Progress 02/06/16 to 01/18/21
Outputs
PROGRESS REPORT Objectives (from AD-416): The long-term objective of this project is to reduce, inhibit, or eliminate toxigenic and pathogenic microbes (i.e., mycotoxigenic fungi or pathogenic bacteria) by utilizing intervention techniques such as biological control. Specifically, during the next five years we will focus on the following interrelated objectives. Objective 1: Develop and implement control measures to reduce, eliminate, or detect contamination of toxin producing fungi of tree nuts, for example the use of host plant- or fungal-derived semiochemicals to attract or control insect pests, or use of sterile insect techniques to decrease insect pest populations. Sub-objective 1A: Use of host plant- or microbe-derived volatile semiochemicals to attract or control insect pests. Sub-objective 1B: Use of sterile insect techniques to decrease insect pest populations. Objective 2: Elucidate principles of microbial ecology and develop biological control measures to inhibit pathogenic and toxigenic microorganisms, particularly fungi, and can include research on the isolation and development of new biocontrol agents and formulations to control or prevent toxigenic microbes, or survey, identify, and determine ecology of microbial populations for control strategies such as competitive microorganisms. Sub-objective 2A: Isolate biocontrol agents that prevent pathogenic/ toxigenic microbes from colonizing crops. Sub-objective 2B: Risk analysis of waste used as fertilizers for pathogen/toxigen contamination. Sub-objective 2C: Develop new biocontrol agents and formulations to control toxigenic fungi, and to survey and characterize populations of Aspergilli. Sub-objective 2D: Determine ecology of black-spored toxigenic Aspergilli and develop control strategies using competitive microorganisms. Objective 3: Discover natural chemical compounds that enhance the efficacy of established microbe intervention strategies, for instance augment the activity of antimicrobial agents/treatments against pathogens via target-based application of natural chemosensitizing agents. Approach (from AD-416): 1A. Tree nuts emit chemicals that attract insect pests that can be used as bait for insect traps. We will analyze volatiles from nuts by GC-MS and test them for pest attraction in electrophysiological and behavioral bioassays. If we are unable to identify volatiles from nuts we will explore volatiles from other biotic and abiotic matrices. 1B. Sterile insect technique can be applied to navel orange worms (NOW) inside discarded nuts on the orchard floor using an X-ray device towed behind a tractor. We will determine the X-ray dose required for sterilization of NOW and adjust this dosage to sterilize NOW inside tree nuts and develop a tractor towed device for field sterilization. If X-ray exposure does not produce sterile NOW other forms of radiation will be used. 2A. Bacteria with agonistic properties to pathogens are present on almond drupes and if applied in large numbers would prevent pathogen contamination. We will isolate bacteria from almonds and test their ability to inhibit pathogen growth in vitro. The bacteria that inhibit pathogen growth in vitro will be examined for the ability to inhibit growth on almonds, then in field trials. If we are not able to identify bacteria that inhibit pathogen growth on almonds we will use other crops. 2B. Applying composted manure to orchards does not represent a food safety threat. We will examine the microbial community structure of soil and fruit before and after the application of manure. We will repeat the analysis for 3 years to determine the effects of manure application. 2C. Atoxigenic Aspergillus flavus strains with deletions in the aflatoxin and CPA genes can be used as biological control agents for toxigenic A. flavus. We will identify atoxigenic A. flavus isolates by PCR and confirm by chemical analysis. We will examine their use as biocontrol agents via growth inhibition assays. Atoxigenic strains that displace the toxigenic strains will be impregnated into biochar and analyzed for as biocontrol agents in green house experiments. If the biochar is not suitable we will examine other matricies such as plastic granula. 2D. Ratios of toxigenic to non-toxigenic Aspergillus sp. fluctuate during the growing season; application of competitive fungal or bacterial strains will reduce mycotoxins in grapes/raisins. Grape/raisin samples will be taken at regular intervals in the growing season and analyzed to determine the ideal time to apply biocontrol agents against toxigenic Aspergillus. At these time points we will isolate bacteria and nontoxigenic Aspergillus sp. from raisin and soil samples and assay their ability to inhibit the growth of Aspergillus sp. If no non-toxigenic strains are not found other sources will be investigated. 3. Natural compounds and derivatives can control the growth of fungal pathogens and the production of toxins. Natural compounds will be tested for the disruption of cell wall integrity and the antioxidant pathway in fungi via genetic and physiologic analysis. We will determine the mode of action of these compounds via microarrays and other genetic tests. If we are unable to identify these compounds we will analyze other chemicals such as benzo derivatives This is the final report for project 2030-42000-039-000D, which was replaced by project 2030-42000-054-000D. The overall goal of Sub-objective 1A was to identify new semiochemicals for attracting insect pests in pistachio orchards. ARS researchers from Albany, California, identified and compared the volatile chemical emissions from pistachios as they developed. Several different chemicals, called terpenes, were identified that attracted female navel orangeworm (NOW) in the lab but were ineffective in field trials. New efforts were undertaken to identify microbe-derived volatiles as NOW attractants. Several volatile chemicals derived from fungi that grew on discarded pistachios in the field (pistachio mummies) were identified that attracted female NOW. Combinations or blends of the volatiles were identified for further testing as lure candidates based on NOW attraction in commercial pistachio and almond orchards under mating disruption conditions. Statistically significant results were obtained for the first NOW flight in April/May 2020. Efforts continued in the development of laboratory-based behavioral assays for NOW attraction to unique pistachio mummy volatiles; these include an assay to determine larval attraction to these volatiles, a Y-tube-based choice test for male and female adults, and an assay to examine the role of volatile attraction in female egg laying preferences. The overall goal under Sub-objective 1B was to develop the use of sterile insect techniques (SIT) to decrease insect pest populations. The work was directed at developing X-ray irradiation technology for sterilization of Navel Orangeworm (NOW), the major pest of California tree nuts. This was approached from the perspective of sterilizing moths for SIT as well as X-ray sterilization of overwintering NOW larvae in pistachio mummies on the orchard floor. A series of X-ray irradiators were developed, with efficacy for insect sterilization demonstrated and published. A patent application was submitted for a novel irradiator design that allows insects to be sterilized with high dose precision as well as dose uniformity. Required doses for X-ray based sterilization of NOW adult moths, larvae and pupae are now published, completing the objective of determining sterilization doses for all life stages of the insect. A mobile X-ray system for in-field irradiation of pistachio mummies was developed and tested but determined to be impractical for real-world use due to regulatory and safety concerns. This approach has been replaced with a new project using high-pressure steam for in-field eradication of overwintering NOW larvae. The goal of Sub-objective 2A was to identify bacteria that normally grow on the surfaces of crop plants that can inhibit the growth of the human pathogenic bacteria Salmonella enterica, Listeria moncytogenes and Escherichia coli on crop plants (i.e. biological control agents). We isolated over 10,000 bacteria from several different types of produce and screened them using an in vitro fluorescence based growth assay for the ability to inhibit pathogen growth. We identified approximately 50 bacterial isolates that were able to inhibit the growth of these pathogens in vitro and identified them using molecular methods. We took the most suitable isolates and assayed them for their ability to grow, persist and inhibit the growth of the pathogens on cantaloupe melons in laboratory and greenhouse studies. Over the life of this project, we were granted one patent, published six peer reviewed manuscripts, have one currently submitted and have two in preparation describing these bacteria and their potential use as biological control agents. The goal of Sub-objective 2B was to determine if using composted dairy cow manure as a fertilizer for almond orchards is a risk factor for the contamination of orchards with Salmonella enterica. To accomplish this, we applied synthetic fertilizer to all plots in a commercial orchard and supplemented some plots with composted dairy cow manure three times a year (October, January and April) for four years. We sampled almond drupes and soil from both types of plots twice a year (April and August) for four years and assayed them for the presence of Salmonella enterica and examined them for differences in their microbiomes using 16S rRNA gene sequence analysis. We observed Salmonella enterica on all groups of almond drupes in the third year but never observed Salmonella enterica in the soil samples. We did not observe any significant difference in the bacterial populations in the two plot types, but the populations did differ more at the end of the fourth year of testing than they did at the start. It is possible that we would have seen statistical differences in the soil bacterial populations if we continued the project. Our overall conclusion is that the use of properly composted dairy cow manure does not represent a significant threat for pathogen contamination and that four years of compost application is not long enough to see significant differences in the soil microbiomes. In support of Sub-objective 2C, researchers investigated Aspergillus flavus strains containing deletions within the aflatoxin biosynthetic gene cluster for use as biocontrol agents. Preliminary experiments with non-aflatoxigenic strains and wild type (aflatoxin-producing) strains in co-culture and in soil indicated that several non-aflatoxigenic strains were effective in reducing total aflatoxin production. Experiments in which biochar was investigated as a substrate for application of non- aflatoxigenic strains into soil showed mixed results in the usefulness of this delivery system. Quantification of wild type and non-aflatoxigenic strains by droplet digital polymerase chain reaction (PCR) indicated that some non-aflatoxigenic strains were less competitive than wild type strains in co-inoculated soil, and that the use of biochar did not significantly change the competitiveness of some non-aflatoxigenic strains relative to wild type strains. For Sub-objective 2D, soil and grape samples were taken from conventional and organic raisin vineyards in California at four time points during two consecutive growing seasons. Quantitative PCR methods were developed to determine the relative population sizes of ochratoxin- producing and non-toxigenic black-spored Aspergillus species that predominate in the grape environment. Results from these studies demonstrated that while fungal populations and the proportions of species fluctuate during the growing season and no significant differences were observed between conventional and organic vineyards. These results suggest that interventions to reduce mycotoxigenic fungal populations would function similarly in conventional and organic regimes. Bacteria were isolated from these vineyard samples to identify strains with antifungal activity against ochratoxigenic and/or non-toxigenic Aspergilli using high-throughput assays to measure fungal inhibition via diffusion in agar and liquid cocultures and via production of inhibitory volatiles. Candidate bacteria with antifungal activity were selected for the development of in-situ soil and fruit microcosm assays to demonstrate the reduction in fungal soil populations and concomitant reduction of fungal colonization and ochratoxin contamination of fruit. Under Objective 3, natural compounds (benzaldehydes and cinnamic acids) have been identified that remove fungal contaminants from food and/or environmental matrices. The identified benzaldehydes are redox-active molecules that disrupt the cellular components for oxidative stress resistance in fungi, inhibit mycotoxin production, and prevent fungal tolerance to commercial fungicides. Cinnamaldehyde rapidly eliminates (= 99.9% fungal death at 2.5 hours) aflatoxin-producing Aspergillus or heat- resistant food-spoilage fungi and can serve as an ecologically-sound active ingredient for antifungal formulation. The natural cinnamic derivatives achieve fungal elimination by disrupting cell wall integrity of fungi. Four cinnamic acids substantially enhance the antifungal efficacy of the commercial drug caspofungin (CAS). CAS alone cannot prevent the growth of filamentous fungi, such as Aspergillus, thus resulting in fungal survival/escape during treatment. Cinnamic acids also overcome fungal resistance to conventional fungicides such as fludioxonil. Notably, 2-hydroxy-4-methoxybenzaldehyde (2H4M) targets both oxidative stress resistance and cell wall integrity systems in fungi. 2H4M effectively disrupts the crosstalk between these two defense systems, where the cytosolic oxidative stress signals, such as superoxide radicals, are transmitted to activate the cell wall integrity pathway. The integration of natural compounds that debilitate fungal defense systems provides the basis for new intervention methods that lower the effective doses of fungicides. These methods reduce the required inputs of these toxic chemicals and ameliorate the environmental and health risks associated with fungicide application. The result of these natural compound studies is the development of a new seed-protection formula that ensures seed viability under abiotic and biotic stress conditions. The high-efficiency formula protects crop seeds during storage or germination and under the pathogen attack. Consequently, the formula can serve as an alternative to the conventional, toxic seed-treatment agents, such as mycotoxin-triggering azole fungicides, facilitating sustainable, precision agriculture. The integration of natural compounds resulted in an invention that directly relates to the objectives of the project to reduce, inhibit, or eliminate toxigenic and pathogenic microbes via new intervention techniques. Altogether, the incorporation of natural compounds in an antifungal formulation established a reliable and effective fungal control strategy, that contributes to an integrated pest management system for stakeholders. Record of Any Impact of Maximized Teleworking Requirement: For Objective 2D the maximized telework requirement had a negative impact on the lab⿿s ability to perform research in development of in-vitro fungal-bacterial competition assays and in-situ microcosm assays, and so progress toward Sub-objective 2D was halted. In addition, no new projects could be initiated, no preliminary experiments could be conducted, and no data could be collected for analysis. These continuing and new experiments will be conducted when we are allowed to return to laboratory operations. Additionally, as a consequence of maximized telework, no data could be collected for preparing the manuscript scheduled as part of Sub- objective 2D. ACCOMPLISHMENTS 01 Biological control agents to reduce pathogen growth on produce. Pathogen contamination of produce remains a substantial food safety challenge. To identify bacterial biological control agents, ARS researchers in Albany, California, created a plant phyllosphere- associated bacterial library containing over 10,000 bacteria and screened it for bacteria that can inhibit the growth of the human pathogenic bacteria Salmonella enterica, Escherichia coli and Listeria monocytogenes, using an in vitro fluorescence-based growth assay. These experiments identified a biological control agent that was effective at preventing the growth of Salmonella enterica on cantaloupe melons (Pantoea agglomerans ASB05), a biological control agent that inhibited the growth of pathogenic Escherichia coli on cantaloupe melons (Enterobacter absuriae AEB30) and a biological control agent that inhibited the growth of Listeria monocytogenes on cantaloupe melons (Bacillus amyloliquefaciens ALB65).
Impacts
(N/A)
Publications
- Tran, T.D., Del Cid, C., Hnasko, R.M., Gorski, L.A., McGarvey, J.A. 2020. Bacillus amyloliquefaciens ALB65 inhibits the growth of Listeria monocytogenes on cantaloupe melons. Applied and Environmental Microbiology. 87(1). Article e01926-20. https://doi.org/10.1128/AEM.01926-20.
- Kim, J., Cheng, L.W., Chan, K.L., Tam, C.C., Mahoney, N.E., Friedman, M., Shilman, M.M., Land, K.M. 2020. Antifungal drug repurposing. Antibiotics. 9(11):812. https://doi.org/10.3390/antibiotics9110812.
- Chellan, P., Avery, V.M., Duffy, S., Land, K., Tam, C.C., Kim, J., Cheng, L.W., Romero-Canelón, I., Sadler, P.J. 2021. Bioactive half-sandwich Rh and Ir bipyridyl complexes containing artemisinin. Inorganic Biochemistry. 219. Article 111408. https://doi.org/10.1016/j.jinorgbio.2021.111408.
- Friedman, M., Tam, C.C., Kim, J., Escobar, S., Gong, S., Liu, M., Yu Mao, X., Do, C., Kuang, I., Boateng, K., Ha, J., Tran, M., Alluri, S., Le, T., Leong, R., Cheng, L.W., Land, K.M. 2021. Anti-parasitic activity of cherry tomato peel powders. Foods. 10(2). Article 230. https://doi.org/10.3390/ foods10020230.
- Friedman, M., Xu, A., Lee, R., Nguyen, D.N., Phan, T.A., Hamada, S.M., Panchel, R., Tam, C.C., Kim, J., Cheng, L.W., Land, K.M. 2020. The inhibitory activity of anthraquinones against pathogenic protozoa, bacteria, and fungi and the relationship to structure. Molecules. 25(13) :3101. https://dx.doi.org/10.3390/molecules25133101.
- Kim, J., Chan, K.L., Tam, C.C., Cheng, L.W., Land, K.M. 2020. Crosstalk between the antioxidant and cell wall integrity systems in fungi by 2- hydroxy-4-methoxybenzaldehyde. Cogent Food & Agriculture. 6(1). Article 1823593. https://doi.org/10.1080/23311932.2020.1823593.
|
Progress 10/01/19 to 09/30/20
Outputs
Progress Report Objectives (from AD-416): The long-term objective of this project is to reduce, inhibit, or eliminate toxigenic and pathogenic microbes (i.e., mycotoxigenic fungi or pathogenic bacteria) by utilizing intervention techniques such as biological control. Specifically, during the next five years we will focus on the following interrelated objectives. Objective 1: Develop and implement control measures to reduce, eliminate, or detect contamination of toxin producing fungi of tree nuts, for example the use of host plant- or fungal-derived semiochemicals to attract or control insect pests, or use of sterile insect techniques to decrease insect pest populations. Sub-objective 1A: Use of host plant- or microbe-derived volatile semiochemicals to attract or control insect pests. Sub-objective 1B: Use of sterile insect techniques to decrease insect pest populations. Objective 2: Elucidate principles of microbial ecology and develop biological control measures to inhibit pathogenic and toxigenic microorganisms, particularly fungi, and can include research on the isolation and development of new biocontrol agents and formulations to control or prevent toxigenic microbes, or survey, identify, and determine ecology of microbial populations for control strategies such as competitive microorganisms. Sub-objective 2A: Isolate biocontrol agents that prevent pathogenic/ toxigenic microbes from colonizing crops. Sub-objective 2B: Risk analysis of waste used as fertilizers for pathogen/toxigen contamination. Sub-objective 2C: Develop new biocontrol agents and formulations to control toxigenic fungi, and to survey and characterize populations of Aspergilli. Sub-objective 2D: Determine ecology of black-spored toxigenic Aspergilli and develop control strategies using competitive microorganisms. Objective 3: Discover natural chemical compounds that enhance the efficacy of established microbe intervention strategies, for instance augment the activity of antimicrobial agents/treatments against pathogens via target-based application of natural chemosensitizing agents. Approach (from AD-416): 1A. Tree nuts emit chemicals that attract insect pests that can be used as bait for insect traps. We will analyze volatiles from nuts by GC-MS and test them for pest attraction in electrophysiological and behavioral bioassays. If we are unable to identify volatiles from nuts we will explore volatiles from other biotic and abiotic matrices. 1B. Sterile insect technique can be applied to navel orange worms (NOW) inside discarded nuts on the orchard floor using an X-ray device towed behind a tractor. We will determine the X-ray dose required for sterilization of NOW and adjust this dosage to sterilize NOW inside tree nuts and develop a tractor towed device for field sterilization. If X-ray exposure does not produce sterile NOW other forms of radiation will be used. 2A. Bacteria with agonistic properties to pathogens are present on almond drupes and if applied in large numbers would prevent pathogen contamination. We will isolate bacteria from almonds and test their ability to inhibit pathogen growth in vitro. The bacteria that inhibit pathogen growth in vitro will be examined for the ability to inhibit growth on almonds, then in field trials. If we are not able to identify bacteria that inhibit pathogen growth on almonds we will use other crops. 2B. Applying composted manure to orchards does not represent a food safety threat. We will examine the microbial community structure of soil and fruit before and after the application of manure. We will repeat the analysis for 3 years to determine the effects of manure application. 2C. Atoxigenic Aspergillus flavus strains with deletions in the aflatoxin and CPA genes can be used as biological control agents for toxigenic A. flavus. We will identify atoxigenic A. flavus isolates by PCR and confirm by chemical analysis. We will examine their use as biocontrol agents via growth inhibition assays. Atoxigenic strains that displace the toxigenic strains will be impregnated into biochar and analyzed for as biocontrol agents in green house experiments. If the biochar is not suitable we will examine other matricies such as plastic granula. 2D. Ratios of toxigenic to non-toxigenic Aspergillus sp. fluctuate during the growing season; application of competitive fungal or bacterial strains will reduce mycotoxins in grapes/raisins. Grape/raisin samples will be taken at regular intervals in the growing season and analyzed to determine the ideal time to apply biocontrol agents against toxigenic Aspergillus. At these time points we will isolate bacteria and nontoxigenic Aspergillus sp. from raisin and soil samples and assay their ability to inhibit the growth of Aspergillus sp. If no non-toxigenic strains are not found other sources will be investigated. 3. Natural compounds and derivatives can control the growth of fungal pathogens and the production of toxins. Natural compounds will be tested for the disruption of cell wall integrity and the antioxidant pathway in fungi via genetic and physiologic analysis. We will determine the mode of action of these compounds via microarrays and other genetic tests. If we are unable to identify these compounds we will analyze other chemicals such as benzo derivatives In support of Sub-objective 1A, research is ongoing to develop lures to attract navel orangeworm (NOW) from volatile compounds unique to pistachio mummies with a high antennal sensitivity for male and female adults. Combinations or blends of volatiles were identified for further testing as lure candidates based on NOW attraction in commercial pistachio and almond orchards under mating disruption conditions. Statistically significant results were obtained for the first NOW flight in April/May. Additional field testing is ongoing. Efforts continued in the development of laboratory-based behavioral assays for NOW attraction to unique pistachio mummy volatiles; these include an assay to determine larval attraction to these volatiles, a Y-tube based choice test for male and female adults, and an assay to examine the role of volatile attraction in female egg laying preferences. In support of Sub-objective 1B, research continues toward implementation of x-ray based sterile insect technique (SIT) for control of NOW in California pistachio and almond orchards. Over the life of the project, a series of x-ray irradiators have been developed, with efficacy as a replacement for gamma-based irradiation demonstrated and published. During fiscal year (FY) 2020, a patent application was submitted for a new irradiator design that allows insects to be irradiated with high dose precision as well as dose uniformity. Also during FY2020, required doses for sterilization of NOW larvae and pupae using x-ray were published, completing the objective of determining sterilization doses for all life stages of the insect. Irradiated moths are currently being supplied to researchers in Parlier, California, for release in ongoing fitness and sterility testing in support of the areawide integrated pest management system for NOW suppression, including SIT. In support of Sub-objective 2B, ARS researchers in collaboration with scientists at the University of California, Davis, continued to evaluate the safety of using composted manure and green waste as a fertilizer/soil conditioner in orchards. Soil and fertilizer samples were obtained after the third year of treatment with composted manure or green waste and analyzed for the presence of E. coli O157:H7 and Listeria monocytogenes by culture methods. The researchers also extracted deoxyribonucleic acid (DNA) from the samples and analyzed them by 16S ribosomal ribonucleic acid (rRNA) gene sequence analysis to determine the effect of applying these substances on the microbial populations and their diversity within the orchard soils. In support of Objective 2, Sub-objective 2D, research continued on the development of microcosm assays to demonstrate whether bacteria from grape surfaces affected the populations of fungi responsible for ochratoxin contamination of grapes (and raisins made from contaminated grapes). Also, ARS researchers continued screening bacteria isolated from vineyard soils for antagonistic effects against target ochratoxin A- producing and nontarget ochratoxin A-nonproducing fungi, using a high- throughput coculture assay previously developed. In support of Objective 3, researchers identified natural compounds 2- hydroxy-4-methoxybenzaldehyde or benzoic acids as potent antimicrobials that disrupted both the redox maintenance and redox sensitive cell structures in fungal and bacterial pathogens. Thus, the antioxidant and cell wall integrity pathways could serve as new antifungal targets. In addition, researchers developed seed protection formulas that increased seed viability under abiotic and biotic stress conditions, improved crop seed protection during storage, germination and reduced Aspergillus spp. contamination. Accomplishments 01 Targeting the crosstalk between the fungal defense systems by natural compounds. The cost for the loss of agricultural products due to mycotoxin contamination has been estimated at $2 to $3 billion per year in the United States. ARS researchers in Albany, California, identified the natural product 2-hydroxy-4- methoxybenzaldehyde (2H4M) as a potent antifungal. 2H4M, a redox-active agent, contains both antioxidant and prooxidant characteristics that disrupted the crosstalk between the antioxidant and cell-wall integrity pathways of fungi. This two-pathway crosstalk serves as a new target for anti-fungal redox-active agents including fungicides. The newly developed intervention using 2H4M provides growers with a valuable tool to protect crops from fungal and mycotoxin contamination. 02 Identification and characterization of an anti-Listeria monocytogenes biocontrol agent. Listeria (L.) monocytogenes is estimated to cause disease in over 2,000 people in the United States every year. The largest outbreak of L. monocytogenes was the 2011 cantaloupe-associated outbreak that resulted in 147 hospitalizations and 33 deaths. ARS researchers in Albany, California, identified a common plant-associated bacterium called Bacillus (B.) amyloliquefaciens, that is able to significantly reduce the growth of L. monocytogenes on cantaloupes in both pre- and post-harvest environments. When applied to melons, B. amyloliquefaciens does not produce any deleterious effects, such as off color or smell, and when applied to cantaloupe plants in the field increases their rate of growth by 50%. This newly identified biological control agent will provide growers and produce packers a new tool to reduce L. monocytogenes growth on produce. 03 High throughput low-energy x-ray irradiators for sterile insect technique. The use of gamma irradiation for insect sterilization in sterile insect technique (SIT) pest control programs can be problematic, and alternatives are needed. Using low energy x-rays is appealing, but creates challenges for the sterilization of large quantities of insects required for SIT. Employing multiple x-ray sources strategically oriented over a rotating platform, such as a conveyor system so that the absorbed dose is consistent, allows for large numbers of irradiated samples. Researchers in Albany, California, have adopted this principle in novel x-ray irradiators that use relatively inexpensive, commercially available, low-energy x-ray tubes that can sterilize insects at a much higher rate than was previously possible. This innovation provides the means for increasing use of x-ray as a sterilization method for SIT.
Impacts
(N/A)
Publications
- Kim, J., Chan, K.L., Mahoney, N.E., Cheng, L.W., Tautges, N., Scow, K. 2020. Rapid elimination of foodborne and environmental fungal contaminants by benzo analogs. Journal of the Science of Food and Agriculture. 100(6) :2800⿿2806.
- McGarvey, J.A., Tran, T.D., Hnasko, R.M., Gorski, L.A. 2019. Use of phyllosphere associated lactic acid bacteria as biocontrol agents to reduce salmonella enterica serovar poona growth on cantaloupe melons. Journal of Food Protection. 82(12):2148-2153.
- Hnasko, R.M., McGarvey, J.A., Lin, A.V. 2019. Rapid detection of staphylococcal enterotoxin-B by lateral flow assay. Monoclonal Antibodies in Immunodiagnosis and Immunotherapy. 38(5):209-212.
- Tran, T.D., Huynh, S., Parker, C., Hnasko, R.M., Gorski, L.A., Khalsa Sat, D., Brown, P., McGarvey, J.A. 2020. Complete genomic sequences of three Salmonella enterica subsp. enterica serovar muenchen strains from an orchard in San Joaquin County, California. Microbiology Resource Announcements. 9(13):e00048-20.
- Toyofuku, N., Mahoney, N.E., Haff, R.P. 2019. Aflatoxin cross- contamination during mixing of shelled almonds. Journal of Food Processing and Preservation. 44(2).
|
Progress 10/01/18 to 09/30/19
Outputs
Progress Report Objectives (from AD-416): The long-term objective of this project is to reduce, inhibit, or eliminate toxigenic and pathogenic microbes (i.e., mycotoxigenic fungi or pathogenic bacteria) by utilizing intervention techniques such as biological control. Specifically, during the next five years we will focus on the following interrelated objectives. Objective 1: Develop and implement control measures to reduce, eliminate, or detect contamination of toxin producing fungi of tree nuts, for example the use of host plant- or fungal-derived semiochemicals to attract or control insect pests, or use of sterile insect techniques to decrease insect pest populations. Sub-objective 1A: Use of host plant- or microbe-derived volatile semiochemicals to attract or control insect pests. Sub-objective 1B: Use of sterile insect techniques to decrease insect pest populations. Objective 2: Elucidate principles of microbial ecology and develop biological control measures to inhibit pathogenic and toxigenic microorganisms, particularly fungi, and can include research on the isolation and development of new biocontrol agents and formulations to control or prevent toxigenic microbes, or survey, identify, and determine ecology of microbial populations for control strategies such as competitive microorganisms. Sub-objective 2A: Isolate biocontrol agents that prevent pathogenic/ toxigenic microbes from colonizing crops. Sub-objective 2B: Risk analysis of waste used as fertilizers for pathogen/toxigen contamination. Sub-objective 2C: Develop new biocontrol agents and formulations to control toxigenic fungi, and to survey and characterize populations of Aspergilli. Sub-objective 2D: Determine ecology of black-spored toxigenic Aspergilli and develop control strategies using competitive microorganisms. Objective 3: Discover natural chemical compounds that enhance the efficacy of established microbe intervention strategies, for instance augment the activity of antimicrobial agents/treatments against pathogens via target-based application of natural chemosensitizing agents. Approach (from AD-416): 1A. Tree nuts emit chemicals that attract insect pests that can be used as bait for insect traps. We will analyze volatiles from nuts by GC-MS and test them for pest attraction in electrophysiological and behavioral bioassays. If we are unable to identify volatiles from nuts we will explore volatiles from other biotic and abiotic matrices. 1B. Sterile insect technique can be applied to navel orange worms (NOW) inside discarded nuts on the orchard floor using an X-ray device towed behind a tractor. We will determine the X-ray dose required for sterilization of NOW and adjust this dosage to sterilize NOW inside tree nuts and develop a tractor towed device for field sterilization. If X-ray exposure does not produce sterile NOW other forms of radiation will be used. 2A. Bacteria with agonistic properties to pathogens are present on almond drupes and if applied in large numbers would prevent pathogen contamination. We will isolate bacteria from almonds and test their ability to inhibit pathogen growth in vitro. The bacteria that inhibit pathogen growth in vitro will be examined for the ability to inhibit growth on almonds, then in field trials. If we are not able to identify bacteria that inhibit pathogen growth on almonds we will use other crops. 2B. Applying composted manure to orchards does not represent a food safety threat. We will examine the microbial community structure of soil and fruit before and after the application of manure. We will repeat the analysis for 3 years to determine the effects of manure application. 2C. Atoxigenic Aspergillus flavus strains with deletions in the aflatoxin and CPA genes can be used as biological control agents for toxigenic A. flavus. We will identify atoxigenic A. flavus isolates by PCR and confirm by chemical analysis. We will examine their use as biocontrol agents via growth inhibition assays. Atoxigenic strains that displace the toxigenic strains will be impregnated into biochar and analyzed for as biocontrol agents in green house experiments. If the biochar is not suitable we will examine other matricies such as plastic granula. 2D. Ratios of toxigenic to non-toxigenic Aspergillus sp. fluctuate during the growing season; application of competitive fungal or bacterial strains will reduce mycotoxins in grapes/raisins. Grape/raisin samples will be taken at regular intervals in the growing season and analyzed to determine the ideal time to apply biocontrol agents against toxigenic Aspergillus. At these time points we will isolate bacteria and nontoxigenic Aspergillus sp. from raisin and soil samples and assay their ability to inhibit the growth of Aspergillus sp. If no non-toxigenic strains are not found other sources will be investigated. 3. Natural compounds and derivatives can control the growth of fungal pathogens and the production of toxins. Natural compounds will be tested for the disruption of cell wall integrity and the antioxidant pathway in fungi via genetic and physiologic analysis. We will determine the mode of action of these compounds via microarrays and other genetic tests. If we are unable to identify these compounds we will analyze other chemicals such as benzo derivatives Under Sub-Objective 1A, researchers in Albany, California, have identified multiple volatile compounds from pistachio mummies and tested them for adult navel orangeworm (NOW) antennal sensitivity and demonstrated that male and female NOW respond to different compounds. NOW lures were developed from high response compounds and are being field tested in commercial pistachio and almond orchards. In addition, researchers continued to make progress on the development of a lab-based behavioral assay for NOW attractancy. Under Sub-Objective 1B, researchers have completed experiments determining the required x-ray doses for NOW sterilization for all larval, pupal, and adult life stages. A new irradiation system has been developed to greatly increase the throughput rate (number of insects per unit time) for irradiation using standard x-ray tubes in which each insect receives the same dose (as opposed to gamma irradiation in which dose uniformity is a major issue). Plans to test x-ray irradiation of pistachio mummies in-field have been deemed impractical and are replaced with ongoing experiments that have demonstrated the feasibility of accomplishing the sub-objective with high pressure steam. Under Sub-objective 2B, ARS researchers in collaborative studies with scientists at the University of California, Davis, have completed the last application of manure to an almond orchard. Samples were obtained and analyzed for the presence of E. coli O157:H7, Salmonella enterica and Listeria monocytogenes by culture methods. Samples were also processed by 16S rRNA gene sequence analysis to determine the effects of manure addition on the microbial population structure of orchard soils. Under Sub-objective 2C, ARS researchers evaluated the benefits of using commercially sourced biochar as a delivery vehicle for atoxigenic Aspergillus flavus as a biocontrol agent to reduce toxigenic A. flavus in soil. Researchers observed that the viable numbers of atoxigenic A. flavus in soil increased over a three-month period. In addition, atoxigenic A. flavus outcompeted the toxigenic strain when grown as a mixture. Under Sub-objective 2D, ARS researchers completed high-throughput screenings of soil bacterial isolate libraries for antifungal activity against ochratoxin-producing Aspergillus carbonarius, as well as other species of Aspergillus, under different media conditions. The bacterial strains showing the strongest antifungal activities (inhibited growth and/ or spore production) were selected for use in soil microcosm assays. Soil microcosm competition assays, in which A. carbonarius on its own or in combination with A. niger, A. welwitschiae, and A. tubingensis, have been added to sterilized soil along with the selected bacterial strains, are underway. In ongoing experiments, the effects of each bacterial strain on the growth of Aspergillus species is monitored by direct plating at three- day intervals, and effects on the airborne movement of fungal spores is monitored at seven-day intervals by wind tunnel air sampling of the same soil microcosms, to mimic the effect of wind on the transfer of fungi from soil (the inoculum source) to the grape surface. In experiments with mixtures of Aspergillus species, the effect of bacterial strains was determined using quantitative polymerase chain reaction (PCR) methods to monitor changes in the proportion of each fungal species within the total population. Under Objective 3, ARS researchers identified natural compounds that can rapidly eliminate fungi from food products or crop field soils. While numerous investigations have focused on the rapid detection of microbial contaminants to ensure food safety or public health, effective intervention tools for the rapid elimination of pathogenic microbes are often limited. Natural compound Ben-1 achieved 99.9 percent fungal death at around 2.5 hours of application in commercial food matrices, or in soils of crop fields, suggesting that Ben-1 could serve as an ecologically sound anti-fungal agent with the potential of cost reductions during food/crop production. A second compound, Ben-2, significantly inhibited the growth of a fungal mutant deficient in the production of chitin, a primary component of fungal cell walls. Ben-2 also negatively affected the survival of two sugar metabolism mutants of fungi. Collectively, these natural benzaldehydes could be used as promising intervention tools for the rapid elimination of food/ environmental fungal contaminants. Under Objective 3, ARS researchers and a consortium of scientists from universities in the U.S., India, South Africa, Australia, and the United Kingdom tested numerous small molecule drugs, natural and bioactive compounds derived from food wastes for anti-microbial and anti-parasitic activities. Promising compounds were identified and will be further studied. New drug derivatives are being designed based on these results to further improve drug potencies. Accomplishments 01 Rapid elimination of fungal contaminants by natural benzaldehydes. Contamination of foods or crop field soils with fungi that either produce toxins or are resistant to conventional fungicide interventions present food safety concerns. ARS researchers in Albany, California, identified natural benzaldehyde compounds that can rapidly remove fungal contaminants from food or environmental matrices. The most potent Ben-1 eliminated 99.9 percent of fungi after about two and a half hours of application, even at low acidity conditions similar to those found at commercial fruit juice processing plants. Another compound, Ben-2, targeted the fungal sugar metabolism pathway, disrupting the growth of fungi defective in cell wall (chitin) production or in sugar utilization. This research represents an improvement over the use of the conventional fungicide thiabendazole, which has similar fungicidal activity to Ben-1, but had the undesirable side-effect of enhancing fungi production of toxins. Altogether, the novel natural compounds identified could serve as sustainable and rapid fungal control agents with great potential for industrial applications. 02 Sensitization of Navel Orangeworm to x-ray irradiation. The use of gamma irradiation for Navel Orangeworm sterilization in Sterile Insect Technique (SIT) pest control programs is problematic, and alternatives are needed. X-ray is an obvious choice, but their low power as compared to gamma sources create challenges for the sterilization of the required large numbers of Navel Orangeworms. Reducing the required x- ray dose for sterilization is one approach to alleviate this shortcoming. ARS researchers have demonstrated that adding certain natural compounds to Navel Orangeworm diets affected their response to x-ray irradiation, both in terms of required doses for sterilization as well as fitness following irradiation. This research demonstrated a proof of concept and provided valuable insight for the selection of natural compounds for future testing.
Impacts
(N/A)
Publications
- Hongsibsong, S., Prapamontol, T., Dong, J., Bever, C.R., Xu, Z., Gee, S.J., Hammock, B.D. 2019. Exposure of consumers and farmers to organophosphate and synthetic pyrethroid insecticides in Northern Thailand. Journal of Consumer Protection and Food Safety. 14(1):17-23.
- Hua, S.T., Sarreal, S.L., Chang, P., Yu, J. 2019. Transcriptional regulation of aflatoxin biosynthesis and conidiation in Aspergillus flavus by Wickerhamomyces anomalus WRL-076 for reduction of aflatoxin contamination. Toxins. 11(2):81.
- Tran, T.D., Huynh, S., Parker, C., Han, R., Hnasko, R.M., Gorski, L.A., McGarvey, J.A. 2018. Complete genome sequence of Lactococcus lactis subsp. lactis strain 14B4, which inhibits the growth of Salmonella enterica serotype Poona in vitro. Microbiology Resource Announcements. 7(19):e01364- 18.
- Kim, J., Chan, K.L., Cheng, L.W., Tell, L.A., Byrne, B.A., Clothier, K., Land, K.M. 2019. High efficiency drug repurposing design for new antifungal agents. Methods and Protocols. 2(2):31.
- Kumar, S., Bains, T., Kim, A., Tam, C.C., Kim, J., Cheng, L.W., Land, K.M., Debnath, A., Kumar, V. 2018. Highly potent 1H-1,2,3-triazole-tethered isatin-metronidazole conjugates against anaerobic foodborne, waterborne, and sexually-transmitted protozoal parasites. Frontiers in Cellular and Infection Microbiology. 8:380.
- Liang, P., Haff, R.P., Zayas, I.Y., Light, D.M., Mahoney, N.E., Kim, J. 2019. Curcumin and quercetin as potential radioprotectors and/or radiosensitizers for x-ray-based sterilization of male navel orangeworm larvae. Scientific Reports. 9:2016.
- McGarvey, J.A., Place, S., Palumbo, J.D., Hnasko, R.M., Mitloehner, F. 2018. Dosage-dependent effects of Monensin on the rumen microbiota of lactating dairy cattle. Advances in Dairy Research. 8(7):e00783.
- Milczarek, R.R., Liang, P., Wong, T., Augustine, M.P., Smith, J.L., Woods, R., Sedej, I., Olsen, C.W., Vilches, A.M., Haff, R.P., Preece, J.E., Breksa, A.P. 2019. Nondestructive determination of the astringency of pollination-variant persimmons (Diospyros kaki) using near-infrared (NIR) spectroscopy and nuclear magnetic resonance (NMR) relaxometry. Postharvest Biology and Technology. 149:50-57.
- Palumbo, J.D., O'Keeffe, T.L., Quejarro, B., Yu, A., Zhao, A. 2019. Comparison of Aspergillus section Nigri species populations in conventional and organic raisin vineyards. Mycotoxin Research. 76(7):848- 854.
- Pennerman, K.K., Gonzalez, J., Chenoweth, L.F., Bennett, J.W., Yin, G., Hua, S.T. 2018. Biocontrol strain Aspergillus flavus WRRL 1519 has differences in chromosomal organization and an increased number of transposon-like elements compared to other strains. Molecular Genetics and Genomics. 293(6):1507-1522.
- Beck, J.J., Gee, W.S., Cheng, L.W., Higbee, B.S., Wilson, H., Daane, K.M. 2018. Investigating host plant-based semiochemicals for attracting the leaffooted bug (Hemiptera: Coreidae), an insect pest of California agriculture. ACS Symposium Series. 1294:143-165.
- Singh, A., Zhang, D., Tam, C.C., Cheng, L.W., Land, K.M., Kumar, V. 2019. Synthesis and antiprotozoal activity of functionalized 1H-1,2,3-triazole- tethered isatin-ferrocene conjugates. Bioorganic and Medicinal Chemistry Letters. 896:1-4.
- Stringer, T., Seldon, R., Liu, N., Warner, D.F., Tam, C.C., Cheng, L.W., Land, K.M., Smith, P.J., Chibale, K., Smith, G.S. 2017. Antimicrobial activity of organometallic isonicotinyl and pyrazinyl ferrocenyl-derived complexes. Dalton Transactions. 46:9875-9885.
- Noritake, S.M., Liu, J., Kanetake, S., Levin, C.E., Tam, C.C., Cheng, L.W., Land, K.M., Friedman, M. 2017. Phytochemical-rich foods inhibit the growth of pathogenic trichomonads. BMC Complementary and Alternative Medicine. 17(1):461.
- Chellan, P., Stringer, T., Shokar, A., Au, A., Tam, C.C., Cheng, L.W., Smith, G.S., Land, K.M. 2019. Antiprotozoal activity of palladium (II) salicylaldiminato thiosemicarbazone complexes on metronidazole resistant Trichomonas vaginalis. Inorganic Chemistry. (102):1-4.
- Chellan, P., Avery, V., Duffy, S., Triccas, J.A., Nagalingam, G., Tam, C.C. , Cheng, L.W., Liu, J., Land, K.M., Clarkson, G.J., Romero, I., Sadler, P. J. 2018. Organometallic conjugates of the drug sulfadoxine for combatting antimicrobial resistance. Chemistry - A European Journal. 24(40):10078- 10090.
- Singh, A., Fong, G., Liu, J., Wu, Y., Chang, K., Park, W., Kim, J., Tam, C. C., Cheng, L.W., Land, K.M., Kumar, V. 2018. Synthesis and preliminary antimicrobial analysis of Isatin-ferrocene and Isatin-ferrocenyl-chalcone conjugates. ACS Omega. 3(5):5808-5813.
- Gumbo, M., Beteck, R.M., Mandizvo, T., Seldon, R., Warner, D.F., Hoppe, H. C., Issacs, M., Laming, D., Tam, C.C., Cheng, L.W., Liu, N., Land, K.M., Khanye, S.D. 2018. Cinnamoyl-oxaborole amides: Synthesis and their in Vitro biological activity. Molecules. 23(8):2038.
- Friedman, M., Huang, V., Quiambao, Q., Noritake, S.S., Liu, J., Kwon, O., Chintalapati, S., Levin, C.E., Tam, C.C., Cheng, L.W., Land, K.M. 2018. Potato peels and their bioactive glycoalkaloids and phenolic compounds inhibit the growth of pathogenic trichomonads. Journal of Agricultural and Food Chemistry. 66(30):7942-7947.
- Hua, S.T., Parfitt, D., Sarreal, S.L., Sidhu, G.K. 2019. Dual culture of atoxigenic and toxigenic strains of Aspergillus flavus to gain insight into repression of aflatoxin biosynthesis and fungal interaction. Mycotoxin Research.
- McGarvey, J.A., Han, R., Tran, T., Hnasko, R.M., Brown, P. 2018. Bacterial population dynamics after foliar fertilization of almond leaves. Journal of Applied Microbiology. 126(3):945-953.
|
Progress 10/01/17 to 09/30/18
Outputs
Progress Report Objectives (from AD-416): The long-term objective of this project is to reduce, inhibit, or eliminate toxigenic and pathogenic microbes (i.e., mycotoxigenic fungi or pathogenic bacteria) by utilizing intervention techniques such as biological control. Specifically, during the next five years we will focus on the following interrelated objectives. Objective 1: Develop and implement control measures to reduce, eliminate, or detect contamination of toxin producing fungi of tree nuts, for example the use of host plant- or fungal-derived semiochemicals to attract or control insect pests, or use of sterile insect techniques to decrease insect pest populations. � Sub-objective 1A: Use of host plant- or microbe-derived volatile semiochemicals to attract or control insect pests. � Sub-objective 1B: Use of sterile insect techniques to decrease insect pest populations. Objective 2: Elucidate principles of microbial ecology and develop biological control measures to inhibit pathogenic and toxigenic microorganisms, particularly fungi, and can include research on the isolation and development of new biocontrol agents and formulations to control or prevent toxigenic microbes, or survey, identify, and determine ecology of microbial populations for control strategies such as competitive microorganisms. � Sub-objective 2A: Isolate biocontrol agents that prevent pathogenic/ toxigenic microbes from colonizing crops. � Sub-objective 2B: Risk analysis of waste used as fertilizers for pathogen/toxigen contamination. � Sub-objective 2C: Develop new biocontrol agents and formulations to control toxigenic fungi, and to survey and characterize populations of Aspergilli. � Sub-objective 2D: Determine ecology of black-spored toxigenic Aspergilli and develop control strategies using competitive microorganisms. Objective 3: Discover natural chemical compounds that enhance the efficacy of established microbe intervention strategies, for instance augment the activity of antimicrobial agents/treatments against pathogens via target-based application of natural chemosensitizing agents. Approach (from AD-416): 1A. Tree nuts emit chemicals that attract insect pests that can be used as bait for insect traps. We will analyze volatiles from nuts by GC-MS and test them for pest attraction in electrophysiological and behavioral bioassays. If we are unable to identify volatiles from nuts we will explore volatiles from other biotic and abiotic matrices. 1B. Sterile insect technique can be applied to navel orange worms (NOW) inside discarded nuts on the orchard floor using an X-ray device towed behind a tractor. We will determine the X-ray dose required for sterilization of NOW and adjust this dosage to sterilize NOW inside tree nuts and develop a tractor towed device for field sterilization. If X-ray exposure does not produce sterile NOW other forms of radiation will be used. 2A. Bacteria with agonistic properties to pathogens are present on almond drupes and if applied in large numbers would prevent pathogen contamination. We will isolate bacteria from almonds and test their ability to inhibit pathogen growth in vitro. The bacteria that inhibit pathogen growth in vitro will be examined for the ability to inhibit growth on almonds, then in field trials. If we are not able to identify bacteria that inhibit pathogen growth on almonds we will use other crops. 2B. Applying composted manure to orchards does not represent a food safety threat. We will examine the microbial community structure of soil and fruit before and after the application of manure. We will repeat the analysis for 3 years to determine the effects of manure application. 2C. Atoxigenic Aspergillus flavus strains with deletions in the aflatoxin and CPA genes can be used as biological control agents for toxigenic A. flavus. We will identify atoxigenic A. flavus isolates by PCR and confirm by chemical analysis. We will examine their use as biocontrol agents via growth inhibition assays. Atoxigenic strains that displace the toxigenic strains will be impregnated into biochar and analyzed for as biocontrol agents in green house experiments. If the biochar is not suitable we will examine other matricies such as plastic granula. 2D. Ratios of toxigenic to non-toxigenic Aspergillus sp. fluctuate during the growing season; application of competitive fungal or bacterial strains will reduce mycotoxins in grapes/raisins. Grape/raisin samples will be taken at regular intervals in the growing season and analyzed to determine the ideal time to apply biocontrol agents against toxigenic Aspergillus. At these time points we will isolate bacteria and nontoxigenic Aspergillus sp. from raisin and soil samples and assay their ability to inhibit the growth of Aspergillus sp. If no non-toxigenic strains are not found other sources will be investigated. 3. Natural compounds and derivatives can control the growth of fungal pathogens and the production of toxins. Natural compounds will be tested for the disruption of cell wall integrity and the antioxidant pathway in fungi via genetic and physiologic analysis. We will determine the mode of action of these compounds via microarrays and other genetic tests. If we are unable to identify these compounds we will analyze other chemicals such as benzo derivatives Under Sub-objective 1A, researchers obtained pistachio mummies (unharvested nuts) and classified them on the level of hull degradation, the presence of a kernel, and whether visible mold was observed. Volatile emissions from these groups were identified and compared with the same mummies in an atmosphere of increased humidity that allowed for the production of microbial volatiles. A group of volatiles unique to the mummies was identified and categorized as originating from pistachio tissues with or without fungal damage. The ability of these volatiles to attract navel orange worm (NOW) adults is being evaluated. Researchers tested various insect traps in commercial orchards to determine the most effective design for use in monitoring the leaf-footed bug (LFB). A colony of LFBs was established from a wild population in a commercial pomegranate orchard and two additional populations from laboratory-reared colonies were maintained. A selection of volatiles common to the host plants of the LFB (i.e. almond, pistachio, pomegranate, citrus, and tomato) was compiled. The sensitivity of LFB antennae to detect each of these host plant volatiles was compared for male and female LFB from both the wild and lab-reared colonies. The most promising host plant volatiles were combined into blends and tested for their ability to lure LFBs to orchard traps. Field trials for the efficacy of these lures in almond, pistachio, and pomegranate orchards are ongoing. Under Sub-objective 1B, researchers have developed a portable x-ray irradiation unit for use in pistachio orchards to sterilize or kill Navel Orangeworm larvae overwintering in pistachio mummies on the orchard floor. A new approach using high pressure steam has also been developed and is being tested for the ability to kill the overwintering larvae, eliminating the need for using irradiation. A novel rearing system has been developed substantially increasing egg production for use in the sterile insect technique (SIT). Irradiation experiments testing the effect of adding certain natural compounds to insect diet, to reduce the required x-ray dose for sterilization, and/or increase their fitness following irradiation, have been completed. Irradiation experiments testing the effect of modified atmospheres on insect radio sensitivity have also been completed, potentially reducing dose requirements for insect sterilization for SIT. Under Sub-objective 2A, researchers have isolated multiple bacteria capable of inhibiting the growth of Salmonella in an in vitro fluorescence assay. Because of California Department of Food and Agriculture regulations restricting the use of experimental bio- pesticides in field trials, the model for growth and persistence on produce was changed from almond drupes to cantaloupe melons, which are available year-round and are suitable for growth in the greenhouse. These bacteria have been shown to grow and persist in cantaloupes, and in addition, inhibit the growth of Salmonella. The genomes of these bacteria have been sequenced and possible antimicrobial compounds have been identified. Two of these organisms have been patented and two different companies have expressed interest in using them in field trials. Under Sub-objective 2B, researchers in collaborative studies with researchers at the University of California, Davis, are in the third year of a project to determine the safety of applying composted cow manure to almond orchards. Samples have been taken every year and analyzed for E. coli O157:H7, Salmonella enterica and Listeria monocytogenes presence by culture methods. Samples are also being analyzed by 16S rRNA gene sequence analysis to describe changes in the microbial population structure in the soils. Under Sub-objective 2C, researchers have isolated genomic DNA from a biocontrol Aspergillus flavus strain followed by DNA sequencing. DNA fragments were assembled to construct a genomic library. Total RNA from this strain was also extracted and converted to cDNA and sequenced to obtain a transcriptomic library. Both sets of data were deposited into National Center for Biotechnology Information (NCBI). In-depth analysis of the genes involved in aflatoxin and cyclopiazonic acid (CPA) synthesis, as well as the genes associated with biocontrol efficacy are in progress. Researchers paired atoxigenic strains of Aspergillus flavus with toxigenic strains and grew them in dual cultures. Fungal material was collected and used to extract RNA for evaluation of the biosynthesis of aflatoxin of toxigenic strains (the toxigenic strain is the cause of aflatoxin contamination in almond, pistachio, peanut and corn). The results demonstrated that the regulatory gene, aflR for the toxin biosynthetic pathway, was inhibited by the presence of the atoxigenic strain. High-performance liquid chromatography analysis of the dual cultures showed significant reduction of aflatoxin presence by more than 90%. Under Sub-objective 2D, researchers described the ecology of black- spored toxigenic Aspergilli. Soil and fruit samples were collected from conventionally and organically farmed raisin grape vineyards in California for the second consecutive year. Samples were taken after berry formation, early in fruit ripening, at full ripeness, and following sun-drying into raisins. DNA from microorganisms present in soil and in washes of fruit surfaces were isolated and used in quantitative polymerase chain reaction (PCR) experiments to determine the relative amounts of the four predominant black-spored Aspergillus species. Results indicated that populations of fungi within vineyards fluctuate during the growing season. However, the relative population sizes and proportions of species (most importantly of ochratoxin-producing A. carbonarius) in soil and on fruit were not significantly different between conventional and organic vineyards, and were similar from year to year. These data suggest that biocontrol interventions would act similarly in conventional and organic vineyards. Also under Sub-objective 2D, researchers initiated studies to identify bacterial populations physically associated with toxigenic and nontoxigenic Aspergillus species in soil. Methods for isolating microbiome components of fungi from soil are in development, and metagenomic analysis of the bacterial species will be performed using next-generation sequencing of 16s ribosomal DNA fragments amplified from fungus-associated bacterial populations. These studies will provide information regarding differences in bacteria associated with toxigenic and non-toxigenic populations of the same Aspergillus species. Bacterial species identified in these studies will be screened for antifungal phenotypes that could be used in biocontrol applications. Under Objective 3, researchers have identified cinnamic acid derivatives that disrupt the cell wall integrity systems of fungi. While disruption of the fungal cell wall is an effective intervention strategy, certain commercial cell wall disruptants cannot completely inhibit the growth of filamentous fungi, including mycotoxin-producing Aspergillus. Four cinnamic acids identified possessed the highest cell wall-disrupting activity. In addition, the efficacy of commercial cell wall disruptants, such as caspofungin, could also be augmented by the co-application of cinnamic acids. Cinnamic acids further overcame resistance to antifungal agents such as fludioxonil. Thus, cinnamic acids can be developed as target-based (namely, disruption of cell wall and/or antioxidant system) intervention catalysts for the inhibition of toxigenic Aspergillus. Accomplishments 01 Mass rearing of navel orangeworm. Mass rearing (i.e. millions of moths per day) is one of the critical elements of an efficient sterile insect technology (SIT) program. The current SIT program operated by Animal and Plant Health Inspection Service (APHIS) in Phoenix, Arizona, utilizes glass jars under simulated daylight for mass rearing. ARS scientists in Albany, California, developed a new rearing system in which the larvae develop without light, resulting in faster insect development and substantially higher egg count. Results indicate that this method significantly increases moth production with minimal added effort or cost. 02 Isolation and characterization of biocontrol agents to reduce the growth of Salmonella on produce. ARS Scientists in Albany, California, created a library of bacteria normally associated with produce. This library was screened to identify bacteria capable of inhibiting the growth of Salmonella. Two isolates inhibited the growth of Salmonella on cantaloupe melons. The genomes of these bacteria were sequenced, and have revealed clues as to the mechanisms of Salmonella growth inhibition. Patents have been filed for these organisms, which could be used as biocontrol agents for farmers interested in inhibiting growth of bacterial pathogens in produce and farm environments. 03 Identification of an atoxigenic strain of Aspergillus flavus for use in the biocontrol of toxigenic fungal species. Aflatoxins, which can be present in nuts, are widely recognized as a major health problem. ARS scientists in Albany, California, identified an effective biocontrol agent that inhibited aflatoxin production by toxigenic Aspergillus flavus. The DNA and RNA of this strain was extracted, sequenced and assembled into two databases for gene discovery and expression studies. The libraries are useful to elucidate the genetic bases of effective biocontrol agents and for the identification of the relevant metabolic capabilities of atoxigenic A. flavus strains that promote biocontrol- related management of toxin contamination. These studies help define the most effective mycotoxin biocontrol agents for nut and farm crops. 04 Identification of natural cinnamic derivatives that interfere with fungal cell wall system. Due to increasing concerns about the safety of certain antifungal drugs that have been in wide use, and the impact of repeated exposure to these compounds on health and fungal resistance, there are constant demands for new antifungals or drug potentiators with improved health and safety profiles. To that end, ARS researchers in Albany, California, identified natural cinnamic derivatives that prevent fungal growth by disrupting the cell wall integrity of pathogens. The efficacy of caspofungin or octyl gallate, commercial cell wall disrupting agents, can be augmented by the co-application of cinnamic acid-1 (CA-1). Cinnamic derivatives can also overcome fludioxonil (fungicide) tolerance of Aspergillus antioxidant mutants. Collectively, natural cinnamic derivatives can be used to enhance current anti-pathogenic fungi treatments, allowing the reduced use of toxic antifungal agents or fungicides, thus leading to better human health and environmental impacts.
Impacts
(N/A)
Publications
- Hua, S.T., Palumbo, J.D., Parfitt, D., Sarreal, S.L., O'Keeffe, T.L. 2018. Development of a droplet digital PCR assay for population analysis of aflatoxigenic and atoxigenic Aspergillus flavus mixtures in soil. Mycotoxin Research. 34(3):187-194.
- Babrak, L.M., McGarvey, J.A., Stanker, L.H., Hnasko, R.M. 2017. Identification and verification of hybridoma-derived monoclonal antibody variable region sequences using recombinant DNA technology and mass spectrometry. Molecular Immunology. 90:287-294.
- Hnasko, R.M., Lin, A.V., McGarvey, J.A., Stanker, L.H. 2018. Enhanced detection of infectious prions by direct ELISA from the brains of asymptomatic animals using DRM2-118 monoclonal antibody and Gdn-HCl. Journal of Immunological Methods. 456:38-43.
- Hnasko, R.M., Lin, A.V., Stanker, L.H., McGarvey, J.A. 2018. A bioassay for the optimization of macrophage conditioned medium (MCM) as culture supplement used to promote hybridoma cell survival and growth. Monoclonal Antibodies in Immunodiagnosis and Immunotherapy. 37(3):126-133.
- Hua, S.T., Parfitt, D.E., Sarreal, S.L., Lee, B.G., Wood, D.F. 2018. First report of an atypical new Aspergillus parasiticus isolates with nucleotide insertion in aflR gene resembling to A. sojae. Mycotoxin Research. 34(2) :151-157.
- Kim, J.H., Chan, K.L., Cheng, L.W. 2018. Octyl gallate as an intervention catalyst to augment antifungal efficacy of caspofungin. Open Access Journal of Multidisciplinary Science. 1(1):19-28.
- Liang, P., Haff, R.P., Hua, S.T., Munyaneza, J.E., Yilmaz, M.T., Sarreal, S.L. 2017. Nondestructive detection of zebra chip disease in potatoes using near-infrared spectroscopy. Biosystems Engineering. 166:161-169.
- Singh, A., Nisha, Bains, T., Hahn, H.J., Liu, N., Tam, C.C., Cheng, L.W., Kim, J.H., Debnath, A., Land, K.M., Kumar, V. 2017. Design, synthesis and preliminary antimicrobial evaluation of n-alkyl chain tethered c-5 functionalized bis-isatins. MedChemComm. 8(10):1982-1992.
- Kim, J., Chan, K.L., Cheng, L.W. 2017. Cinnamic acid analogs as intervention catalysts for overcoming antifungal tolerance. Molecules. 22(10):1783.
- Tran, T., Huynh, S., Parker, C., Hnasko, R.M., Gorski, L.A., McGarvey, J.A. 2018. Complete genome sequences of three Bacillus amyloliquefaciens strains that inhibit the growth of Listeria monocytogenes in vitro. Genome Announcements. 6(25):e00579-18.
- Yin, G., Hua, S.T., Pennerman, K.K., Yu, J., Bu, L., Sayre, R.T., Bennett, J. 2018. Genome sequence and comparative analyses of atoxigenic Aspergillus flavus WRRL 1519. Mycologia. 110(3):482-493.
|
Progress 10/01/16 to 09/30/17
Outputs
Progress Report Objectives (from AD-416): The long-term objective of this project is to reduce, inhibit, or eliminate toxigenic and pathogenic microbes (i.e., mycotoxigenic fungi or pathogenic bacteria) by utilizing intervention techniques such as biological control. Specifically, during the next five years we will focus on the following interrelated objectives. Objective 1: Develop and implement control measures to reduce, eliminate, or detect contamination of toxin producing fungi of tree nuts, for example the use of host plant- or fungal-derived semiochemicals to attract or control insect pests, or use of sterile insect techniques to decrease insect pest populations. � Sub-objective 1A: Use of host plant- or microbe-derived volatile semiochemicals to attract or control insect pests. � Sub-objective 1B: Use of sterile insect techniques to decrease insect pest populations. Objective 2: Elucidate principles of microbial ecology and develop biological control measures to inhibit pathogenic and toxigenic microorganisms, particularly fungi, and can include research on the isolation and development of new biocontrol agents and formulations to control or prevent toxigenic microbes, or survey, identify, and determine ecology of microbial populations for control strategies such as competitive microorganisms. � Sub-objective 2A: Isolate biocontrol agents that prevent pathogenic/ toxigenic microbes from colonizing crops. � Sub-objective 2B: Risk analysis of waste used as fertilizers for pathogen/toxigen contamination. � Sub-objective 2C: Develop new biocontrol agents and formulations to control toxigenic fungi, and to survey and characterize populations of Aspergilli. � Sub-objective 2D: Determine ecology of black-spored toxigenic Aspergilli and develop control strategies using competitive microorganisms. Objective 3: Discover natural chemical compounds that enhance the efficacy of established microbe intervention strategies, for instance augment the activity of antimicrobial agents/treatments against pathogens via target-based application of natural chemosensitizing agents. Approach (from AD-416): 1A. Tree nuts emit chemicals that attract insect pests that can be used as bait for insect traps. We will analyze volatiles from nuts by GC-MS and test them for pest attraction in electrophysiological and behavioral bioassays. If we are unable to identify volatiles from nuts we will explore volatiles from other biotic and abiotic matrices. 1B. Sterile insect technique can be applied to navel orange worms (NOW) inside discarded nuts on the orchard floor using an X-ray device towed behind a tractor. We will determine the X-ray dose required for sterilization of NOW and adjust this dosage to sterilize NOW inside tree nuts and develop a tractor towed device for field sterilization. If X-ray exposure does not produce sterile NOW other forms of radiation will be used. 2A. Bacteria with agonistic properties to pathogens are present on almond drupes and if applied in large numbers would prevent pathogen contamination. We will isolate bacteria from almonds and test their ability to inhibit pathogen growth in vitro. The bacteria that inhibit pathogen growth in vitro will be examined for the ability to inhibit growth on almonds, then in field trials. If we are not able to identify bacteria that inhibit pathogen growth on almonds we will use other crops. 2B. Applying composted manure to orchards does not represent a food safety threat. We will examine the microbial community structure of soil and fruit before and after the application of manure. We will repeat the analysis for 3 years to determine the effects of manure application. 2C. Atoxigenic Aspergillus flavus strains with deletions in the aflatoxin and CPA genes can be used as biological control agents for toxigenic A. flavus. We will identify atoxigenic A. flavus isolates by PCR and confirm by chemical analysis. We will examine their use as biocontrol agents via growth inhibition assays. Atoxigenic strains that displace the toxigenic strains will be impregnated into biochar and analyzed for as biocontrol agents in green house experiments. If the biochar is not suitable we will examine other matricies such as plastic granula. 2D. Ratios of toxigenic to non-toxigenic Aspergillus sp. fluctuate during the growing season; application of competitive fungal or bacterial strains will reduce mycotoxins in grapes/raisins. Grape/raisin samples will be taken at regular intervals in the growing season and analyzed to determine the ideal time to apply biocontrol agents against toxigenic Aspergillus. At these time points we will isolate bacteria and nontoxigenic Aspergillus sp. from raisin and soil samples and assay their ability to inhibit the growth of Aspergillus sp. If no non-toxigenic strains are not found other sources will be investigated. 3. Natural compounds and derivatives can control the growth of fungal pathogens and the production of toxins. Natural compounds will be tested for the disruption of cell wall integrity and the antioxidant pathway in fungi via genetic and physiologic analysis. We will determine the mode of action of these compounds via microarrays and other genetic tests. If we are unable to identify these compounds we will analyze other chemicals such as benzo derivatives Under Objective 1A, ARS researchers from Albany, California performed research on the volatile profiles of stored almond and pistachio products as they await processing. Humidity (moisture) played a large role in the activation of fungi on both almonds and pistachios. Distinct volatile biomarkers can be identified based on humidity levels and could be used as signals for early detection of fungal contamination. In order to identify new semiochemicals for attracting insect pests in pistachios, ARS researchers from Albany, California identified and compared the volatile emissions from several categories of developing pistachios. During the growing season, pistachios are susceptible to navel orangeworm (NOW) infestation when the hull splits open. Early split pistachios open when the fruit is immature, while regular split and tattered hull pistachios occur as part of the normal ripening process, close to the time of harvest. All of the pistachio types allow for insect access through the hull and are at risk for NOW infestation. The volatile profiles of the split and unsplit pistachios consisted almost exclusively of terpenes. Insect assays showed that NOW adult females responded to these terpenes; however, blends of these compounds were ineffective as lures in pistachio orchards. Because these plant-derived volatiles were ineffective as field lures in pistachio orchards, a new effort was undertaken to identify microbe-derived volatiles as navel orangeworm attractants. These microbes (fungi) are most abundant in almond stocks; thus, stockpiled almonds were sorted to identify samples with field- generated mold contamination. Moldy almonds were incubated under elevated humidity to allow for the production of fungal volatiles, but not additional fungal growth. The presence of mold contamination increased the complexity and level of volatiles from the almond tissues, and resulted in the production of many compounds that tested well in NOW evaluations. These compounds will be used to produce new blends to test as NOW lures under field conditions. Another insect pest that vectors pathogenic fungi is the leaffooted bug (LFB), which has a wide range of host plants. In order to identify semiochemicals for use in pest lures, volatile extracts were prepared from a variety of fruits that are susceptible to LFB damage such as ripe pomegranates, moldy and split pomegranates, oil stock pistachios, oil stock almonds, and ripe oranges. Volatile profiles (either from pure compounds or blends) of these extracts were evaluated for LFB attraction in the laboratory. 30 different blends of these volatiles were tested for field attractiveness to LFB. Unfortunately, the field results could not be adequately evaluated due to the lack of effective traps for LFB (unlike for NOW, LFB adults escaped traps easily). Research is ongoing to develop an effective field test and better insect traps to evaluate volatile blends as lures for LFB. Under Objective 1B, a portable x-ray irradiation unit for use in pistachio fields has been completed, but due to equipment failure, implementation has been temporarily delayed. A novel rearing system has been developed in which NOW are �trained� to form their cocoons on flat sheets of paper, which are then attached to a cylinder that rotates below an x-ray source. This allows a high throughput of irradiated NOW pupae and overcomes the problem of non-uniformity of absorbed dose when irradiating insects in containers. New methodologies for rearing NOW have been developed to increase insect production with less required labor in anticipation of the need to provide sufficient quantities of sterile insects for sterile insect technique (SIT) implementation. Dosimetry methodologies have been improved and upgraded allowing more precise determination of absorbed dose. Under Objective 2A, a produce-associated bacterial library, containing over 80,000 isolates, was screened for the ability to inhibit Salmonella enterica growth using our recently developed in vitro fluorescence assay. Researchers identified 30 isolates that inhibited the growth of S. enterica between 36- and 164-fold after 48 hours, as compared to a phosphate buffered saline negative control. The isolates were members of the phyla Firmicutes (genera: Aerococcus, Bacillus, Carnobacterium, Enterobacter, Lactococcus and Weissella), and Proteobacteria (genera: Citrbacter, Hafnia, Klebsiella, Pantoea, Pseudomonas and Serratia). The entire library (80,000 isolates) was screened for the ability to inhibit the growth of E. coli O157:H7 and Listeria monocytogenes. Isolates that can inhibit these pathogens were identified and are currently being characterized. S. enterica growth inhibiting isolates are also being tested to determine if they can grow, persist and inhibit the growth of S. enterica on fresh produce. Under Objective 2B, researchers studied whether manure application affected pathogenic bacterial populations. Manure was applied to an orchard floor for two years and the microbial population structure was examined. Soil and composted manure were tested for the presence of the pathogens E. coli O157:H7, Salmonella enterica, and Listeria monocytogenes by culture methods. Increased microbial diversity was observed in the soils receiving manure and pathogens have not been idenfitied in any of the soils tested. Samples will be collected again at the end of this year and manure will be reapplied one more time. Under Objective 2C, in order to develop new Aspergilli biocontrol agents, ten atoxigenic Aspergillus flavus isolates, with deletions in both aflatoxin and cyclopiazonic biosynthesis genes were isolated and characterized. The efficacy of the atoxigenic strains to reduce aflatoxin production were evaluated in a dual cultural system. Different ratios of atoxigenic and toxigenic A. flavus strains were co-cultured for five days and the amount of toxin produced was determined. Several mixtures of atoxigenic and toxigenic A. flavus produced significantly lower amounts of aflatoxin, some by as much as 90%. Under Objective 2D, to determine the ecology of black-spored toxigenic Aspergilli, soil and fruit samples were taken from conventionally and organically farmed raisin grape vineyards in California at four stages during the growing season: after berries formed, at the early stages of fruit ripening, at fruit harvest, and following sun-drying into raisins. DNA from soil and from microorganisms adhering to fruit surfaces was isolated and used in quantitative polymerase chain reaction (PCR) experiments to determine fungal population size and structure (relative amounts of four predominant black-spored Aspergillus species) in soil and on fruit. Results from the first year of this study indicate that there are no significant differences in the size or composition of Aspergillus populations between conventional and organic vineyards, especially with regard to ochratoxin A (OTA)-producing A. carbonarius populations. This suggests that biocontrol interventions should act the same on these vineyards regardless of the farming practice. A second year of sampling of these vineyards to determine year-to-year variation in fungal populations is ongoing. The same vineyard samples were used to isolate bacterial strains, to test for antifungal activity against A. carbonarius under lab conditions. Researchers developed assays to identify bacteria that inhibit fungal growth on agar plates (diffusion of antifungal compounds), in liquid media (diffusion of antifungal compounds or direct cell-cell interactions) , and via production of volatile compounds (gas-phase antifungal compounds). Analysis of antifungal activities and identification of the antifungal compounds and the bacteria that produce them are ongoing. Under Objective 3, natural products have been identified that inhibited fungal growth (by disrupting fungal metabolism). One of the compounds identified acts as a natural fumigant and can enhance the potency of commercial fungicides (e.g. fludioxonil). This chemical possesses both antifungal and herbicidal activities; thus, the compound not only eliminates pathogens from raw tree nuts, but also controls the growth of weeds in orchards (a reservoir for pathogenic fungi). The development of a fungal pathogen control methodology for use in orchards using nutshell particles as a delivery matrix for these newly identified volatile compounds is underway. Accomplishments 01 New natural fumigant that inhibits pathogenic fungal growth identified. ARS researchers at Albany, California, identified benzaldehyde-1 (BA-1) as a natural fumigant that can effectively prevent fungal growth in tree nuts. Co-application of BA-1 with a conventional fungicide fludioxonil (FD) inhibited growth of FD resistant strains of Aspergillus sp. and Penicillium sp. Benzaldehydes prevented fungal growth by disrupting metal chelation in the metabolism processes. BA-1 also inhibited the germination of weed seeds, a natural reservoir for fungal pathogens. Thus, natural products such as BA-1, when used alone or in combination with existing fungicides, could serve as potent antifungals by controlling the growth of pathogenic fungi and their reservoirs. This research benefits crop industries by identifying safe and natural antifungals that can replace or reduce the use of existing fungicides. 02 Improved X-ray irradiation technique for generating sterile Navel Orangeworm (NOW). Insects reared for SIT (sterile insect techniques) programs are generally irradiated in relatively large containers, resulting in a non-uniform distribution of absorbed dose (i.e. insects closer to the edge of the container receive a higher dose than those at the center). Since it is essential to sterilize all insects in the container, the applied dose tends to over radiate some proportion of the insects, resulting in reduced fitness. ARS scientists at Albany, California, have developed a rearing-irradiating system in which NOW larvae are manipulated to form their cocoons on flat sheets of paper, which are subsequently attached to a rotating cylinder below an x-ray source. Since the insects are thus presented to the x-ray source in a single plane, the distribution of absorbed dose is much more uniform than that achieved using traditional irradiation techniques. This provides the means for producing sterile insects with higher overall fitness. This technique contributes toward controlling NOW, a major agricultural pest of fruits and nuts. 03 Development of a host plant volatile blend that attracts navel orangeworm in almonds. Damage of fruit nuts by navel orangeworm (NOW) has been associated with increases in Aspergillus infection, a risk factor for aflatoxin contamination. ARS researchers in Albany, California, developed and patented (U.S. Patent No. 9,655,366) a new blend of host plant volatiles that attract the navel orangeworm to almonds. The efficacy of this blend in attracting both male and female NOW was demonstrated in orchards and used in the monitoring of NOW infestation and as an aid in pest management. The NOW blend is being developed as a product for commercialization. This new technology will help farmers monitor and reduce NOW infestation in orchards and thus eliminate aflatoxin contamination.
Impacts
(N/A)
Publications
- Adams, M., Stringer, T., De Kock, C., Smith, P.J., Land, K.M., Liu, N., Tam, C.C., Cheng, L.W., Njoroge, M., Chibale, K., Smith, G.S. 2016. Bioisosteric ferrocenyl-containing quinolines with antiplasmodial and antitrichomonal properties. Dalton Transactions. 45(47):19086-19095.
- Kim, J.H., Hart-Cooper, W.M., Chan, K.L., Cheng, L.W., Orts, W.J., Johnson, K. 2016. Antifungal efficacy of octylgallate and 4-isopropyl-3- methylphenol for control of Aspergillus. Microbiology Discovery. 4:2.
- Liang, P., Haff, R.P., Moscetti, R., Light, D.M., Massintini, R. 2017. Detection of pit fragments in fresh cherries using near infrared spectroscopy. Near Infrared Spectroscopy Journal. 25(3):196-202.
- 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.
- Beck, J.J., Willett, D.S., Mahoney, N.E., Gee, W.S. 2016. Silo-stored pistachios at varying humidity levels produce distinct volatile biomarkers. Journal of Agricultural and Food Chemistry. 65:551-556.
- Beck, J.J., Willett, D.S., Gee, W.S., Mahoney, N.E., Higbee, B.S. 2016. Differentiation of volatile profiles of stockpiled almonds at varying relative humidity levels using benchtop and portable GC-MS. Journal of Agricultural and Food Chemistry. 64:9286-9292. doi: 10.1021/acs.jafc. 6b04220.
- Buckley, H.L., Hart-Cooper, W.M., Kim, J.H., Faulkner, D.N., Cheng, L.W., Chan, K.L., Vulpe, C.D., Orts, W.J., Amrose, S.E., Mulvihill, M.J. 2017. Design and testing of safer, more effective preservatives for consumer products. ACS Sustainable Chemistry & Engineering. 5(5):4320-4331. doi: 10. 1021/acssuschemeng.7b00374.
- Hnasko, R.M., Lin, A.V., Stanker, L.H., Bala, K., McGarvey, J.A. 2016. Prion extraction methods: comparison of bead beating, ultrasonic disruption and repeated freeze-thaw methodologies for the recovery of functional renilla-prion fusion protein from bacteria. In: Micic, M., editor. Sample Preparation Techniques for Soil, Plant, and Animal Samples. New York, NY: Humana Press. p. 389-399.
- Hua, S.T., Chang, P., Palumbo, J.D. 2017. Mycotoxins. In: Witczak, A., Sikorski, Z., editors. Toxins and Other Harmful Compounds in Foods. Boca Raton, FL: CRC Press. p. 153-168.
- Yin, G., Zhang, Y., Pennerman, K.K., Wu, G., Hua, S.T., Yu, J., Jurick II, W.M., Guo, A., Bennett, J.W. 2017. Characterization of blue mold Penicillium species isolated from stored fruits using multiple highly conserved loci. The Journal of Fungi. 3(1):12. doi:10.3390/jof3010012.
- Light, D.M., Grant, J., Haff, R.P., Knight, A.L. 2017. Addition of pear ester enhances disruption of mating by female codling moth (Lepidoptera: Tortricidae) in walnut orchards treated with meso dispensers. Environmental Entomology. 46(2):319-327.
|
Progress 10/01/15 to 09/30/16
Outputs
Progress Report Objectives (from AD-416): The long-term objective of this project is to reduce, inhibit, or eliminate toxigenic and pathogenic microbes (i.e., mycotoxigenic fungi or pathogenic bacteria) by utilizing intervention techniques such as biological control. Specifically, during the next five years we will focus on the following interrelated objectives. Objective 1: Develop and implement control measures to reduce, eliminate, or detect contamination of toxin producing fungi of tree nuts, for example the use of host plant- or fungal-derived semiochemicals to attract or control insect pests, or use of sterile insect techniques to decrease insect pest populations. � Sub-objective 1A: Use of host plant- or microbe-derived volatile semiochemicals to attract or control insect pests. � Sub-objective 1B: Use of sterile insect techniques to decrease insect pest populations. Objective 2: Elucidate principles of microbial ecology and develop biological control measures to inhibit pathogenic and toxigenic microorganisms, particularly fungi, and can include research on the isolation and development of new biocontrol agents and formulations to control or prevent toxigenic microbes, or survey, identify, and determine ecology of microbial populations for control strategies such as competitive microorganisms. � Sub-objective 2A: Isolate biocontrol agents that prevent pathogenic/ toxigenic microbes from colonizing crops. � Sub-objective 2B: Risk analysis of waste used as fertilizers for pathogen/toxigen contamination. � Sub-objective 2C: Develop new biocontrol agents and formulations to control toxigenic fungi, and to survey and characterize populations of Aspergilli. � Sub-objective 2D: Determine ecology of black-spored toxigenic Aspergilli and develop control strategies using competitive microorganisms. Objective 3: Discover natural chemical compounds that enhance the efficacy of established microbe intervention strategies, for instance augment the activity of antimicrobial agents/treatments against pathogens via target-based application of natural chemosensitizing agents. Approach (from AD-416): 1A. Tree nuts emit chemicals that attract insect pests that can be used as bait for insect traps. We will analyze volatiles from nuts by GC-MS and test them for pest attraction in electrophysiological and behavioral bioassays. If we are unable to identify volatiles from nuts we will explore volatiles from other biotic and abiotic matrices. 1B. Sterile insect technique can be applied to navel orange worms (NOW) inside discarded nuts on the orchard floor using an X-ray device towed behind a tractor. We will determine the X-ray dose required for sterilization of NOW and adjust this dosage to sterilize NOW inside tree nuts and develop a tractor towed device for field sterilization. If X-ray exposure does not produce sterile NOW other forms of radiation will be used. 2A. Bacteria with agonistic properties to pathogens are present on almond drupes and if applied in large numbers would prevent pathogen contamination. We will isolate bacteria from almonds and test their ability to inhibit pathogen growth in vitro. The bacteria that inhibit pathogen growth in vitro will be examined for the ability to inhibit growth on almonds, then in field trials. If we are not able to identify bacteria that inhibit pathogen growth on almonds we will use other crops. 2B. Applying composted manure to orchards does not represent a food safety threat. We will examine the microbial community structure of soil and fruit before and after the application of manure. We will repeat the analysis for 3 years to determine the effects of manure application. 2C. Atoxigenic Aspergillus flavus strains with deletions in the aflatoxin and CPA genes can be used as biological control agents for toxigenic A. flavus. We will identify atoxigenic A. flavus isolates by PCR and confirm by chemical analysis. We will examine their use as biocontrol agents via growth inhibition assays. Atoxigenic strains that displace the toxigenic strains will be impregnated into biochar and analyzed for as biocontrol agents in green house experiments. If the biochar is not suitable we will examine other matricies such as plastic granula. 2D. Ratios of toxigenic to non-toxigenic Aspergillus sp. fluctuate during the growing season; application of competitive fungal or bacterial strains will reduce mycotoxins in grapes/raisins. Grape/raisin samples will be taken at regular intervals in the growing season and analyzed to determine the ideal time to apply biocontrol agents against toxigenic Aspergillus. At these time points we will isolate bacteria and nontoxigenic Aspergillus sp. from raisin and soil samples and assay their ability to inhibit the growth of Aspergillus sp. If no non-toxigenic strains are not found other sources will be investigated. 3. Natural compounds and derivatives can control the growth of fungal pathogens and the production of toxins. Natural compounds will be tested for the disruption of cell wall integrity and the antioxidant pathway in fungi via genetic and physiologic analysis. We will determine the mode of action of these compounds via microarrays and other genetic tests. If we are unable to identify these compounds we will analyze other chemicals such as benzo derivatives This is a new project established in February 2016, and continues research from 2030-42000-037, "Chemical Approaches to Eliminate Fungal Contamination and Mycotoxin Production in Plant Products" and 2030-42000- 038-00D, "Environmental and Ecological Approaches to Eliminate Fungal Contamination and Mycotoxin Production in Plant Products." A bacterial library containing 80,000 phyllosphere associated bacterial isolates from various types of produce was constructed and we are beginning to screen the isolates for the ability to inhibit growth of the pathogens Salmonella enterica, Escherichia coli, and Listeria monocytogenes using our in vitro fluorescent assay. Preliminary results suggest we have over 400 isolates that are able to inhibit the growth of Salmonella enterica by over 50-fold. Volatile profiles of varying treatments of pomegranate, almond, and pistachio matrices were obtained and candidate volatiles were investigated for responses of leaffooted bug via laboratory-based electrophysiological and behavioral bioassays. Field trials for attracting the leaffooted bug in pomegranate, almond, and pistachio orchards was performed by an industry stakeholder collaborator. Conventional and organic raisin vineyards have been identified and fruit and soil sampling has begun. Bacteria and fungi have been isolated from these samples and identification and characterization of these microorganisms is underway. We have identified chemical compounds that enhance the efficacy of established antimicrobe intervention strategies, such as augmenting the activity of commercial antifungal reagents or treatments against filamentous or model fungi via target-based application of chemosensitizing agents. In particular, benzo analogs identified in this study modulate/debilitate the cell wall integrity of fungi, which significantly lower the effective doses, and thus costs, of commercial antimycotic agents. Of note, certain ecologically benign compounds also functioned as heat-sensitizing agents, which further lowered the costs of antifungal treatments. Multiple x-ray irradiation units have been constructed and are available for use at: Otis Air Force Base (Animal and Plant Health Inspection Service), Massachusetts; Hilo, Hawaii (ARS); and in Albany, California (ARS). The x-ray emitter system is installed and operational in the x-ray bunker in Albany, and dose rate experiments have begun. Required x-ray irradiation doses for sterilization of adult male navel orangeworm moths have been determined and published. Preliminary experiments have been conducted establishing the required x-ray doses for sterilization of female adults and larvae, but are not yet completed or published. Lab based experiments simulating in-field irradiation of pistachio mummies have begun. Polymerase chain reaction (PCR) primers have been designed for aflatoxin and cyclopiazonic acid biosynthetic genes for use in multiplex PCR to detect the presence or absence of the genes in isolates of A. flavus collected from almond and pistachio orchards in California. A few variants of A. flavus were identified and found to have deletions in both aflatoxin biosynthetic and CPA genes. Screening for additional deletion variants is essential for the success of the project. Molecular markers have been tested to characterize the deletion Aspergillus flavus strains. Accomplishments 01 Natural compounds that enhance activity of fungicides. Mycotoxin producing fungi such as Asperegilli are increasingly developing resistance to current fungicides. ARS researchers at Albany, California, discovered that the natural compound 2-hydroxy-4-methoxybenzaldehyde (2H4M) can act as a chemosensitizer when used together with monoterpenoid phenol compounds. 2H4M acts by weakening the cell wall integrity of fungi. Of particular interest, 2H4M overcame the tolerance of Aspergillus mutants to fludioxonil (a conventional fungicide) as well as inhibited aflatoxin production by a toxin producing fungi. Application of this natural compound with mild heat (57.5oC) for a short time period (90 seconds), resulted in more than 99.999% bacterial and fungal reduction, thus allowing safe, rapid, and energy/cost- effective pathogen elimination in agricultural and food processing environments. The use of this natural compound could lead to better mycotoxin inhibition methods and reduce use of fungicides. 02 Sterile insect technique for navel orangeworm (NOW). Using custom built x-ray irradiators, ARS scientists at Albany, California have determined and reported the required x-ray dose for sterilization of adult male NOW moths. The reproductive fitness of the sterilized moths has been demonstrated through mating studies. The efficacy of conventional x-ray tube based irradiators has thus been demonstrated as a potential substitute for radioisotopes for insect sterilization. Given the increasing difficulties in obtaining and maintaining radioactive sources, a practical substitute for gamma irradiation for insect sterilization could positively impact sterile insect technique programs worldwide.
Impacts
(N/A)
Publications
- Kim, J.H., Chan, K.L., Mahoney, N.E. 2015. Augmenting the activity of monoterpenoid phenols against fungal pathogens using 2-hydroxy-4- methoxybenzaldehyde that target cell wall integrity. International Journal of Molecular Sciences. (16)26850-26870. doi: 10.3390/ijms161125988.
- Haff, R.P., Jackson, E.S., Gomez, J., Light, D.M., Follett, P.A., Simmons, G.S., Higbee, B.S. 2015. Building lab-scale x-ray tube based irradiators. Journal of Radiation Physics and Chemistry. 121:43-49.
- Light, D.M., Ovchinnikova, I., Jackson, E.S., Haff, R.P. 2015. Effects of x-ray irradiation on male navel orangeworm (Lepidoptera: Pyralidae) on mating, fecundity, fertility, and inherited sterility. Journal of Economic Entomology. 8(5):2200-2212. doi: 10.1093/JEE/TOV201.
|
|