Project : USDA ARS

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Research Project: Novel Methods for the Mitigation of Human Pathogens and Mycotoxin Contamination of High Value California Specialty Crops

Location: Foodborne Toxin Detection and Prevention Research

2024 Annual Report

Objectives
Successful execution of these Objectives will contribute to field by: improving our knowledge of how microbial populations can affect and impact food safety and public health and delineating how pathogens are transmitted and disseminated in and among plant crops allowing for future development of improved/alternate interventions and control strategies (Objectives 1-4); developing novel intervention strategies using sustainable, natural fungicide alternatives that eliminate aflatoxigenic fungi; enhancing our knowledge regarding the prevalence of azole-resistant aspergilli with enhanced aflatoxin production (Objective 5); and developing novel methods to control invasive insect pests and reducing the need for the use of radioisotopes for irradiation (Objective 6). These Objectives, if successful, will allow growers to produce a safer food supply and reduce the use of toxic chemicals (pesticides) and enhance environmental quality. Objective 1: Identify and characterize agricultural soils that suppress the persistence of the human pathogenic bacteria Salmonella enterica, Listeria monocytogenes and Escherichia coli O157:H7. Objective 2: Examine the microbiomes, potential for human pathogen colonization, and effectiveness of biological control agents on lettuces grown in indoor vertical hydroponic systems. Objective 3: Examine the effects of bacterial biocontrol candidate strains on population dynamics of black Aspergillus spp. on grapes and raisins. Objective 4: Identification and utilization of antifungal metabolites from microbial sources as interventions. • Sub-objective 4A: Identification of antifungal metabolites from candidate biocontrol bacteria collected from raisin grape vineyards. • Sub-objective 4B: Isolation and characterization of bacteria with antifungal activities from pistachio orchards. Objective 5: Development of resistance management augmenting fungal and mycotoxin elimination. • Sub-objective 5A: Determine the prevalence of azole-resistant aspergilli (A. flavus, A. parasiticus) that produce increased levels of mycotoxins in California tree nut orchards. • Sub-objective 5B: Develop new intervention strategies for the control of azole-resistant Aspergillus species utilizing natural products/derivatives as fungicide alternatives. Objective 6: Investigate novel methods to address mycotoxin contamination of tree nuts through control of fungal and insect vectors. • Sub-objective 6A: Evaluate X-ray based irradiation as an alternative to gamma irradiation for SIT. • Sub-objective 6B: Investigate high pressure steam as a tool for orchard sanitation through destruction of overwintering NOW larvae in pistachio mummies. Objective 7: The use of previously approved natural products as an accelerated chemical interventions strategy to inhibit food-associated mycotoxins, fungal pathogens, and their insect pest transmitters. • Sub-objective 7A: Identify previously approved natural products that inhibit mycotoxins and fungal pathogens frequently found in food contaminations. • Sub-objective 7B: Identify previously approved natural products that immunosuppress insect pests and increase their sensitivity to microbes.

Approach
1. Bacteria with agonistic properties to pathogens are present in soils and if applied in large numbers would prevent pathogen persistence. We will isolate bacteria from soils and test their ability to inhibit pathogen growth in vitro. The bacteria that inhibit pathogen growth will be examined for the ability to inhibit pathogen persistence in soils. 2. In vitro hydroponic systems (IVHS) are used to grow leafy greens indoors. Little is known about the effects of IVHS on the plant microbiome. We will compare the microbiomes of leafy greens grown in IVHS and conventional outdoor cropping systems (COCS). We will also compare the ability of pathogens to grow on IVHS and COCS leafy greens. 3. Bacterial collections from vineyards will be screened for antifungal activity against ochratoxin-producing Aspergilli using a spore pour plate/stamp method. Effects on mixed-species Aspergillus populations in culture and on grapes after bacterial treatment will be measured by quantitative PCR for each fungal species. 4A. Cell-free extracts of bacteria identified in Obj. 3 will be screened for antifungal activity against the same Aspergillus species using similar spore plate method. Antifungal products will be separated and identified by LC-MS. Genome sequences of antifungal bacteria will help to identify potentially novel antifungal products. 4B. Bacteria will be collected from pistachio fruits and soil during growing season and screened as in Obj. 3 against ochratoxin-producing Aspergillus species associated with pistachio. Cell-free extracts will be screened and antifungal products identified as in Obj. 4A. 5A. Azole resistance testing for aspergilli will be performed on soil, dust and orchard debris samples. Resistant fungi will have their target genes sequenced to determine the mechanism. Increased levels of aflatoxins will be determined via liquid chromatography, that will be compared to the newly developed imaging system having high throughput capacity. 5B. The pest control efficacy of the natural fumigant Benzoic-1, possessing both fungicidal and herbicidal properties, will be performed via soil solarization. The use of plant-derived compound, Benzoic-2, will be examined as an alternative to azole fungicides for seed sanitation during seed soaking using corn and Brassica inoculated with aspergilli. 6A. The current custom irradiator configuration comprises moths in Ziploc bags rotated on the surface of a drum adjacent to the x-ray sources. An automated moth collection process will be developed based on vacuum and LED lights. Various x-ray tube and drum configurations will be tested to increase throughput of sterilized moths. 6B. Infested pistachio nuts will be refrigerated to induce overwintering. An autoclave will be used to determine conditions required for mortality. An electric powered steam unit will be employed in a simulated orchard environment to demonstrate feasibility. Finally, a steam unit will be used to treat actual orchard rows. 7. We will screen the library of approved natural products that inhibit i) the cytotoxicity of mycotoxins in cell survival assays, ii) the growth of A. flavus and parasiticus, and iii) Navel Orangeworm immunity.

Progress Report
This report documents progress for project 2030-42000-054-000D, titled, “Novel Methods for the Mitigation of Human Pathogens and Mycotoxin Contamination of High Value California Specialty Crops”, which started in January 2021. In support of Objective 1, ARS researchers in Albany, California, completed the screen of approximately 20,000 bacteria isolates from soils that were found to be suppressive for the persistence of the human pathogens Salmonella enterica, Escherichia coli and Listeria monocytogenes. Among the bacterial isolates identified as suppressive to pathogens in vitro were Citrobacter braakii and Enterococcus casseliflavus. Researchers found that both bacteria do not inhibit plant growth and can persist and inhibit the pathogens in soil. Researchers are now performing experiments to identify the genes involved in pathogen suppression. For Objective 2, ARS researchers have completed the microbiome analysis and pathogen growth experiments of leafy greens grown under conventional outdoor and indoor hydroponic growth methodologies. The researchers have also initiated studies to determine if biological control agents identified in previous studies are effective for pathogen suppression on indoor vertical hydroponic grown leafy greens. In support of Objective 3, ARS researchers in Albany, California, identified candidate bacterial strains for biocontrol of ochratoxin- and aflatoxin-producing Aspergillus species from environmental bacterial libraries. Laboratory experiments to demonstrate effects of biocontrol strains on mixed populations of black-spored Aspergillus species in culture and on grape and raisin surfaces have been initiated. For Sub-objective 4.A, ARS researchers previously identified candidate bacterial strains that showed antifungal activity against black-spored Aspergillus species relevant to the grape environment, yellow-spored ochratoxin-producing Aspergillus species relevant to the tree nut environment, and aflatoxin-producing Aspergillus flavus. Screens to determine antifungal activities present in bacterial culture filtrates were developed and used to determine breadth of antifungal activity produced by candidate bacterial strains. Identification of active components of culture filtrates, and genome sequencing of candidate bacterial strains are ongoing. In support of Sub-objective 4.B, ARS researchers in Albany, California, have compiled a library of fast- and slow-growing bacterial strains isolated from commercial almond and walnut vineyards and experimental pistachio vineyards. This library has been screened for antifungal activity against ochratoxin-producing Aspergillus species relevant to tree nut and grape environments (A. ochraceus, A. westerdijkiae, A. steynii, A. carbonarius) and aflatoxin-producing A. flavus. Candidate biocontrol strains have been identified and further characterization of antifungal activities is ongoing. For Sub-objective 5.A, ARS researchers in Albany, California, performed baseline determination of azole-resistant aspergilli in California farms and identified the correlation between azole resistance and aflatoxin (AF) production. It’s determined that fungicide resistance of Aspergillus field isolates was unique to azoles where one A. flavus isolate exhibited increased level of AF production compared to the non-resistant strains. AF over-production by A. flavus was not linked to stress-dependent fungal fitness. Under Sub-objective 5.B, ARS researchers performed antifungal “drug/compound repurposing” which is a repositioning process of already marketed, off-patent pharmaceutical drugs/compounds to control fungi, such as agricultural fungal pathogens. Results indicated that the sensitivity of the drug/compound repurposing could be augmented by ARS-developed “chemosensitization” method, where co-application of a second compound (namely, chemosensitizer; natural or synthetic) with a commercial drug/compound significantly enhanced antifungal efficacy of the co-applied drugs/compounds. Supporting Sub-objective 6.A, ARS researchers in Albany, California, have delivered and installed an x-ray-based irradiation cabinet at the University of California, Kearney Agricultural Research Extension Center in Parlier, California, for use in the ongoing navel orangeworm sterile insect technique program. The unit is being evaluated as a replacement for gamma irradiation as the method of sterilization. This work has urgency, since the 2019 Nuclear Defense Authorization Act (Section 3141) set a goal of replacing all gamma irradiators with x-ray devices by 2027 and suitable replacements for insect sterilization are still elusive. For Sub-objective 6.B, ARS researchers in Albany, California, have changed direction, as the original plan to deploy a full-sized highway ready steam unit in California orchards is problematic due to space limitations and irrigation systems. The unit has been stripped down and is being rebuilt in a more compact and maneuverable format. The work is ongoing, but completion is anticipated before the beginning of FY25. In support of Sub-objective 7.A, ARS researchers in Albany, California, identified four natural volatile chemicals capable of inhibiting the growth of five mycotoxin-producing fungi: Aspergillus flavus, Aspergillus parasiticus, Penicillium expansum, Fusarium verticillioides, and Fusarium oxysporum. Antifungal concentration ranges of the identified natural chemicals have been determined. Two of the chemicals synergized to effectively prevent the growth of all five fungi. The efficacy of these chemicals against the human fungal pathogens, Candida albicans and Candida auris, has also been determined. Under Sub-objective 7.B, ARS researchers identified 20 additional natural products that rapidly kill navel orangeworms (NOW). Overall, the researchers identified 40 compounds that effectively kill adult NOW moths. Effective insecticidal concentration ranges have been determined for some of the chemicals. The antennae-dependent insecticidal mechanism of action has been determined for some of the chemicals. The efficacy of several of these chemicals has been shown individually and in combination in mini-fumigation tests using NOW-infested almonds.

Accomplishments
1. Biocontrol of Escherichia coli O157:H7 by Enterobacter asburiae AEB30. Escherichia coli O157:H7 causes more than 73,000 foodborne illnesses in the United States annually, many of which have been associated with fresh ready-to-eat produce. There are currently no pre-harvest and few post-harvest methods to prevent E. coli growth and persistence on produce. ARS scientists in Albany, California, demonstrated that E. asburiae AEB30 was able to grow, persist and inhibit the growth of E. coli on cantaloupe melons under pre-harvest conditions and significantly reduce the levels of E. coli on contaminated melons under post-harvests conditions. ARS scientists demonstrated that mode of action of E. asburiae AEB30 against E. coli was due to a contact-dependent growth inhibition (CDI) system. This discovery provides a new method to prevent E. coli contamination of cantaloupe melons in both pre- and post-harvest environments and demonstrates for the first time that bacteria containing CDI systems can be used as biocontrol agents.

U.S. DEPARTMENT OF AGRICULTURE

Foodborne Toxin Detection and Prevention Research: Albany, CA