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

2025 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 FY 2025 progress for project 2030-42000-054-000D, “Novel Methods for the Mitigation of Human Pathogens and Mycotoxin Contamination of High Value California Specialty Crops”, which began in January 2021. In support of Objective 1, ARS researchers in Albany, California, sequenced the genomes of the previously identified biological control agents Enterococcus casseliflavus and Citrobacter braakii that inhibit the persistence of the pathogens Escherichia coli and Salmonella enterica in soil. Analysis of the genomes failed to identify secondary metabolite biosynthetic genes, so transposon mutant libraries containing greater than 10,000 mutants were constructed to determine their mode of inhibition. Mutations in genes responsible for lactic acid production in E. casseliflavus could no longer inhibit the pathogens, thus lactic acid is responsible for pathogen inhibition. Mutations in genes responsible for general fitness in C. braakii could no longer inhibit the pathogens and it was concluded that bacterium's ability to outcompete the pathogens was responsible for inhibition. For Objective 2, ARS researchers in Albany, California, in collaboration with scientists and engineers at the University of California, Berkeley, designed and constructed a bespoke ebb and flow hydroponic plant growth system. Studies to determine the suitability of previously identified biological control agents for the suppression of human pathogen colonization of lettuce plant roots grown in the hydroponic system are ongoing. Under Objective 3, ARS researchers in Albany, California, performed laboratory experiments to demonstrate differences in antifungal activity of each bacterial culture against different fungal species. Experiments to determine antifungal activity on grapes and raisins artificially infested with mixtures of Aspergillus species are ongoing. In support of Sub-objective 4A, ARS researchers in Albany, California, continued whole genome sequence analysis of 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 to determine breadth of antifungal activity produced by candidate bacterial strains were continued. Identification of active components of culture filtrates are ongoing. For Sub-objective 4B, ARS researchers in Albany, California, continued to isolate bacterial strains from commercial almond and walnut vineyards and experimental pistachio vineyards. Libraries were compiled using multiple isolation media to increase the variety of bacterial species recovered. Libraries have 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. Additional experiments to confirm anti-fungal activities and to demonstrate differential activity among combinations of bacterial candidate strains and fungal target species are in development. Under Sub-objective 5A, ARS researchers in Albany, California, collaborated with scientists at the University of California, Los Angeles, to develop optimum pulsed terahertz imaging system. The multi-well plates containing almond samples were mounted on a motorized two-axis translational stage and placed on the terahertz beam path in a way that the focused terahertz radiation transmitted through them. Results showed the terahertz scanner can offer adequate sensitivity to detect the maximum allowed aflatoxin contamination level regulated by the U.S. Food and Drug Administration (20 ppb). Pilot scale, high throughput pulsed terahertz imaging system is currently under development which will enable high efficiency aflatoxin analysis. In support of Sub-objective 5B, ARS researchers in Albany, California, examined new natural antifungal compounds for their ability to inhibit the growth of aflatoxigenic and fungicide resistant Aspergillus species isolated from a California farm. The selected compounds exhibited potent antifungal and anti-aflatoxigenic activity against aspergilli including azole resistant Aspergillus flavus (up to 77.5 percent aflatoxin reduction); they also inhibited the growth of Fusarium and Penicillium species infecting food crops. Yeast gene deletion mutant bioassay indicated that one of the molecules targets cell wall integrity system of fungi. Formulation study is being progressed to create reliable antifungal products to be used for tree nut industry. For Sub-objective 6A, ARS researchers in Albany, California, have incorporated a conveyor-based insect handling system for the previously developed thin layer X-ray irradiator, demonstrating that insects can be sterilized in large numbers with these systems. This development confirms that thin layer irradiation, which allows the use of low energy X-rays for high dose rate irradiation with superior dose precision and uniformity, is an appropriate alternative for traditional radioactive cesium or cobalt sources. Congress has called for replacing all gamma irradiators with X-ray devices by 2027. Under Sub-objective 6B, ARS researchers in Albany, California, have performed lab-based experiments with infested pistachio mummies to demonstrate that high-pressure stem is an effective means for orchard sanitation even when the mummies are buried up to four centimeters deep. Development of a portable system for use in the orchard is ongoing. In support of Sub-objective 7A, ARS researchers in Albany, California, identified a new plant-derived chemical that inhibits five mycotoxin-producing fungi: Aspergillus flavus, A. parasiticus, Penicillium expansum, Fusarium verticillioides, and F. oxysporum. This compound synergized with two previously discovered natural volatile antifungal chemicals to suppress fungal growth. Two of the three chemicals also reduced aflatoxin production. For Sub-objective 7B, ARS researchers in Albany, California, identified a new plant-derived chemical and two previously discovered natural volatile antifungal chemicals that rapidly kill the navel orangeworm. They were combined with two previously identified plant-made insecticides to form a five-compound blend. This new formulation exhibited strong, synergistic, broad-spectrum activity against three key pests of California tree nuts: navel orangeworm, Carpophilus truncatus, and carob moth. Efficacy was demonstrated in both spray and fumigation trials.

Accomplishments
1. New utility of natural polyphenol as a fungal and mycotoxin control agent. Identification of a new utility of various marketed, non-antifungal compounds, such as food additives/supplements or medicinal agents, as novel fungicides is a sustainable, cost-effective strategy for fungal pathogen control. The merit of compound reutilization is that the mechanisms of action or safety of the candidate compounds has already been characterized, which reduces the time for regulatory approval. Researchers in Albany, California, developed a reutilization method wherein combined application of a second component (termed “potentiator”) with a non-antifungal compounds (termed “candidates”) greatly enhanced antifungal potency of the candidates by weakening fungal defense system. Using this method to test eighty-eight natural medicinal reagents, researchers found one polyphenol compound, which has been used as herb/nutritional supplement, exhibited potent antifungal activity against fungal pathogens infecting food crops; the polyphenol also inhibited aflatoxin production by foodborne fungi including fungicide-resistant field isolate. The selected compound, along with previously identified candidate compounds, will be investigated further for field application via biopolymer-based controlled release. The developed method could be applied for high-efficiency, large-scale screening of candidate compounds for new, safe fungicides that expand new market opportunities through safer, aflatoxin-free food production/exports, deliver regulatory relief by lowering dependance on toxic fungicides, and overcome fungicide resistance, which benefits stakeholders and consumers.

2. Natural fumigant agent for fungal and aflatoxin control. Fungal infections and aflatoxin contamination thereof in food crops are serious food safety and trade concern since effective agents for treating fungal pathogens are often very limited. The long-term application of conventional fungicides to crop fields also triggers fungal resistance to commercial fungicides. Researchers in Albany, California, identified that safe natural compounds or structural derivatives, such as salicylaldehyde (SLD), were highly effective for fungal and aflatoxin control as a fumigant; the treatment effectively disrupted the proliferation of aflatoxin-producing fungi in crops, such as tree nuts or corn. The application of SLD as a fumigant warrants minimal deposition of the compound on the crop surfaces, which can lower fungicide burden and minimize the development of fungicide resistance. The developed method can ensure the safe production/supply of food crops for stakeholders and consumers as well as prevent the European Union’s border rejections directed at US nut products due to aflatoxin contaminations.

3. Discovery and development of a natural pesticide blend targeting insect, fungal, and mycotoxin contaminations in tree nuts. Almonds, pistachios, and walnuts are among California’s most valuable crops but suffer significant losses from multiple insect pests. Despite the use of integrated pest management strategies and insecticides, these pests continue to cause damage, and rising insecticide resistance underscores the urgent need for alternative control methods. In addition to the direct damage they cause, feeding by these pests enables the entry of mycotoxin-producing fungi, primarily Aspergillus flavus, which contaminates the nuts with aflatoxins, a potent carcinogen with serious food safety implications. In response to the need for an alternative, ARS researchers discovered a blend of five natural compounds that shows exceptional, synergistic, and broad-spectrum insecticidal activity against all three pests. Three of these compounds also inhibit the growth of multiple mycotoxin-producing fungi, with two chemicals specifically blocking aflatoxin production. This novel, plant-based blend offers a safe and effective solution to protect tree nuts from insect pests, fungal infestation, and mycotoxin contamination.

4. A superior bug zapper. Replacement of radioactive sources, such as cobalt or cesium, with x-ray technology is a high priority due to increased government regulation in light of Homeland Security concerns, safety considerations, suppliers going out of business, and the aging of existing sources in radiation facilities. In fact, the 2019 Nuclear Defense Authorization Act (Section 3141) set a goal of replacing all gamma irradiators with x-ray devices by 2027. ARS researchers at Albany, California, have developed thin layer x-ray irradiation facilitating the use of low energy x-rays for high dose rate irradiation with superior dose precision and uniformity. They have recently incorporated a conveyor-based insect handling system and demonstrated that insects can be sterilized in large numbers with this technology, affirming the suitability as a replacement for gamma irradiation for Sterile Insect Technique programs, in which pest insects are reared, sterilized in large numbers, and released to compete sexually with the existing population.

U.S. DEPARTMENT OF AGRICULTURE

Foodborne Toxin Detection and Prevention Research: Albany, CA