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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

2021 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
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.

1. 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).

Review 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.
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.
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.
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.
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.
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.