Location: Foodborne Toxin Detection and Prevention Research
2021 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.
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.
Progress Report
This is a new project that started in January 2021 and replaces expired project 2030-42000-039-000D, “Biocontrol Interventions for High-Value Agricultural Commodities.” For additional information, see the expired project’s report.
In support of Objective 1, researchers in Albany, California, in collaboration with scientists at the University of California, Davis, and the University of California, Berkeley, collected soil samples from multiple crop fields in northern and central California that are managed under organic, conventional or hybrid cropping methodologies. The soils were assayed for their ability to suppress the persistence of the pathogenic bacteria Salmonella enterica, Escherichia coli and Listeria monocytogenes. Work has begun to construct a bacterial library from the most suppressive soils in order to identify the bacteria that are responsible for the suppression of the pathogens.
In support of Sub-objective 5A, ARS researchers isolated Aspergillus flavus, Aspergillus parasiticus and other fungi from contaminated soil, dust and orchard debris samples collected from tree nut orchards in California, and stored them for further analysis. Preliminary fungicide resistance assays identified several azole resistant aspergilli. The azole resistant prevalence (6.4%) was determined and was comparable to that reported for other fungal pathogens.
In support of Sub-objective 5B, ARS researchers completed laboratory scale analysis of Benzoic-1 (BA-1) and determined the optimum concentrations of BA-1 for weed control. BA-1 at 0.8 to 1.6 M possessed both pre- and post-emergent herbicidal fumigant activity, that can be used as a sustainable alternative to conventional weed control systems. BA-1 also helped overcome fludioxonil tolerance of Aspergillus mutants.
In support of Sub-objective 6A, a novel X-ray irradiation unit is under construction that will allow insect sterilization in the field. Upon completion, the unit can be transported and operated from the bed of a pickup truck or the equivalent. Design and component testing are complete, and final tests of shielding are underway.
In support of Sub-objective 6B, a “highway ready” steam unit has been acquired and is capable of operating in pistachio orchards. Preliminary testing on-site at Albany, California, has demonstrated the unit’s efficacy for killing Navel Orangeworm larvae in pistachio kernels.
Accomplishments
1. Determining parameters that affect antifungal efficacy of redox-active natamycin. The clinical polyene drug natamycin (NAT; a.k.a. pimaricin) has been used in the food and agricultural industries for treating fungi contaminated food and crops. Since invasive fungal infections (and mycotoxin uptake) in humans are also acquired from contaminated foods/crops, there have been discussions on whether the broad utility of NAT can trigger the emergence of fungal pathogens cross-resistant to other types of polyenes or antifungal drugs. ARS researchers in Albany, California, identified the differential antifungal activity of NAT between the human pathogen (Aspergillus fumigatus) and foodborne, mycotoxin-producing fungi (Aspergillus flavus, Aspergillus parasiticus, etc.). Altogether, NAT shows promise for protecting crops and preserving foods at acidic pH against foodborne fungi; the proposed mechanism is targeting the fungal antioxidant defense system. The results provided new information on how to apply NAT to the fields or commercial food matrices.
Review Publications
Chellan, P., Avery, V.M., Duffy, S., Land, K., Tam, C.C., Kim, J., Cheng, L.W., Romero-Canelón, I., Sadler, P.J. 2021. Bioactive half-sandwich Rh and Ir bipyridyl complexes containing artemisinin. Inorganic Biochemistry. 219. Article 111408. https://doi.org/10.1016/j.jinorgbio.2021.111408.
Friedman, M., Tam, C.C., Kim, J., Escobar, S., Gong, S., Liu, M., Yu Mao, X., Do, C., Kuang, I., Boateng, K., Ha, J., Tran, M., Alluri, S., Le, T., Leong, R., Cheng, L.W., Land, K.M. 2021. Anti-parasitic activity of cherry tomato peel powders. Foods. 10(2). Article 230. https://doi.org/10.3390/foods10020230.
