Progress 02/06/11 to 02/05/16
Outputs
Progress Report Objectives (from AD-416): The overall objective of this project is to develop commercially viable methods for the control of pathogens in tree nuts (almonds, pistachios and walnuts) and raisins which lead to foodborne illness in humans and animals. This includes both the control of fungal pathogens (mycotoxins) as well as bacterial pathogens such as E. coli and Salmonella. One approach will be the development of biocontrol agents using bacteria, yeast and non-toxigenic Aspergillus carbonarius. In addition, a better understanding of organic and conventional farming systems will provide new insights on mycotoxin control. The specific objectives for the period covered by this project plan are as follows: Objective 1: Define the critical control points for pathogen contamination during the production stream. Place particular emphasis on agricultural water sources including dairy waste water. Using both cultural and non-cultural based methods we will identify the points in the developmental process, as well as the processing cycle where tree nuts are most likely to become contaminated with human pathogenic bacteria and mycotoxigenic fungi. Objective 2: Evaluate the microbial ecology of organic v. conventional practices. Although several surveys have reported that consumers equate organically grown food stuffs with higher levels of food safety, little is known about how these practices affect the microbial population structure or mycotoxin levels in tree nuts. We will address the influence of phyllosphere microbial community on the population diversity of A. flavus in tree nut orchards, and A. carbonarius on grape surfaces in both farming systems. Objective 3: Delineate the factors affecting cross-contamination during processing and develop a potential intervention strategy during storage. A number of experiments point to a strong possibility that cross- contamination of toxigenic fungi is possible during processing and storage, although no research appears to have been done on the transfer in actual (not laboratory) processing and storage conditions. Development of novel approaches to prevent the growth of storage fungi and production of harmful toxins is a high priority in the almond industry. Edible films and coatings (EFC) containing antimicrobial natural compounds will be tested for their efficiency to reduce mycotoxin in stored almonds. Objective 4: Develop biological-control/intervention technologies using competitive or antagonistic microorganisms such as yeasts or bacteria that can be mass-produced and effectively utilized in a variety of pre- or post-harvest environments. Bacterial and yeast biocontrol agents will be tested in almond orchards and vineyards to control A. flavus and A. carbonarius, as well as human pathogenic bacteria, E. coli and Salmonella. EPA registration of the patented yeast, Pichia anomala will be pursued for commercial application. Methods to enhance the biocontrol efficacy will be developed. Approach (from AD-416): Develop methods to control insect pests and toxic fungi of tree nuts. Insects include naval orangeworm, codling moth and peach twig borer. Feeding damage by these insects leads to infection by aflatoxigenic aspergilli. Control methods for insects are to be environmentally benign and employ semiochemicals to disrupt insect behavior. Control of toxic fungi focuses on biological control using competitive or antagonistic microorganisms. These microorganisms include either yeasts or bacteria that can be mass-produced and effectively utilized in a variety of pre- or post harvest environments. This is the final report for the project 2030-42000-038-00D, which terminated in February 2016. Research continues under project 2030-42000- 039-00D, "Biocontrol Interventions for High-Value Agricultural Commodities." Substantial results were realized over the five years of the project. Previously it was thought that almonds become contaminated by Salmonella enterica after they are shaken from trees and are stored in windrows on the orchard floor. However by monitoring the microbial populations on almond drupes, the critical point of infection was identified as right after hull split. This information is being used as the basis for the development of biological control strategies to prevent contamination of almond drupes while they are still in trees. In a second project, ARS scientists examined the bacterial population structure of almond drupes from three organic orchards and three conventional orchards that were located within five miles from each other. The organically grown drupes contained significantly more bacteria in the Class Alphaproteobacteria and the conventionally grown drupes contained significantly more bacteria within the class Gammaproteobacteria and the phylum Actinobacteria. We also observed Salmonella enterica, a member of the Gammaproteobacteria on the almond dupes from one of the conventionally grown orchards but not from any of the organically grown almonds. This was the first study to show that orchard management practices may have an effect on the types of bacteria that are on the surfaces of almonds. Regarding fungal population on organic and conventionally grown raisins, the majority of isolates belonged to four Aspergillus species: A. niger, A. welwitschiae, A. tubingensis, and A. carbonarius. Of these species, the only isolates that produced ochratoxin A were A. carbonarius, which were isolated less frequently than the other species. In contrast, A. niger and A. welwitschiae are much more abundant, and the majority of these isolates produce fumonisin B2. Initial analyses showed no significant differences in fungal populations or mycotoxin production rates between conventional and organic vineyards. Ochratoxin and fumonisin contamination in almonds was examined. Rejected almond samples were analyzed to determine whether indigenous black-spored Aspergillus species could contaminate almonds with mycotoxins in addition to aflatoxin. The major species recovered was A. tubingensis, which does not produce ochratoxin or fumonisin. Fumonisin was produced by 72% of the isolated strains of A. niger and A. welwitschiae, but no ochratoxin- producing fungi were isolated. Almonds contained no detectable fumonisin, but three of 21 samples contained low levels of ochratoxin. These results indicate that properly dried and stored almonds are not susceptible to fumonisin contamination, and that ochratoxin contamination is sporadic, and possibly caused by other Aspergillus species. Genetic analysis of fumonisin-nonproducing Aspergillus niger and Aspergillus welwitschiae strains showed that in A. welwitschiae strains, a large section of the fumonisin biosynthetic gene cluster is deleted, resulting in loss of fumonisin production. In A. niger, fumonisin- nonproducing strains contain the entire biosynthetic gene cluster, indicating that fumonisin nonproduction is due to other genetic factors. These differences in mechanism of fumoninsin nonproduction may be useful in distinguishing these two very closely related fungal species. Species-specific PCR primer sets were developed for detection and differentiation of the four major Aspergillus species based on DNA sequence differences in the calmodulin gene. These primers can be used to detect these species in soil containing mixtures of Aspergillus species. This analysis will allow faster throughput of field samples to detect the presence of ochratoxin- and fumonisin-producing species. Quantitative droplet digital PCR methods were developed to measure relative population sizes of the four major Aspergillus species in vineyard soil and on fruit, beginning at flowering and fruit set, continuing through fruit development and ripening, and ultimately to grape harvesting and drying. This analysis will determine points at which interventions may be useful for mycotoxin control. Bacterial strains that produce antibiotic compounds and fungal cell wall- degrading enzymes were shown to inhibit growth and sporulation of A. flavus in soil. Additionally, bacteria that inhibit growth of A. carbonarius have been isolated from vineyard soils, and screened for antifungal properties in coculture experiments. Small-scale field trials to determine the efficacy of these candidate bacterial biocontrol agents on corn for reducing aflatoxin contamination showed that the bacterial strains that performed best in laboratory assays did not produce measurable reduction of aflatoxin on corn. Therefore, large-scale field trials were not initiated. Methods of application of biocontrol agents (to soil, for example), their effects on fungal populations in soil and on fruit, and their impact on total mycotoxins in fruit are currently being evaluated. Aflatoxin cross-contamination from nut to nut during processing of shelled almonds and potential transfer of aflatoxin to processing equipment was monitored. The results indicated an approximate 16% transfer of aflatoxin to the clean almonds. The amount of aflatoxin correlated strongly with the number of hot nuts in each sample, as expected. It was found that very little aflatoxin transferred to the equipment. Experiments were conducted to evaluate films containing natural antimicrobial essential oils as an alternative approach for external protection of almonds. By applying the film on the lid facing inside of the container during transport and storage, the vapor can reduce surface microbial populations on the nuts. Usually fungal growth in storage bin starts near the lid due to moisture condensation. The most effective film is tomato film with 3% oregano oil. Tests on agar plates showed both aflatoxin biosynthesis and fungal growth were inhibited. Raw almond, stored in jars with oregano film on the lid showed no visible fungal growth for six months in contrast with the control without the film. The research will be continued as a collaborative effort with investigators at University of California, Davis. DNA sequences of ribosomal intergenic spacer 1 (IGS1) region of the species Pichia anomala were analyzed. Strains from agricultural fields including P. anomala WRL-076 and pathogenic strains from the Center for Disease Control were used for the sequence analysis. Primers of IGS1 region were designed and used for PCR reactions. DNA fragments were sequenced. Phylogenic analysis based on ribosomal IGS1 nucleotide sequences clearly demonstrated that the clinical strains and agricultural ones were separated into two clusters. Growth media and formulations were designed to prolong the shelf life of Pichia anomala WRL076. Intracellular concentrations of sorbitol and trehalose synergistically protect the yeast cells to a survival rate of 83% after storage for 12 months. A patent was issued for the invention. The patented yeast, Pichia anomala WRL076 and formulation invention were licensed to Verdesian Life Sciences LLC as a biological control system. Biological control provides a good alternative to the use of fungicides in agriculture. Biological control can reduce the harmful effect of phytopathogenic or mycotoxigenic fungi while having a minimal impact on the environment. Commercial products of Pichia anomala are needed for the control of toxigenic aspergilli to eliminate mycotoxin contamination on food. The overall impact of the accomplishments provide almond, pistachio, raisin producers and industries with new information on which to make decisions concerning production systems to maximize profits while sustaining food safety. Accomplishments 01 Phyllosphere associated lactic acid bacteria as biocontrol agents. ARS scientists in Albany, California identified two naturally occurring lactic acid bacteria that are capable of growing and persisting on the rinds of cantaloupe melons. Once on the melons, these bacteria reduce the ability of Salmonella enterica to grow on the surfaces of the melons by over 40-fold. Furthermore, the addition of these bacteria to the surfaces of the melons did not result in rot or any other undesirable characteristic (i.e. smell, off color, etc.). A patent of these bacteria is being pursued and a search is underway for a partner to license the technology. 02 Quantification of mycotoxigenic Aspergillus species by digital droplet polymerase chain reaction (ddPCR). Several species of black-spored Aspergillus fungi, in addition to causing food spoilage, have the potential to produce toxic compounds, including ochratoxin and fumonisin. ARS researchers in Albany, California, previously developed calmodulin gene-based PCR methods to detect four of these Aspergillus species. They used those discoveries to develop ddPCR methods to measure the population sizes of these species on grapes and in soil during the grape growing season. This provides a rapid, powerful way to measure the development of mycotoxin-producing species from field samples, in order to predict when control measures should be used, and show whether those measures are effective at reducing mycotoxin- producing fungi on at-risk foods. This research has given growers and packers another tool to help prevent human illnesses caused by mycotoxins.
Impacts
(N/A)
Publications
- Mcgarvey, J.A., Hnasko, R.M., Stanker, L.H., Han, R., Connel, J. 2015. Bacterial populations on the surfaces of organic and conventionally grown almond drupes. Journal of Applied Microbiology. 119:529-538.
- Babrak, L.M., Lin, A.V., Stanker, L.H., McGarvey, J.A., Hnasko, R.M. 2016. Rapid microfluidic assay for the detection of botulinum neurotoxin in animal sera. Toxins. 8(1):13.
- Chang, P., Hua, S.T., Sarreal, S.L., Li, R.W. 2015. Suppression of aflatoxin biosynthesis in Aspergillus flavus by 2-phenylethanol is associated with stimulated growth and decreased degradation of branched- chain amino acids. Toxins. 7:3887-3902. doi: 10.3390/toxins7103887.
- Palumbo, J.D., O Keeffe, T.L., Ho, Y.S., Fidelibus, M.W. 2016. Population dynamics of Aspergillus section Nigri species on vineyard samples of grapes and raisins. Journal of Food Protection. 79:448-453. doi: 10.4315/ 0362-028X.JFP-15-437.
- Hnasko, R.M., McGarvey, J.A. 2015. Affinity purification of antibodies. In: Hnasko, R., editor. ELISA Methods and Protocols. New York, NY: Humana Press. p. 29-41. doi: 10.1007/978-1-4939-2742-5.
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Progress 10/01/14 to 09/30/15
Outputs
Progress Report Objectives (from AD-416): The overall objective of this project is to develop commercially viable methods for the control of pathogens in tree nuts (almonds, pistachios and walnuts) and raisins which lead to foodborne illness in humans and animals. This includes both the control of fungal pathogens (mycotoxins) as well as bacterial pathogens such as E. coli and Salmonella. One approach will be the development of biocontrol agents using bacteria, yeast and non-toxigenic Aspergillus carbonarius. In addition, a better understanding of organic and conventional farming systems will provide new insights on mycotoxin control. The specific objectives for the period covered by this project plan are as follows: Objective 1: Define the critical control points for pathogen contamination during the production stream. Place particular emphasis on agricultural water sources including dairy waste water. Using both cultural and non-cultural based methods we will identify the points in the developmental process, as well as the processing cycle where tree nuts are most likely to become contaminated with human pathogenic bacteria and mycotoxigenic fungi. Objective 2: Evaluate the microbial ecology of organic v. conventional practices. Although several surveys have reported that consumers equate organically grown food stuffs with higher levels of food safety, little is known about how these practices affect the microbial population structure or mycotoxin levels in tree nuts. We will address the influence of phyllosphere microbial community on the population diversity of A. flavus in tree nut orchards, and A. carbonarius on grape surfaces in both farming systems. Objective 3: Delineate the factors affecting cross-contamination during processing and develop a potential intervention strategy during storage. A number of experiments point to a strong possibility that cross- contamination of toxigenic fungi is possible during processing and storage, although no research appears to have been done on the transfer in actual (not laboratory) processing and storage conditions. Development of novel approaches to prevent the growth of storage fungi and production of harmful toxins is a high priority in the almond industry. Edible films and coatings (EFC) containing antimicrobial natural compounds will be tested for their efficiency to reduce mycotoxin in stored almonds. Objective 4: Develop biological-control/intervention technologies using competitive or antagonistic microorganisms such as yeasts or bacteria that can be mass-produced and effectively utilized in a variety of pre- or post-harvest environments. Bacterial and yeast biocontrol agents will be tested in almond orchards and vineyards to control A. flavus and A. carbonarius, as well as human pathogenic bacteria, E. coli and Salmonella. EPA registration of the patented yeast, Pichia anomala will be pursued for commercial application. Methods to enhance the biocontrol efficacy will be developed. Approach (from AD-416): Develop methods to control insect pests and toxic fungi of tree nuts. Insects include naval orangeworm, codling moth and peach twig borer. Feeding damage by these insects leads to infection by aflatoxigenic aspergilli. Control methods for insects are to be environmentally benign and employ semiochemicals to disrupt insect behavior. Control of toxic fungi focuses on biological control using competitive or antagonistic microorganisms. These microorganisms include either yeasts or bacteria that can be mass-produced and effectively utilized in a variety of pre- or post harvest environments. We have performed extensive microbial analysis of the bacterial population structure of almond drupes grown under organic and conventional growing methodologies. We found that these growing practices do alter the microbial populations on the almond drupes. Specifically we found that the conventionally grown drupes contained higher levels of Gammaproteobacteria, which include members of the genus Salmonella. Of the 3 organically and 3 conventionally farmed orchards we examined, we only observed two 16S rRNA gene clones associated with the genus Salmonella in one of the conventionally grown orchards during hull split. This is consistent with our previous findings in 2014. We have published these results in the Journal of Applied Microbiology. In 2015, we also set up a randomized plot experiment in a working almond orchard and have fertilized some plots with manure, some with green waste, and others with only synthetic fertilizers. We are currently analyzing these samples. PCR primers and probes were developed to distinguish between four species of Aspergillus present in grapes and vineyard soil. Using droplet digital PCR, relative population sizes for each species has been determined for fruit and soil samples from berry set, fruit ripening, grape harvest, and raisin drying from two raisin vineyards in Fresno, California, during two consecutive growing seasons. Comparisons of quantitative PCR results and culture-based population analysis are underway, and manuscripts resulting from this work are being prepared for submission. In order to determine whether ochratoxin-nonproducing strains of Aspergillus carbonarius could be used in biological control via competitive displacement of toxin-producing populations, A. carbonarius strains are being collected from raisin vineyard soils. Analysis of collected strains for ochratoxin production is ongoing, and in-vitro and soil microcosm assays will be designed using toxin-nonproducing strains and toxin-producing strains from the same vineyards to determine the extent of strain displacement and ochratoxin reduction following co- inoculation. Varying volatiles were studied for inhibitory activity to prevent growth of Aspergillus flavus and aflatoxin production. These volatiles are being investigated for their potential use in post-harvest storage for improvement of food safety and quality of high valued crops, including almonds. Several hundred yeast isolates collected from California orchards were screened for finding potential yeast strains to use for biocontrol of Aspergillus flavus. A colored visual bioassay was used to identify yeast species, which can simultaneously inhibit aflatoxin and fungal growth. Multiplex PCR Primers were designed to screen natural variants in Aspergillus flavus population, deficient in aflatoxin biosynthesis and cyclopiazonic acid biosynthesis. The multiples primers were used to score for deletion in aflatoxin and cyclopiazonic acid biosynthesitic gene cluster. These variants are potential strains for developing next generation biocontrol agents so as to displace toxigenic A. flavus in tree nut orchards. For a better understanding of the biocontrol mechanisms of the patented yeast, Pichia anomala, total RNA-Seq protocol was applied to define yeast genes expressed for controlling A. flavus. Analysis of the big data is currently underway to discover new genes important in biocontrol efficacy. Accomplishments 01 Determination of ochratoxin contamination in dried fruits and nuts. Ochratoxins are produced by several fungal species, and are sometimes found as toxic contaminants in a wide variety of foods, including dried fruits and tree nuts. The purpose of the study was to provide information regarding ochratoxin contamination in foods in the United States, so that health risks may be assessed. ARS researchers in Albany, California, in collaboration with researchers at the University of Idaho, Michigan State University, University of Nebraska, North Dakota State University, and Illinois Institute of Technology collected samples of dried fruits (raisins, dates, figs, prunes) and tree nuts (almonds, pistachios, walnuts) from retail sources. Analysis of samples collected over two years showed ochratoxin contamination in 44% of raisin samples, dates, figs and pistachios were sporadically contaminated with ochratoxin, and no ochratoxin was found in almonds, walnuts and prunes. Two raisin samples exceeded the European Union regulatory limit for ochratoxin of 10 parts per billion, indicating the need to develop monitoring and control strategies to limit frequencies and levels of ochratoxin contamination, particularly in raisins. In addition, these results are contributing to ongoing risk assessment for ochratoxin exposure in dried fruits and nuts (especially raisins), both for domestic consumption and for export. 02 Culture-independent detection of mycotoxigenic Aspergillus species by polymerase chain reaction (PCR). Several species of black-spored Aspergillus fungi, in addition to causing food spoilage, have the potential to produce toxic compounds, including ochratoxin and fumonisin. ARS researchers in Albany, California, developed PCR primer sets to specifically amplify a target fragment of calmodulin gene from each of four closely-related black-spored Aspergillus species. These species are difficult to differentiate visually and by microscopic comparisons, and are typically identified by DNA sequence analysis of the calmodulin gene and other genes. Using the newly developed PCR- based identification is equally accurate and faster, and can be used to detect each of these species in mixed fungal populations. This provides new strategies for monitoring of ochratoxin- and fumonisin-producing Aspergillus species in at-risk environments and foods. 03 New yeast strain discovered for biocontrol of Aspergillus flavus. Aflatoxin contamination, produced by the fungus Aspergillus flavus, remains a serious food safety problem. A. flavus has a broad ecological niche and reproduces with abundant and genetically diverse populations. One biocontrol strategy to inhibit toxigenic A. flavus growth, and thus reduce the chance of aflatoxin contamination, is to identify and use populations of innocuous and currently present strains of microorganisms to competitively displace A. flavus. An ARS scientist in Albany, California, recently discovered a second yeast species, Candida railenensis that in laboratory studies reduced aflatoxin biosynthesis by 96%, and inhibited the growth of A. flavus; a similar result as the previously identified Pichia anomala. Both yeasts can be mass-produced as a biocontrol agent for application in orchards where A. flavus exists. 04 Trehalose and sorbitol synergistically promote Pichia anomala yeast cell viability. An important requirement for the use of biocontrol agents is the production of large quantities of the microorganisms and the development of formulations to prolong the shelf-life of the organisms. Maintaining the viability of biocontrol agents in formulated products is a great challenge. A stable liquid formulation is highly desirable for P. anomala WRL-076 because the biocontrol yeast products can be thereby easily dispersed in water and delivered by spraying or dipping to the agricultural and horticultural crops and commodities. ARS scientists in Albany, California, demonstrated that intracellular and trehalose and sorbitol enhanced the cell viability. Trehalose has a remarkable capacity to protect cells and their component macromolecules against an array of environmental stresses. Sorbitol in the growth media increased intracellular accumulation of the sugar to amplify the effect of trehalose on protecting yeast cells. 05 Molasses inhibiting aflatoxin biosynthesis. Cane molasses is a thick syrup obtained as a byproduct of the manufacture or refining of sucrose from sugar cane. More than 35 million metric tons of molasses are produced annually worldwide. ARS scientists in Albany, California, demonstrated that when Aspergillus flavus was cultured in 4, 8 or 12% molasses media, aflatoxin was not detected in the fugal culture by HPLC (high performance liquid chromatography) analysis. Total RNA was extracted from fungal cultures for gene expression studies. The majority of genes in the aflatoxin biosynthetic pathway were silent and not expressed. The finding suggests the potential of new application of molasses in food and feed products in aflatoxin control.
Impacts
(N/A)
Publications
- Hua, S.T., Hernlem, B.J., Yokoyama, W.H., Sarreal, S.L. 2015. Intracellular trehalose and sorbitol synergistically promoting cell viability of a biocontrol yeast Pichia anomala for aflatoxin reduction. World Journal of Microbiology and Biotechnology. doi: 10.1007/s11274-015- 1824-3..
- Hua, S.T., Beck, J.J., Sarreal, S.L., Gee, W.S. 2014. The major volatile compound 2-phenylethanol from the biocontrol yeast Pichia anomala inhibits growth and expression of aflatoxin biosynthetic genes of Aspergillus flavus. Mycotoxin Research. 30(2):71-78. doi: 10.1007/s12550-014-0189-Z.
- Chang, P.-K., Scharfenstein, L.L., Solorzano, C.D., Abbas, H.K., Hua, S.S., Jones, W.A., Zablotowicz, R.M. 2015. High sequence variations in the region containing genes encoding a cellular morphogenesis protein and the repressor of sexual development help to reveal origins of Aspergillus oryzae. International Journal of Food Microbiology. 200:66-71.
- Crespo-Sempere, A., Selma-Lazaro, C., Palumbo, J.D., Gonzalez-Candelas, L., Martinez-Culebras, P.V. 2015. Effect of oxidant stressors and phenolic antioxidants on the ochratoxigenic fungus aspergillus carbonarius. Journal of the Science of Food and Agriculture. doi: 10.1002/JSFA.7077.
- Palumbo, J.D., O Keeffe, T.L., Ho, Y.S., Santillan, C.J. 2015. Occurrence of ochratoxin a contamination and detection of ochratoxigenic aspergillus species in retail samples of dried fruits and nuts. Journal of Food Protection. 78(4):836-842. doi: 10.4315/0362-028X.JFP-14-471.
- Palumbo, J.D., O Keeffe, T.L. 2015. Detection and discrimination of four aspergillus section nigri species by pcr. Letters in Applied Microbiology. 60(2):188-195. doi: 10.1111/LAM.12358.
- Palumbo, J.D., Mahoney, N.E., Light, D.M., Siegel, J.P., Ryan, P., Themis, M. 2014. Spread of Aspergillus flavus by navel orangeworm (Amyelois transitella) on almonds. Plant Disease. 98(9):1194-1199.
- Ching, K.H., He, X., Stanker, L.H., Lin, A.V., Mcgarvey, J.A., Hnasko, R.M. 2015. Detection of shiga toxins by lateral flow assay. Toxins. 7:1163- 1173. doi: 10.3390/Toxins7041163.
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Progress 10/01/13 to 09/30/14
Outputs
Progress Report Objectives (from AD-416): The overall objective of this project is to develop commercially viable methods for the control of pathogens in tree nuts (almonds, pistachios and walnuts) and raisins which lead to foodborne illness in humans and animals. This includes both the control of fungal pathogens (mycotoxins) as well as bacterial pathogens such as E. coli and Salmonella. One approach will be the development of biocontrol agents using bacteria, yeast and non-toxigenic Aspergillus carbonarius. In addition, a better understanding of organic and conventional farming systems will provide new insights on mycotoxin control. The specific objectives for the period covered by this project plan are as follows: Objective 1: Define the critical control points for pathogen contamination during the production stream. Place particular emphasis on agricultural water sources including dairy waste water. Using both cultural and non-cultural based methods we will identify the points in the developmental process, as well as the processing cycle where tree nuts are most likely to become contaminated with human pathogenic bacteria and mycotoxigenic fungi. Objective 2: Evaluate the microbial ecology of organic v. conventional practices. Although several surveys have reported that consumers equate organically grown food stuffs with higher levels of food safety, little is known about how these practices affect the microbial population structure or mycotoxin levels in tree nuts. We will address the influence of phyllosphere microbial community on the population diversity of A. flavus in tree nut orchards, and A. carbonarius on grape surfaces in both farming systems. Objective 3: Delineate the factors affecting cross-contamination during processing and develop a potential intervention strategy during storage. A number of experiments point to a strong possibility that cross- contamination of toxigenic fungi is possible during processing and storage, although no research appears to have been done on the transfer in actual (not laboratory) processing and storage conditions. Development of novel approaches to prevent the growth of storage fungi and production of harmful toxins is a high priority in the almond industry. Edible films and coatings (EFC) containing antimicrobial natural compounds will be tested for their efficiency to reduce mycotoxin in stored almonds. Objective 4: Develop biological-control/intervention technologies using competitive or antagonistic microorganisms such as yeasts or bacteria that can be mass-produced and effectively utilized in a variety of pre- or post-harvest environments. Bacterial and yeast biocontrol agents will be tested in almond orchards and vineyards to control A. flavus and A. carbonarius, as well as human pathogenic bacteria, E. coli and Salmonella. EPA registration of the patented yeast, Pichia anomala will be pursued for commercial application. Methods to enhance the biocontrol efficacy will be developed. Approach (from AD-416): Develop methods to control insect pests and toxic fungi of tree nuts. Insects include naval orangeworm, codling moth and peach twig borer. Feeding damage by these insects leads to infection by aflatoxigenic aspergilli. Control methods for insects are to be environmentally benign and employ semiochemicals to disrupt insect behavior. Control of toxic fungi focuses on biological control using competitive or antagonistic microorganisms. These microorganisms include either yeasts or bacteria that can be mass-produced and effectively utilized in a variety of pre- or post harvest environments. Extensive microbial analyses were performed to determine the bacterial population structure of almond drupes and the microbial population dynamics that occur during their development. The analysis also indicated that almond drupe split was the critical control point for drupes contamination by Salmonella enterica. The results of these studies have been published in the Journal of Applied Microbiology. The research will be continued to include cantaloupe melons which are also often contaminated by Salmonella enterica. Research proposed for sub objective 3.1 has been completed and a manuscript is being prepared for submission. For objective 2, research to develop species-specific PCR primer sets for detection and differentiation of four Aspergillus species is continued from previous year. These primers have been used to detect fungal species in soil inoculated with mixtures of Aspergillus species. Primer sets have been adapted for use in quantitative PCR assays to determine the relative amounts of each of the four species in environmental samples. Grape, raisin and soil samples have been collected for two consecutive growing seasons at the stages of berry set, fruit ripening, grape harvest, and raisin drying, from two commercial vineyards in Fresno, California. Quantitative PCR assays will be used to determine variations in Aspergillus species populations during the growing season, and between consecutive growing seasons. DNA sequence information of aflatoxin biosynthetic genes of the Pistachio Wounding Experiment (PWE) strains is applied for identifying potential new aflatoxigenic species. Both Aspergillus flavus Link and A. parasiticus Speare cause aflatoxin contamination in crops such as corn, cottonseed, peanuts and tree-nuts. These two fungal species are saprophytes which infect plants through woundings. A. parasiticus strains produce aflatoxins B1, B2, G1 and G2. The lack of aflatoxin-producing ability of domesticated A. sojae isolates is due to primarily from an early termination point mutation in the pathway-specific AflR regulatory gene. Fungal strains isolated from pistachio orchard were designated as PWE strains in our lab collections. The analysis indicated that new aflatoxingenic strains were present in the orchard environment. Towards objective 4, a strain of A. carbonarius that does not produce detectable amounts of ochratoxin has been isolated from packaged raisins. This strain and strains of fumonisin-nonproducing A. niger and A. welwitschiae are being used to develop soil and fruit microcosm assays to examine their potential use as biocompetitive agents for control of mycotoxins on grapes. DNA sequences of ribosomal intergenic spacer 1 (IGS1) region of Pichia anomala (P. anomala) have been analyzed. Strains are from agricultural fields including P. anomala WRL-076 and pathogenic strains from the Center for Disease Control. DNA extraction of yeast cells were carried out by treating the yeast cells with 5% Chelex solution. Primers of IGS1 region were designed and used for PCR reactions. DNA fragments were sequenced. Phylogenic analysis based on ribosomal IGS1 nucleotide sequences clearly demonstrated that the clinical strains and agricultural ones were separated into two clusters. Accomplishments 01 Transfer of aflatoxin during almond processing. It was previously unknown whether aflatoxin is transferred between nuts during almond processing, either directly from nut to nut or indirectly through contamination of processing equipment. ARS scientists in Albany, California, introduced known quantities of aflatoxin to uncontaminated almonds in a simulated processing environment, and subsequent distribution of the aflatoxin was monitored. It was found that substantial cross contamination can occur through both paths. These results directly impact processing methods and imply a need for equipment decontamination at regular intervals. 02 Spread of Aspergillus flavus on almonds by navel orangeworm. Navel orangeworm larvae were shown to transport A. flavus spores to uncontaminated almonds under laboratory conditions. Feeding on almond kernels by A. flavus-carrying larvae was strongly correlated with aflatoxin contamination of the almonds. ARS researchers in Albany, California, collected adult navel orangeworm moths from almond orchards during the growing season, and from almond mummies following overwintering. A significant incidence of A. flavus contamination was evident in the insects. These results indicate that navel orangeworm larvae and adults are likely to be vectors of A. flavus on almonds, leading to aflatoxin contamination of the crop. This reinforces the need for measures to control insect infestation and to monitor and sort almonds for insect damage, in order to reduce aflatoxin contamination of the product. 03 Volatile compound from biocontrol yeast, Pichia anomola WRL-076. Plants and microbes are able to produce volatile compounds that act as a defense mechanism against other organisms. ARS scientists in Albany, California, identified 2-phenylethanol (2- PE) as the major volatile compound produced by Pichia anomola WRL-076. The 2-PE inhibited spore germination and aflatoxin production of A. flavus. Inhibition of aflatoxin formation by 2-PE was correlated with the significant down regulation of aflatoxin biosynthesis genes. This research validates the use of Pichia anomala WRL-076 to reduce aflatoxin contamination of tree nuts and other affected crops. 04 Molecular marker differentiates beneficial Pichia anomola WRL-076 from clinical pathogenic strains. A safety assessment of WRL-076 is essential for its application as a biocontrol agent and is required for Environmental Protection Agency registration in the U.S. ARS scientists in Albany, California, sequenced the ribosomal DNA intergenic spacer of P. anomala isolates and determined that it is possible to differentiate beneficial P. anomala strains from clinical pathogenic ones. In addition, growth temperature studies showed that WRL-076 and P. anomala isolates from plants did not grow at 37 �C or 40�C in nutrient broth. This suggests the yeast will not proliferate in humans and animals with body temperatures of 37�C and above. This research showed that the use and spread of WRL-076 as a biocontrol agent can be easily monitored and is safe to humans and animals. 05 New strains of Aspergillus section Flavi isolated. Molecular genetic data indicate that fungal strains from pistachio nut fruits share certain genetic sequences with both A. parasiticus and A. sojae. These pistachio nut fruit fungal strains produce aflatoxins B1, B2, G1 and G2 and could potentially contaminate food crops. ARS scientists in Albany, California, performed genomic analyses and built phylogenetic trees to illustrate the differences among the strains from pistachio nut fruits, A. parasiticus, and A. soja. Documenting the evolutionary changes of these different pathogenic fungi species will allow scientists to predict future strains that may become problematic to food crops.
Impacts
(N/A)
Publications
- Hua, S.T. 2013. Biocontrol of Aspergillus flavus by Pichia anomala. In: Mendez-Vilas, A., editor. Microbial Pathogens and Strategies for Combating Them: Science Technology and Education. Vol. 2. Spain. Formatex Research Center. p 1067-1072.
- Peretto, G., Du, W., Avena Bustillos, R.D., Sarreal, S.L., Hua, S.T., Sambo, P., McHugh, T.H. 2013. Increasing strawberry shelf-life with carvacrol and methyl cinnamate antimicrobial vapors released from edible films. Postharvest Biology and Technology. DOI: 10.1016/j.postharvbio.2013. 11.003.
- McGarvey, J.A., Connell, J., Stanker, L.H., Hnasko, R.M. 2014. Bacterial population structure and dynamics during the development of almond drupes. Journal of Applied Microbiology. 116:1542-1552.
- McGarvey, J.A., Franco, R., Palumbo, J.D., Hnasko, R.M., Stanker, L.H., Mitloehner, F. 2013. Bacterial population dynamics during the ensiling of Medicago sativa (alfalfa) and subsequent exposure to air. Journal of Applied Microbiology. 114:1661-1670.
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Progress 10/01/12 to 09/30/13
Outputs
Progress Report Objectives (from AD-416): The overall objective of this project is to develop commercially viable methods for the control of pathogens in tree nuts (almonds, pistachios and walnuts) and raisins which lead to foodborne illness in humans and animals. This includes both the control of fungal pathogens (mycotoxins) as well as bacterial pathogens such as E. coli and Salmonella. One approach will be the development of biocontrol agents using bacteria, yeast and non-toxigenic Aspergillus carbonarius. In addition, a better understanding of organic and conventional farming systems will provide new insights on mycotoxin control. The specific objectives for the period covered by this project plan are as follows: Objective 1: Define the critical control points for pathogen contamination during the production stream. Place particular emphasis on agricultural water sources including dairy waste water. Using both cultural and non-cultural based methods we will identify the points in the developmental process, as well as the processing cycle where tree nuts are most likely to become contaminated with human pathogenic bacteria and mycotoxigenic fungi. Objective 2: Evaluate the microbial ecology of organic v. conventional practices. Although several surveys have reported that consumers equate organically grown food stuffs with higher levels of food safety, little is known about how these practices affect the microbial population structure or mycotoxin levels in tree nuts. We will address the influence of phyllosphere microbial community on the population diversity of A. flavus in tree nut orchards, and A. carbonarius on grape surfaces in both farming systems. Objective 3: Delineate the factors affecting cross-contamination during processing and develop a potential intervention strategy during storage. A number of experiments point to a strong possibility that cross- contamination of toxigenic fungi is possible during processing and storage, although no research appears to have been done on the transfer in actual (not laboratory) processing and storage conditions. Development of novel approaches to prevent the growth of storage fungi and production of harmful toxins is a high priority in the almond industry. Edible films and coatings (EFC) containing antimicrobial natural compounds will be tested for their efficiency to reduce mycotoxin in stored almonds. Objective 4: Develop biological-control/intervention technologies using competitive or antagonistic microorganisms such as yeasts or bacteria that can be mass-produced and effectively utilized in a variety of pre- or post-harvest environments. Bacterial and yeast biocontrol agents will be tested in almond orchards and vineyards to control A. flavus and A. carbonarius, as well as human pathogenic bacteria, E. coli and Salmonella. EPA registration of the patented yeast, Pichia anomala will be pursued for commercial application. Methods to enhance the biocontrol efficacy will be developed. Approach (from AD-416): Develop mentods to control insect pests and toxic fungi of tree nuts. Insects include naval orangeworm, codling moth and peach twig borer. Feeding damage by these insects leads to infection by aflatoxigenic aspergilli. Control methods for insects are to be environmentally benign and employ semiochemicals to disrupt insect behavior. Control of toxic fungi focuses on biological control using competitive or antagonistic microorganisms. These microorganisms include either yeasts or bacteria that can be mass-produced and effectively utilized in a variety of pre- or post harvest environments. Sub-objective 4.4: Develop commercial products for control A. flavus and aflatoxin on food products. DNA was extracted from almond surfaces obtained from farms that used both conventional and organic practices and the microbial population structure on them determined using 16S rRNA DNA sequence analysis. Significant differences were at different stages of almond development. Sequences associated with Salmonella enterica were observed in fully mature almonds obtained from one of 3 farms using conventional growing methodologies; while none were obtained from those using organic practices. Species-specific PCR primer sets for detection and differentiation of four Aspergillus species (based on DNA sequence differences in the calmodulin gene) were developed. These primers have been used to detect these species in soil inoculated with mixtures of Aspergillus species. This analysis will allow faster throughput of field samples to detect the presence of ochratoxin- and fumonisin-producing species. We have initiated an analysis of Aspergillus population diversity in raisin vineyards beginning at flowering and fruit set, continuing through fruit development and ripening, and ultimately to grape harvesting and drying. Samples are being analyzed for the presence and distribution of mycotoxin-producing Aspergillus species, to determine points at which interventions may be useful for mycotoxin control. This project investigates aflatoxin cross-contamination from nut to nut during processing of shelled almonds and potential transfer of aflatoxin to processing equipment. The results indicated an approximate 16% transfer of aflatoxin to the Clean almonds. Amount of aflatoxin correlated strongly with the number of Hot nuts in each sample, as expected. It was found that very little aflatoxin transferred to the equipment. Further analysis is pending and a manuscript is under preparation. Mini-storage bins (250 ml glass jar with caps) were filled with 50 g of raw almonds. Sterile distilled water was added to adjust the water activity of almonds to 0.83. Individual jar was lined with an edible film on the bottom and another film glued to the inside of the lid. Visual observation on fungal growth was conducted monthly. Tomato edible film was found to be effective in preventing fungal growth on almonds. Fungal cell walls were prepared and added to growth medium. Growth of Pichia anomala cells was monitored. The colony forming units (CFU) were determined when yeast cells reached to stationary phase. Chitosan solution was prepared and its effect on aflatoxin production by Aspergillus flavus was tested. These biopolymers may be useful to increase the yield of biocontrol yeast cells and to inhibit aflatoxin production. The biocontrol yeast, Pichia anomala WRL076 U. S. Patent No. 7,579,183) was licensed to Verdesian Life Sciences, LLC. for commercial products development. The technology transfer is anticipated to mitigate Mycotoxin reduction in food chain. Accomplishments 01 Ochratoxin and fumonisin contamination in almonds. Rejected almond samples were analyzed to determine whether indigenous black-spored Aspergillus species could contaminate almonds with mycotoxins. Isolated species included atoxigenic strains of A. tubingensis and toxigenic strains of A. niger, and A. awamori, which accounted for 72% of fumonisin production. No ochratoxin-producing fungi were isolated. In contrast, almonds contained no detectable fumonisin, but 3 of 21 samples contained low levels of ochratoxin. These results indicate that properly dried and stored almonds are not conducive to fumonisin contamination, and that ochratoxin contamination is sporadic, and possibly caused by other Aspergillus species. This analysis points out future research focus for solving ochratoxin contamination in almonds. 02 Effect of biopolymer on yeast cells growth and inhibition of aflatoxin biosynthesis. Mass production of biocontrol yeast cells is essential. Fungal cell walls were prepared and added to growth medium. Yeast cells grown in the supplemented medium were stimulated and colony forming units (CFU) were increased by 40%. The biopolymer chitosan was found to be effective in inhibit aflatoxin biosynthesis of Aspergillus flavus by 43% at concentration of 0.05%. Chitosan is a GRAS (generally regarded as safe) biopolymer and can be used in conjuction with biocontrol yeast. The findings can be applied to solve problems of aflatoxin contamination in the almond and corn industries. 03 Development of commercial products for control of A. flavus and aflatoxin. The patented yeast, Pichia anomala WRL076 has been licensed by Verdesian Life Sciences LLC as a biological control agent antagonistic to mycotoxigenic fungi. Biological control provides a viable alternative to the use of fungicides in agriculture. Moreover, to reduce harmful fungi while having a minimal adverse impact on the environment. Commercial products of Pichia anomala are needed for the control of toxigenic aspergilli and eliminate Mycotoxin contamination on food.
Impacts
(N/A)
Publications
- Palumbo, J.D., O Keeffe, T.L., Gorski, L.A. 2013. Multiplex PCR analysis of fumonisin biosynthetic genes in fumonisin-nonproducing Aspergillus niger and A. awamori strains. Mycologia. 105:277-284.
- Palumbo, J.D., O Keeffe, T.L. 2013. Distribution and mycotoxigenic potential of Aspergillus section Nigri species in naturally-contaminated almonds. Journal of Food Protection. 76:702-706.
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report Objectives (from AD-416): The overall objective of this project is to develop commercially viable methods for the control of pathogens in tree nuts (almonds, pistachios and walnuts) and raisins which lead to foodborne illness in humans and animals. This includes both the control of fungal pathogens (mycotoxins) as well as bacterial pathogens such as E. coli and Salmonella. One approach will be the development of biocontrol agents using bacteria, yeast and non-toxigenic Aspergillus carbonarius. In addition, a better understanding of organic and conventional farming systems will provide new insights on mycotoxin control. The specific objectives for the period covered by this project plan are as follows: Objective 1: Define the critical control points for pathogen contamination during the production stream. Place particular emphasis on agricultural water sources including dairy waste water. Using both cultural and non-cultural based methods we will identify the points in the developmental process, as well as the processing cycle where tree nuts are most likely to become contaminated with human pathogenic bacteria and mycotoxigenic fungi. Objective 2: Evaluate the microbial ecology of organic v. conventional practices. Although several surveys have reported that consumers equate organically grown food stuffs with higher levels of food safety, little is known about how these practices affect the microbial population structure or mycotoxin levels in tree nuts. We will address the influence of phyllosphere microbial community on the population diversity of A. flavus in tree nut orchards, and A. carbonarius on grape surfaces in both farming systems. Objective 3: Delineate the factors affecting cross-contamination during processing and develop a potential intervention strategy during storage. A number of experiments point to a strong possibility that cross- contamination of toxigenic fungi is possible during processing and storage, although no research appears to have been done on the transfer in actual (not laboratory) processing and storage conditions. Development of novel approaches to prevent the growth of storage fungi and production of harmful toxins is a high priority in the almond industry. Edible films and coatings (EFC) containing antimicrobial natural compounds will be tested for their efficiency to reduce mycotoxin in stored almonds. Objective 4: Develop biological-control/intervention technologies using competitive or antagonistic microorganisms such as yeasts or bacteria that can be mass-produced and effectively utilized in a variety of pre- or post-harvest environments. Bacterial and yeast biocontrol agents will be tested in almond orchards and vineyards to control A. flavus and A. carbonarius, as well as human pathogenic bacteria, E. coli and Salmonella. EPA registration of the patented yeast, Pichia anomala will be pursued for commercial application. Methods to enhance the biocontrol efficacy will be developed. The flowchart shown in Figure 1 illustrates the integration of the four objectives to achieve the ultimate goal of eliminating mycotoxins and human bacterial pathogen contamination (i.e. Salmonella, E. coli O157:H7, etc.) in tree nuts and raisins. Approach (from AD-416): Develop mentods to control insect pests and toxic fungi of tree nuts. Insects include naval orangeworm, codling moth and peach twig borer. Feeding damage by these insects leads to infection by aflatoxigenic aspergilli. Control methods for insects are to be environmentally benign and employ semiochemicals to disrupt insect behavior. Control of toxic fungi focuses on biological control using competitive or antagonistic microorganisms. These microorganisms include either yeasts or bacteria that can be mass-produced and effectively utilized in a variety of pre- or post harvest environments. Fungal populations from raisins produced under different management practices have been isolated and characterized. Analysis of ochratoxin and fumonisin production by Aspergillus isolates has been performed. Molecular analysis of fumonisin biosynthesis genes in fumonisin- nonproducing strains has been performed. We have examined almonds grown in 3 conventional and 3 organic orchards at 4 different time points: 1. When the nut first forms; 2. When the nut is set; 3. When the nut is mature; and, 4. When the hull splits, using small subunit ribosomal sequence analysis of DNA extracted from the nut surfaces. Our results indicate that there are significant differences in the types of bacteria present on the surfaces of almonds grown under conventional and organic methodologies. We did not observe any sequences representative of Salmonella enterica in the organically grown almonds at any point in the maturation of the nut. However we did observe 2 sequences representative of Salmonella enterica in the nuts grown under conventional methodologies. Mixing and collection of samples for analysis for sub-objectives 3.1 and 3.2 are complete. Aflatoxin analysis is ongoing and nearing completion. A visual screening method for anti-fungal activity of essential oil edible films has been developed to screen for effective edible films. Oregano oil was shown to inhibit both the growth and aflatoxin production of A. flavus. Allspice oil and thyme oil were demonstrated to be effective as well. These edible films are being tested on mini-bins to prevent fungal growth in stored almond nuts. Production of antibiotic compounds and fungal cell wall-degrading enzymes by candidate bacterial biocontrol agents has been demonstrated in solid and liquid culture. Small-scale field trials to determine efficacy of bacterial biocontrol agents on corn for reducing aflatoxin contamination showed that the bacterial strains that performed best in laboratory assays did not produce measurable reduction of aflatoxin on corn. Large-scale field trials were not initiated. Methods of application of biocontrol agents (to soil, for example) are currently being re-evaluated. Stimulation of Pichia anomala growth and cell yield by Aspergillus flavus was observed in a dual culture system. Medium composition influenced the increases. Cell yield determined by colony forming unit (CFU) showed that yeast grown in the presence of A. flavus had 30% higher CFUs than the control without A. flavus. Accomplishments 01 Ochratoxin and fumonisin production in conventional vs. organic orchards Black Aspergillus isolates have been collected and identified from vineyard samples of raisins. These isolates have been screened for ochratoxin and fumonisin production. Initial analysis showed no significant differences in population diversity of black Aspergillus species between conventional and organic vineyards. This result supports the hypothesis that organic farming practices do not lead to greater ris of ochratoxin or fumonisin contamination. 02 Genetic basis of fumonisin production. Genetic analysis of fumonisin- nonproducing Aspergillus Niger and Aspergillus awamori strains showed th in A. awamori strains, a larger section of the fumonisin bysynthetic gen cluster is deleted. This deletion in the gene cluster resulted in a loss of fumonisin production. In A. Niger, fumonisin-nonproducing strains contain the entire biosynthetic gene cluster, indicating that the nonproduction is due to other genetic factors. This research increases t fundamental understanding of the genetic basis of fumonisin production i these species. 03 Repression of mycotoxin genes in Aspergillus flavus. The biocontrol yea Pichia anomala WRL-076 has been demonstrated to repress the expression aflatoxin and cyclopiazonic acid biosynthetic genes from Aspergillus flavus by quantitative reverse transcriptase PCR ( qRT PCR ). Genes demonstrated to be repressed in aflatoxin biosynthesis are: aflR (coding for transcription activator), aflJ (coding for transcription enhancer), omtB (coding for 0-methyltranferase B) and pksA (coding for polyketide synthase). Genes demonstrated to be repressed in cyclopianzonic (CPA) ac biosynthesis are: pks-npls1 (coding for polyketide synthase and nonribosomal peptide synthase), hydA (coding for CPA amidohydrolase and ctfR1 (coding for CPA C6-type transcription factor). The molecular data validated that the patented P. anomala is a suitable biocontrol agent fo reducing both aflatoxin and cyclopiazonic acid in plant food products.
Impacts
(N/A)
Publications
- Hua, S.T., Mcalpin, C.E., Chang, P., Sarreal, S.L. 2011. Characterization of toxigenic and atoxigenic Aspergillus flavus isolates from pistachio. Mycotoxin Research. Vol 28:67-75.
- Du, W., Avena Bustillos, R.D., Hua, S.T., Mchugh, T.H. 2011. Antimicrobial volatile essential oils in edible films for food safety. In: Science against Microbial Pathogens: Communicating Current Research and Technological Advances. Badajoh, Spain: Formatex. p. 1124-1134.
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report Objectives (from AD-416) The overall objective of this project is to develop commercially viable methods for the control of pathogens in tree nuts (almonds, pistachios and walnuts) and raisins which lead to foodborne illness in humans and animals. This includes both the control of fungal pathogens (mycotoxins) as well as bacterial pathogens such as E. coli and Salmonella. One approach will be the development of biocontrol agents using bacteria, yeast and non-toxigenic Aspergillus carbonarius. In addition, a better understanding of organic and conventional farming systems will provide new insights on mycotoxin control. The specific objectives for the period covered by this project plan are as follows: Objective 1: Define the critical control points for pathogen contamination during the production stream. Place particular emphasis on agricultural water sources including dairy waste water. Using both cultural and non-cultural based methods we will identify the points in the developmental process, as well as the processing cycle where tree nuts are most likely to become contaminated with human pathogenic bacteria and mycotoxigenic fungi. Objective 2: Evaluate the microbial ecology of organic v. conventional practices. Although several surveys have reported that consumers equate organically grown food stuffs with higher levels of food safety, little is known about how these practices affect the microbial population structure or mycotoxin levels in tree nuts. We will address the influence of phyllosphere microbial community on the population diversity of A. flavus in tree nut orchards, and A. carbonarius on grape surfaces in both farming systems. Objective 3: Delineate the factors affecting cross-contamination during processing and develop a potential intervention strategy during storage. A number of experiments point to a strong possibility that cross- contamination of toxigenic fungi is possible during processing and storage, although no research appears to have been done on the transfer in actual (not laboratory) processing and storage conditions. Development of novel approaches to prevent the growth of storage fungi and production of harmful toxins is a high priority in the almond industry. Edible films and coatings (EFC) containing antimicrobial natural compounds will be tested for their efficiency to reduce mycotoxin in stored almonds. Objective 4: Develop biological-control/intervention technologies using competitive or antagonistic microorganisms such as yeasts or bacteria that can be mass-produced and effectively utilized in a variety of pre- or post-harvest environments. Bacterial and yeast biocontrol agents will be tested in almond orchards and vineyards to control A. flavus and A. carbonarius, as well as human pathogenic bacteria, E. coli and Salmonella. EPA registration of the patented yeast, Pichia anomala will be pursued for commercial application. Methods to enhance the biocontrol efficacy will be developed. The flowchart shown in Figure 1 illustrates the integration of the four objectives to achieve the ultimate goal of eliminating mycotoxins and human bacterial pathogen contamination (i.e. Salmonella, E. coli O157:H7, etc.) in tree nuts and raisins. Approach (from AD-416) Develop methods to control insect pests and toxic fungi of tree nuts. Insects include naval orangeworm, codling moth and peach twig borer. Feeding damage by these insects leads to infection by aflatoxigenic aspergilli. Control methods for insects are to be environmentally benign and employ semiochemicals to disrupt insect behavior. Control of toxic fungi focuses on biological control using competitive or antagonistic microorganisms. These microorganisms include either yeasts or bacteria that can be mass-produced and effectively utilized in a variety of pre- or post harvest environments. Replacing 5325-42000-036-00D (1/11). This report documents progress for the parent project 5325-42000-038-00D ENVIRONMENTAL AND ECOLOGICAL APPROACHES TO ELIMINATE FUNGAL CONTAMINATION AND MYCOTOXIN PRODUCTION IN PLANT PRODUCTS which started Feb 2011 and continues research from project 5325-42000-036-00D BIOLOGICAL CONTROL OF INSECTS AND MICROORGANISMS TO PREVENT MYCOTOXIN CONTAMINATION. In cooperation with scientists from University of California, three organic and three conventional almond orchards were identified and selected as sources for almonds for the research. Immature almonds from all 6 sites have already been collected and additional samples will be collected as the nuts mature on the trees. Fungal populations from raisins produced under different management practices have been isolated. Isolated strains have been identified to the species level, and the ability of each strain to produce mycotoxins (ochratoxin and/or fumonisin) are ongoing. Small-scale field trials to determine efficacy of bacterial biocontrol agents on corn for reducing aflatoxin contamination showed that the bacterial strains that performed best in laboratory assays did not produce measurable reduction of aflatoxin on corn. Collection and identification of black Aspergillus isolates from vineyard samples of raisins. These isolates are currently being screened for ochratoxin and fumonisin production. Frequency of species and mycotoxin production within each sample will be analyzed relative to production method to determine whether patterns of correlation exist. Almond nut samples have been obtained from commercial sources and half of them have been �spiked� artificially with aflatoxin. Mixing experiments are ready to proceed to determine the mode of aflatoxin cross contamination. A variety of films have been fabricated and screened in order to find films that are effective in preventing fungal growth. Edible tomato and apple based films containing cinnamon oil, allspice oil, oregano oil, garlic oil, bay laurel oil, bay leaf oil, cedarwood oil, lemomgrass oil, and thyme oil are being tested. Interactions of yeast cells with Aspergillus flavus or cell walls of the fungus were investigated. The study is to identify factors which may be used to enhance the efficacy of biocontrol activities of the yeast, Pichia anomala. Yeast cells grown in the presence of fungi or fungal cell walls, were collected for gene expression studies. Parameters influencing real time polymerase chain reaction (RT-PCR) were examined. A procedure to determine intracellular sugars and polyols by high pressure liquid chromatography (HPLC) analysis is being developed. Accomplishments 01 Evaluation of antifungal films for use in almond storage. Usually funga growth on stored almonds in the bin starts near the lid due to moisture condensation. The use of films as carriers of natural antimicrobials (su as plant essential oils) constitutes an alternative approach for externa protection of almonds, especially raw almonds, to reduce surface microbi populations. ARS scientists at Albany, CA, have developed a method to screen edible films which can inhibit both aflatoxin production and fung growth. Several edible films have been identified so far. These films ca be easily applied on the lid of the container and protect raw almonds during transport. 02 Molecular mechanisms of biocontrol yeast, Pichia (P.) anomala. Understanding the modes of action is important for maximizing the potential use of microbial biocontrol agents. ARS scientists at Albany, California, have identified two genes in P. anomala that produce enzymes that break down the cell wall of Aspergilles (A.) flavus. They discovere that expression of these genes can be increased four to six-fold upon exposure to A. flavus. This finding validates P. anomala as a promising biocontrol agent against A. flavus. 03 Improve shelf-life of biocontrol yeast, Pichia anomala. Maintaining the viability of this biocontrol agent in formulated products is a challenge A wet formulation has been developed by ARS scientists in Albany, CA, by adding certain compounds to the formulation. The shelf-life of this formulation was up to fourteen months with very little change in viabili of yeast cells stored at 40c. The results were validated by flow cytomet analysis and florescent microscopy using viability stains. A stable liqu formulation is highly desirable because most of the bicontrol products a dispersed in water and delivered by spraying or dipping to protect agricultural and horticultural crops.
Impacts
(N/A)
Publications
- Hernlem, B.J., Hua, S.T. 2010. Dual fluorochrome flow cytometric assessment of yeast viability. Current Microbiology. 61:57-63.
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