Location: Foodborne Toxin Detection and Prevention Research
2023 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, have constructed a bacterial isolate library containing approximately 20,000 bacterial isolates from the most suppressive soils identified in our study and have screened it to identify isolates that inhibit the growth of Salmonella enterica, Escherichia coli and Listeria monocytogenes in our in vitro fluorescence inhibition assay. We are now performing 16S rRNA gene sequence analysis on the most suppressive isolates to identify them.
In support of Objective 2, ARS researchers in Albany, California, have completed the construction of the 16S rRNA gene libraries to compare the microbiomes of leafy greens grown under conventional outdoor management and indoor vertical hydroponic management. The libraries have been sequenced and analysis is ongoing. The researchers have also completed experiments to examine the ability of the human pathogenic bacteria Salmonella enterica, and Listeria monocytogenes to grow on leafy greens grown under these different methodologies. The researchers are initiating studies to determine if previously identified biological control agents can inhibit the growth of the pathogens on the indoor vertical hydroponic grown leafy greens.
In support of Objective 3, ARS researchers in Albany, California, continued experiments to screen bacterial libraries for antifungal activity against Aspergillus carbonarius and other black-spored Aspergillus species relevant to the grape environment. Antifungal screens were expanded to identify activity against aflatoxin-producing A. flavus as well as against ochratoxin-producing A. ochraceus and other yellow-spored species of Aspergillus. Identification of candidate bacterial strains for in vitro and in situ biocontrol experimentation are ongoing.
In support of Sub-objective 4.A, ARS researchers in Albany, California, have identified candidate bacteria with broad-spectrum antifungal activity against mycotoxigenic Aspergillus species for further characterization. Antifungal activity assays of bacterial culture filtrates and fractionation of active filtrates are ongoing.
In support of Sub-objective 4.B, ARS researchers in Albany, California, have developed multiplex digital polymerase chain reaction (PCR) methods for characterizing populations of ochratoxin-producing Aspergillus species (A. ochraceus, A. westerdijkiae, A. steynii) and aflatoxin-producing Aspergillus species (A. flavus, A. parasiticus). Orchard soil and nut sample collection and digital PCR-based detection and characterization of target mycotoxigenic fungal populations from those samples have been initiated. In parallel, isolation of bacteria from the orchard samples and screening for antifungal activity against mycotoxigenic Aspergillus species have been initiated.
In support of Sub-objective 5.A, ARS researchers in Albany, California, designed specific PCR primers and amplified Aspergillus parasiticus CYP51A and CYP51B genes. The gene sequences are currently under investigation to determine whether the azole resistance of A. parasiticus field-isolates is triggered by mutations in CYP51A and CYP51B genes.
In support of Sub-objective 5.B, ARS researchers in Albany, California, determined in soil testing that 0.8 to 1.6 M of Benzoic-1 effectively control both mono- and dicotyledonous weed plants. The ethanol has been determined as a sustainable solvent for Benzoic-1 field application, where the natural Benzoic-1 completely prevented weed growth without solarization and/or mild heat.
In support of Sub-objective 6.A, ARS researchers in Albany, California, have repurposed a four-tube x-ray based irradiation cabinet previously built for Animal and Plant Health Inspection Service (APHIS) quarantine research for use in the navel orangeworm sterile insect technique program. The unit is in final preparation and testing for relocation to the ARS facility in Parlier, California, where the sterile insect releases are being coordinated, for evaluation as a potential to supplement and/or eventually replace gamma irradiation as the method of sterilization.
In support of Sub-objective 6.B, ARS researchers in Albany, California, have continued testing various high-pressure steam systems to determine efficacy and efficiency in sterilizing/killing navel orangeworm larvae in pistachio mummy nuts in a simulated orchard floor environment.
In support of Sub-objective 7.A, ARS researchers in Albany, California, have identified several natural products that rapidly kill navel orangeworm, Drosophila suzukii, Drosophila melanogaster, potato tubeworm, and stinkbugs. Minimal efficacious concentrations of the identified natural chemicals have been determined. Several aspects of the mechanism of insecticidal action have been determined.
In support of Sub-objective 7.B, ARS researchers in Albany, California, have identified several natural molecules capable of inhibiting the growth of mycotoxin-producing fungi: Aspergillus flavus, Aspergillus parasiticus, Penicillium expansum, Fusarium verticillioides, and Fusarium oxysporum. Minimal efficacious concentrations of the identified natural chemicals have been determined for insects and fungi. Several aspects of the mechanism of insecticidal action have also been determined.
Accomplishments
1. Discovery of antifungal natural products. Because of the rise in mycotoxigenic fungi that are resistant to conventional antifungal compounds, ARS scientists in Albany, California, identified novel antifungal compounds and combinations of them are highly effective, broad-spectrum, and safe. These natural compounds inhibit mycotoxin-producing Aspergillus flavus, Aspergillus parasiticus, Penicillium expansum, Fusarium verticillioides, and Fusarium oxysporum. The chemicals are natural and approved by the Food and Drug Administration as food additives, and some of the chemicals are also registered by the Environmental Protection Agency as pesticides for other agricultural pests and are exempt from pesticide tolerance determination. This may simplify the regulatory approval path for these molecules for treating mycotoxin-producing fungal contaminations.
2. Natural compounds that accelerate development of navel orangeworm larvae. The navel orangeworm (NOW) is the predominant pest of California tree nuts and the main cause of mold and subsequent aflatoxin contamination. Sterile insect technique (SIT), in which the pest is reared in mass quantities, sterilized, then released to sexually compete with the native population is the latest control strategy being used against this pest. ARS researchers in Albany, California, have discovered natural compounds that accelerate development of NOW larvae. This has the potential to increase the throughput of insects in the NOW SIT rearing program.
3. New antifungal formulations with natural chitosan derivatives. The control of fungal pathogens, especially those producing mycotoxins, in crop fields is important for the agricultural industry including the tree nut industry. ARS researchers in Albany, California, in collaboration with the pesticide industry, developed natural chitosan derivatives as new antifungal compounds. ARS scientists developed an efficient screening system to optimize formulations possessing antifungal activity against mycotoxigenic and phytopathogenic fungi. Among 33 formulations screened, ARS scientists selected the most effective formulation for field testing. Further development of formulations is underway for improving their antifungal efficacy, such as the identification of synergism with known antifungal agents, thus, making the formulations broad-spectrum antifungal products.
4. Discovery of insecticidal natural products. The navel orangeworm contributes to the spread of mycotoxin-producing fungi in the field, resulting in the contamination of the crops with mycotoxins. ARS scientists in Albany, California, identified new insecticidal molecules that are highly effective, broad-spectrum, safe and inhibit the navel orangeworm. Inhibition of navel orangeworm in the field reduces mycotoxin-producing fungi and their toxins contaminations of food products. Moreover, the same chemicals destroy other agricultural insect pests including: Drosophila suzukii, potato tubeworm, and stinkbugs. The discovered chemicals are natural and approved by the Food and Drug Administration as food additives, and some of the chemicals are also registered by the Environmental Protection Agency as pesticides for agricultural pests and are exempt from pesticide tolerance determination. This pre-existing approval status may simplify the regulatory approval path for these molecules for treating navel orangeworm infestation of tree nut orchards and other types of crop fields.
5. Development of the trap for Drosophila melanogaster and Drosophila suzukii. Drosophila is used as a model organism to screen insecticidal compounds. ARS researchers in Albany, California, discovered numerous natural insecticidal chemicals that kill fruit flies effectively. Drosophila melanogaster and D. suzukii feed on soft-fleshed fruits, resulting in economic losses to growers and packers. Natural insecticidal molecules and more efficient traps are needed to reduce infestations. The researchers developed a novel trap that contains fermenting yeast and vinegar to lure fruit flies, and newly discovered chemicals to rapidly kill them. They observed that this trap is also selective and does not attract and kill other beneficial insects.
Review Publications
Wang, Y., Hart-Cooper, W.M., Rasooly, R., Carter, M.Q., Orts, W.J., Gu, Y.Q., He, X. 2022. Effect of an eco-friendly cuminaldehyde guanylhydrazone disinfectant on Shiga toxin production and global transcription of Escherichia coli. Toxins. 14(11). Article 752. https://doi.org/10.3390/toxins14110752.
Beteck, R.M., Isaacs, M., Legoabe, L.J., Hoppe, H.C., Tam, C.C., Kim, J., Petzer, J.P., Cheng, L.W., Quiambao, Q., Land, K.M., Khanye, S.D. 2023. Synthesis and in vitro antiprotozoal evaluation of novel metronidazole–schiff base hybrids. Archive of Pharmacy. 356(3). Article 2200409. https://doi.org/10.1002/ardp.202200409.
Hnasko, R.M., Lin, A.V., McGarvey, J.A., Mattison, C.P. 2022. Sensitive and selective detection of peanut allergen Ara h 1 by ELISA and lateral flow immunoassay. Food Chemistry. 396. Article 133657. https://doi.org/10.1016/j.foodchem.2022.133657.
Kim, J., Cheng, L.W., Land, K.M. 2022. Advances in antifungal development: Discovery of new drugs and drug repurposing. Pharmaceuticals. 15(7). Article 787. https://doi.org/10.3390/ph15070787.
Kim, J., Cheng, L.W., Land, K.M. 2022. Advances in antifungal development: Discovery of new drugs and drug repurposing. Complete Book. 282 p. https://doi.org/10.3390/books978-3-0365-4767-1.
