Research Project: Biocontrol Interventions for High-Value Agricultural Commodities

Location: Foodborne Toxin Detection and Prevention Research

2016 Annual Report

Objectives
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
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 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
1. 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.

2. 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.

None.

Review 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.