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

2022 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
In support of Objective 1, ARS researchers in Albany, California, in collaboration with scientists at the University of California, Davis, and University of California, Berkeley, collected soils from over 80 crop fields in northern and central California. The soils were examined for the ability suppress the persistence of Salmonella enterica, Escherichia coli and Listeria monocytogenes. The researchers performed 16S ribosomal RNA (rRNA) gene sequence analysis to identify the microbiological differences between suppressive and non-suppressive soils and created a bacterial isolate library containing ~20,000 bacterial isolates from the most suppressive soils. In Support of Objective 2, ARS researchers have begun extracting bacterial DNA from leafy greens grown under conventional outdoor management and a commercial indoor vertical hydroponic system in order to construct 16S rRNA gene libraries from them to determine the microbiological differences between leafy greens grown under these systems. Researchers also began to examine the ability of the human pathogenic bacteria Salmonella enterica, and Listeria monocytogenes to grow on leafy greens grown under these different methodologies. In support of Objective 3, ARS researchers initiated experiments to screen bacterial libraries for antifungal activity against Aspergillus carbonarius and other black-spored Aspergillus species relevant to the grape environment. Identification of candidate bacterial strains for further experimentation are ongoing. In support of Sub-objective 4B, ARS researchers have initiated experiments to screen existing bacterial libraries (from grape environments) for antifungal activity against ochratoxin-producing Aspergillus species (Aspergillus ochraceus, Aspergillus westerdijkiae, Aspergillus steynii) in parallel with experiments in support of Objective 3. In support of Sub-objective 5A, ARS researchers analyzed Aspergillus flavus and Aspergillus parasiticus environmental collections for different levels of azole resistance. The standard VIP-check testing (Voriconazole 2 ppm, Itraconazole 4 ppm, Posaconazole 0.5 ppm) revealed that several fungi (A. flavus, A. parasiticus) exhibited differential resistance to all azoles (VIP) tested. In support of Sub-objective 5B, ARS researchers determined the efficacy of Benzoic-2 as a seed sanitizer via seed-soaking and mild heat. Co-application of Benzoic-2 and mild heat (50 degree Celsius) for 20 to 30 minutes completely inhibited the germination of aflatoxin-producing A. flavus contaminated on Brassica rapa Pekinensis seeds, whereas the seed germination rate was unaffected. In support of Sub-objective 6A, a novel x-ray irradiation unit has been implemented that can be transported and operated from the bed of a pickup truck or the equivalent. Rigorous tests of the shielding, cooling system and irradiator stability are complete. Dosimetry testing is ongoing. In support of Sub-objective 6B, a portable electric steam unit has been acquired and used to treat pistachio larvae, further demonstrating their susceptibility to steam. In support of Objective 7, ARS researchers organized and established a library of 600 approved natural products. Concurrently, the researchers established a sustainable colony of Drosophila melanogaster (vinegar flies). The researchers developed an insect infection assay, where flies are infected and killed with orally administered fungi. Using this infection assay, they developed a parallel screening approach that can be used to identify molecules capable of: i) affecting the immunity of insects; ii) affecting the longevity of insects; and iii) inhibiting fungi used to infect insects. The scientists screened 170 of 600 approved natural products in the insect infection assay and identified several compounds capable of positively and negatively affecting the survival of insects and new antifungal molecules.

Accomplishments
1. A novel portable x-ray irradiator system for sterile insect technique. Sterile insect technique is a well-established insect suppression strategy. However, insects sterilized through irradiation in large facilities must be transported to the release site, often affecting insect fitness and overall effectiveness. On-site sterilization using traditional means is not practical. ARS researchers in Albany, California, have developed a novel x-ray based irradiation unit that can be transported on a small truck and operate in the field. This technology supports mobile on-site rearing and release programs for rapid response to newly discovered invasive species.

2. Developed a model of fungal intestinal infections in insects. For most studies where insects are exposed to microbial pathogens, the microbes are delivered in a model of a septic injury, using a needle previously dipped in a concentrated microbial solution. However, this model does not represent the physiological way the insects are exposed to microbes. Insects like flies and moths naturally accumulate microbes in their intestines after eating microbe-rich food, such as decaying fruits. Many insect pests of agricultural importance beneficially co-exist with fungal pathogens and participate in fungal transmission. ARS researchers in Albany, California, developed a new model of fungal intestinal infection of insects using two surrogate organisms: vinegar fly as a host and baker's yeast as a fungus. This model is useful for identifying new chemical inhibitors of insect and fungal pests and understanding interactions of insects with fungi on the molecular and genetic levels. The model is also useful for discovering chemical inhibitors of the navel orangeworm moth, which coexists with and contributes to the transmission of mycotoxins-producing Aspergillus fungi.

3. Natural salicylaldehyde for fungal and pre- and post-emergent weed control. The control of weeds in crop fields is an important task for the agricultural industry. Uncontrolled weed growth engenders diverse flora in crop fields, which not only compete with crops for water and nutrients but are also harmful pests that damage and contaminate crops, especially fungicide-resistant fungi that produce mycotoxins. ARS researchers in Albany, California, developed a sustainable weed control strategy using salicylaldehyde (SA), a natural, generally recognized as safe food additive, as the active ingredient. Researchers showed that SA possesses both pre- and post-emergent herbicidal activity, and that the emitted SA completely prevented the germination of plant seeds and/or the growth of the germinated plants. SA also possesses an intrinsic antifungal activity that overcomes fungicide resistance of fungal pathogens. Tree nutshell particles were used as SA delivery vehicles for use in orchards, thus contributing to the growers’ sustainable by-product recycling program. Soil covering (i.e. soil pasteurization) was determined to be one of the optimum practices to effectively deliver SA to the soil.

Review Publications
Kim, J., Tam, C.C., Chan, K.L., Mahoney, N., Cheng, L.W., Friedman, M., Land, K.M. 2022. Antimicrobial efficacy of edible mushroom extracts: assessment of fungal resistance. Applied Sciences. 12(9). Article 4591. https://doi.org/10.3390/app12094591.
Kim, J., Cheng, L.W., Land, K.M., Gruhlke, M.C. 2021. Editorial: Redox-active molecules as antimicrobials: Mechanisms and resistance. In: Gruhlke, M.C.H., Kim, J.H., Land, K.M., Cheng, L., editors. Redox-Active Molecules as Antimicrobials: Mechanisms and Resistance. Lausanne, Switzerland: Frontiers Media SA. p. 4-6. https://doi.org/10.3389/978-2-88971-637-1.
Kim, J., Chan, K.L. 2022. Natural salicylaldehyde for fungal and pre- and post-emergent weed control. Applied Sciences. 12(8). Article 3749. https://doi.org/10.3390/app12083749.
Jenkins, D.M., Watanabe, S., Haff, R.P., Melzer, M.J., Jackson, E.S., Liang, P. 2021. Dose response of coconut rhinoceros beetle (Coleoptera: Scarabaeidae) to 92 kV x-ray irradiation. Journal of Applied Entomology. 145(10):1039-1049. https://doi.org/10.1111/jen.12930.
Kim, J., Tam, C.C., Chan, K.L., Cheng, L.W., Land, K.M., Friedman, M., Chang, P. 2021. Antifungal efficacy of redox-active natamycin against some foodborne fungi—comparison with Aspergillus fumigatus. Foods. 10(9). Article 2073. https://doi.org/10.3390/foods10092073.
Palumbo, J.D., O'Keeffe, T.L. 2021. Method for high-throughput antifungal activity screening of bacterial strain libraries. Journal of Microbiological Methods. 189. Article 106311. https://doi.org/10.1016/j.mimet.2021.106311.
Samaddar, S., Karp, D., Schmidt, R., Devarajan, J., McGarvey, J.A., Pires, A., Scow, K. 2021. Role of soil in the regulation of human and plant pathogens: Soils' contributions to people. Philosophical Transactions of the Royal Society B. 376. Article 20200179. https://doi.org/10.1098/rstb.2020.0179.
Tam, C.C., Nguyen, K., Nguyen, D., Hamada, S., Kwon, O., Kuang, I., Gong, S., Escobar, S., Liu, M., Kim, J., Hou, T., Tam, J., Cheng, L., Kim, J., Land, K.M., Friedman, M. 2021. Antimicrobial properties of tomato leaves, stems, and fruit and their relationship to chemical composition. BMC Complementary Medicine and Therapies. 21. Article 229. https://doi.org/10.1186/s12906-021-03391-2.
Tran, T., Hnasko, R.M., Huynh, S., Parker, C.T., Gorski, L.A., McGarvey, J.A. 2021. Complete genome sequence of Enterobacter asburiae strain AEB30, determined using Illumina and PacBio sequencing. Microbiology Resource Announcements. 10(31). Article e00562-21. https://doi.org/10.1128/MRA.00562-21.