prior to the AF36 application), which also shows similar effects after AF36 was applied in commercial pistachio orchards. This demonstrates displacement of the aflatoxin producers. This is the mechanism through which AF36 effects aflatoxin management and displacement observed in the almond orchard is very similar to that previously observed in pistachio orchards. Applications of AF36 in a research almond orchard successfully reduced the percentage of aflatoxin-producing strains within the fungal population. Before applying the AF36 product, 92% of the isolates in the soil of a research almond orchard belonged to either A. parasiticus or the A. flavus S strain, both of which consistently produce high levels of aflatoxins. However, after applying the AF36 product, the frequency of A. parasiticus and the A. flavus S strain in the fungal population steadily decreased to the point that only 2% of the isolates belonged to A. parasiticus or the A. flavus S strain. Furthermore, the application of AF36 in the treated areas resulted in increased levels in the non-treated areas. This suggests an area-wide spread. We found this both in the experimental almond orchard and in a commercial pistachio orchard where the untreated areas showed increased levels of AF36 after application in treated areas. Most importantly, the percentages of almond hulls decayed by A. flavus/A. parasiticus in treated areas in a research almond orchard were not significantly different from untreated areas; thus, the application of the atoxigenic AF36 strain did not increase the incidence of decay of almonds, results similar to those obtained with pistachios. The sorghum-AF36 product performed similarly to the wheat-AF36 product. The percentages of seeds of the AF36 products with visible sporulation of A. flavus was similar over time between the sorghum-AF36 product and the wheat-AF36 product after application of the products to the soil surface in a research almond orchard. In inoculations studies, when the AF36 was co-inoculated with highly toxigenic isolates of A. flavus and A. parasiticus on living almond kernels under conducive conditions for aflatoxin formation, the accumulation of aflatoxin B1 was reduced over 95% in comparison to those levels accumulated on almonds inoculated only with the highly toxigenic isolates. Several other atoxigenic isolates were evaluated in these experiments. In all cases, AF36 was among the most effective isolates in reducing aflatoxin accumulation regardless of the toxigenic isolate used in the co-inoculations studies. This demonstrates AF36's superior ability in inhibiting aflatoxin formation on living almond tissues.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Donner, M., Lichtemberg, P. S. F., Doster, M., Picot, A., Cotty, P. J., Puckett, R. D., and Michailides, T. J. Community structure of Aspergillus flavus and A. parasiticus in major almond-producing areas of California, United States. Plant Disease 99:1161-1169.