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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Foodborne Toxin Detection and Prevention Research » Research » Research Project #430817

Research Project: Biocontrol Interventions for High-Value Agricultural Commodities

Location: Foodborne Toxin Detection and Prevention Research

2020 Annual Report

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.

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

Progress Report
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.

1. 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.

2. 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.

3. 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.

Review 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).