Research Project: Biocontrol Interventions for High-Value Agricultural Commodities

Location: Foodborne Toxin Detection and Prevention Research

2019 Annual Report

Objectives
The long-term objective of this project is to reduce, inhibit, or eliminate toxigenic and pathogenic microbes (i.e., mycotoxigenic fungi or pathogenic bacteria) by utilizing intervention techniques such as biological control. Specifically, during the next five years we will focus on the following interrelated objectives. Objective 1: Develop and implement control measures to reduce, eliminate, or detect contamination of toxin producing fungi of tree nuts, for example the use of host plant- or fungal-derived semiochemicals to attract or control insect pests, or use of sterile insect techniques to decrease insect pest populations. • Sub-objective 1A: Use of host plant- or microbe-derived volatile semiochemicals to attract or control insect pests. • Sub-objective 1B: Use of sterile insect techniques to decrease insect pest populations. Objective 2: Elucidate principles of microbial ecology and develop biological control measures to inhibit pathogenic and toxigenic microorganisms, particularly fungi, and can include research on the isolation and development of new biocontrol agents and formulations to control or prevent toxigenic microbes, or survey, identify, and determine ecology of microbial populations for control strategies such as competitive microorganisms. • Sub-objective 2A: Isolate biocontrol agents that prevent pathogenic/toxigenic microbes from colonizing crops. • Sub-objective 2B: Risk analysis of waste used as fertilizers for pathogen/toxigen contamination. • Sub-objective 2C: Develop new biocontrol agents and formulations to control toxigenic fungi, and to survey and characterize populations of Aspergilli. • Sub-objective 2D: Determine ecology of black-spored toxigenic Aspergilli and develop control strategies using competitive microorganisms. Objective 3: Discover natural chemical compounds that enhance the efficacy of established microbe intervention strategies, for instance augment the activity of antimicrobial agents/treatments against pathogens via target-based application of natural chemosensitizing agents.

Approach
1A. Tree nuts emit chemicals that attract insect pests that can be used as bait for insect traps. We will analyze volatiles from nuts by GC-MS and test them for pest attraction in electrophysiological and behavioral bioassays. If we are unable to identify volatiles from nuts we will explore volatiles from other biotic and abiotic matrices. 1B. Sterile insect technique can be applied to navel orange worms (NOW) inside discarded nuts on the orchard floor using an X-ray device towed behind a tractor. We will determine the X-ray dose required for sterilization of NOW and adjust this dosage to sterilize NOW inside tree nuts and develop a tractor towed device for field sterilization. If X-ray exposure does not produce sterile NOW other forms of radiation will be used. 2A. Bacteria with agonistic properties to pathogens are present on almond drupes and if applied in large numbers would prevent pathogen contamination. We will isolate bacteria from almonds and test their ability to inhibit pathogen growth in vitro. The bacteria that inhibit pathogen growth in vitro will be examined for the ability to inhibit growth on almonds, then in field trials. If we are not able to identify bacteria that inhibit pathogen growth on almonds we will use other crops. 2B. Applying composted manure to orchards does not represent a food safety threat. We will examine the microbial community structure of soil and fruit before and after the application of manure. We will repeat the analysis for 3 years to determine the effects of manure application. 2C. Atoxigenic Aspergillus flavus strains with deletions in the aflatoxin and CPA genes can be used as biological control agents for toxigenic A. flavus. We will identify atoxigenic A. flavus isolates by PCR and confirm by chemical analysis. We will examine their use as biocontrol agents via growth inhibition assays. Atoxigenic strains that displace the toxigenic strains will be impregnated into biochar and analyzed for as biocontrol agents in green house experiments. If the biochar is not suitable we will examine other matricies such as plastic granula. 2D. Ratios of toxigenic to non-toxigenic Aspergillus sp. fluctuate during the growing season; application of competitive fungal or bacterial strains will reduce mycotoxins in grapes/raisins. Grape/raisin samples will be taken at regular intervals in the growing season and analyzed to determine the ideal time to apply biocontrol agents against toxigenic Aspergillus. At these time points we will isolate bacteria and nontoxigenic Aspergillus sp. from raisin and soil samples and assay their ability to inhibit the growth of Aspergillus sp. If no non-toxigenic strains are not found other sources will be investigated. 3. Natural compounds and derivatives can control the growth of fungal pathogens and the production of toxins. Natural compounds will be tested for the disruption of cell wall integrity and the antioxidant pathway in fungi via genetic and physiologic analysis. We will determine the mode of action of these compounds via microarrays and other genetic tests. If we are unable to identify these compounds we will analyze other chemicals such as benzo derivatives

Progress Report
Under Sub-Objective 1A, researchers in Albany, California, have identified multiple volatile compounds from pistachio mummies and tested them for adult navel orangeworm (NOW) antennal sensitivity and demonstrated that male and female NOW respond to different compounds. NOW lures were developed from high response compounds and are being field tested in commercial pistachio and almond orchards. In addition, researchers continued to make progress on the development of a lab-based behavioral assay for NOW attractancy. Under Sub-Objective 1B, researchers have completed experiments determining the required x-ray doses for NOW sterilization for all larval, pupal, and adult life stages. A new irradiation system has been developed to greatly increase the throughput rate (number of insects per unit time) for irradiation using standard x-ray tubes in which each insect receives the same dose (as opposed to gamma irradiation in which dose uniformity is a major issue). Plans to test x-ray irradiation of pistachio mummies in-field have been deemed impractical and are replaced with ongoing experiments that have demonstrated the feasibility of accomplishing the sub-objective with high pressure steam. Under Sub-objective 2B, ARS researchers in collaborative studies with scientists at the University of California, Davis, have completed the last application of manure to an almond orchard. Samples were obtained and analyzed for the presence of E. coli O157:H7, Salmonella enterica and Listeria monocytogenes by culture methods. Samples were also processed by 16S rRNA gene sequence analysis to determine the effects of manure addition on the microbial population structure of orchard soils. Under Sub-objective 2C, ARS researchers evaluated the benefits of using commercially sourced biochar as a delivery vehicle for atoxigenic Aspergillus flavus as a biocontrol agent to reduce toxigenic A. flavus in soil. Researchers observed that the viable numbers of atoxigenic A. flavus in soil increased over a three-month period. In addition, atoxigenic A. flavus outcompeted the toxigenic strain when grown as a mixture. Under Sub-objective 2D, ARS researchers completed high-throughput screenings of soil bacterial isolate libraries for antifungal activity against ochratoxin-producing Aspergillus carbonarius, as well as other species of Aspergillus, under different media conditions. The bacterial strains showing the strongest antifungal activities (inhibited growth and/or spore production) were selected for use in soil microcosm assays. Soil microcosm competition assays, in which A. carbonarius on its own or in combination with A. niger, A. welwitschiae, and A. tubingensis, have been added to sterilized soil along with the selected bacterial strains, are underway. In ongoing experiments, the effects of each bacterial strain on the growth of Aspergillus species is monitored by direct plating at three-day intervals, and effects on the airborne movement of fungal spores is monitored at seven-day intervals by wind tunnel air sampling of the same soil microcosms, to mimic the effect of wind on the transfer of fungi from soil (the inoculum source) to the grape surface. In experiments with mixtures of Aspergillus species, the effect of bacterial strains was determined using quantitative polymerase chain reaction (PCR) methods to monitor changes in the proportion of each fungal species within the total population. Under Objective 3, ARS researchers identified natural compounds that can rapidly eliminate fungi from food products or crop field soils. While numerous investigations have focused on the rapid detection of microbial contaminants to ensure food safety or public health, effective intervention tools for the rapid elimination of pathogenic microbes are often limited. Natural compound Ben-1 achieved 99.9 percent fungal death at around 2.5 hours of application in commercial food matrices, or in soils of crop fields, suggesting that Ben-1 could serve as an ecologically sound anti-fungal agent with the potential of cost reductions during food/crop production. A second compound, Ben-2, significantly inhibited the growth of a fungal mutant deficient in the production of chitin, a primary component of fungal cell walls. Ben-2 also negatively affected the survival of two sugar metabolism mutants of fungi. Collectively, these natural benzaldehydes could be used as promising intervention tools for the rapid elimination of food/environmental fungal contaminants. Under Objective 3, ARS researchers and a consortium of scientists from universities in the U.S., India, South Africa, Australia, and the United Kingdom tested numerous small molecule drugs, natural and bioactive compounds derived from food wastes for anti-microbial and anti-parasitic activities. Promising compounds were identified and will be further studied. New drug derivatives are being designed based on these results to further improve drug potencies.

Accomplishments
1. Rapid elimination of fungal contaminants by natural benzaldehydes. Contamination of foods or crop field soils with fungi that either produce toxins or are resistant to conventional fungicide interventions present food safety concerns. ARS researchers in Albany, California, identified natural benzaldehyde compounds that can rapidly remove fungal contaminants from food or environmental matrices. The most potent Ben-1 eliminated 99.9 percent of fungi after about two and a half hours of application, even at low acidity conditions similar to those found at commercial fruit juice processing plants. Another compound, Ben-2, targeted the fungal sugar metabolism pathway, disrupting the growth of fungi defective in cell wall (chitin) production or in sugar utilization. This research represents an improvement over the use of the conventional fungicide thiabendazole, which has similar fungicidal activity to Ben-1, but had the undesirable side-effect of enhancing fungi production of toxins. Altogether, the novel natural compounds identified could serve as sustainable and rapid fungal control agents with great potential for industrial applications.

2. Sensitization of Navel Orangeworm to x-ray irradiation. The use of gamma irradiation for Navel Orangeworm sterilization in Sterile Insect Technique (SIT) pest control programs is problematic, and alternatives are needed. X-ray is an obvious choice, but their low power as compared to gamma sources create challenges for the sterilization of the required large numbers of Navel Orangeworms. Reducing the required x-ray dose for sterilization is one approach to alleviate this shortcoming. ARS researchers have demonstrated that adding certain natural compounds to Navel Orangeworm diets affected their response to x-ray irradiation, both in terms of required doses for sterilization as well as fitness following irradiation. This research demonstrated a proof of concept and provided valuable insight for the selection of natural compounds for future testing.

Review Publications
Hongsibsong, S., Prapamontol, T., Dong, J., Bever, C.R., Xu, Z., Gee, S.J., Hammock, B.D. 2019. Exposure of consumers and farmers to organophosphate and synthetic pyrethroid insecticides in Northern Thailand. Journal of Consumer Protection and Food Safety. 14(1):17-23. https://doi.org/10.1007/s00003-019-01207-7.
Hua, S.T., Sarreal, S.L., Chang, P., Yu, J. 2019. Transcriptional regulation of aflatoxin biosynthesis and conidiation in Aspergillus flavus by Wickerhamomyces anomalus WRL-076 for reduction of aflatoxin contamination. Toxins. 11(2):81. https://doi.org/10.3390/toxins11020081.
Tran, T.D., Huynh, S., Parker, C., Han, R., Hnasko, R.M., Gorski, L.A., McGarvey, J.A. 2018. Complete genome sequence of Lactococcus lactis subsp. lactis strain 14B4, which inhibits the growth of Salmonella enterica serotype Poona in vitro. Microbiology Resource Announcements. 7(19):e01364-18. https://doi.org/10.1128/MRA.01364-18.
Kim, J., Chan, K.L., Cheng, L.W., Tell, L.A., Byrne, B.A., Clothier, K., Land, K.M. 2019. High efficiency drug repurposing design for new antifungal agents. Methods and Protocols. 2(2):31. https://doi.org/10.3390/mps2020031.
Kumar, S., Bains, T., Kim, A., Tam, C.C., Kim, J., Cheng, L.W., Land, K.M., Debnath, A., Kumar, V. 2018. Highly potent 1H-1,2,3-triazole-tethered isatin-metronidazole conjugates against anaerobic foodborne, waterborne, and sexually-transmitted protozoal parasites. Frontiers in Cellular and Infection Microbiology. 8:380. https://doi.org/10.3389/fcimb.2018.00380.
Liang, P., Haff, R.P., Zayas, I.Y., Light, D.M., Mahoney, N.E., Kim, J. 2019. Curcumin and quercetin as potential radioprotectors and/or radiosensitizers for x-ray-based sterilization of male navel orangeworm larvae. Scientific Reports. 9:2016. https://doi.org/10.1038/s41598-019-38769-3.
McGarvey, J.A., Place, S., Palumbo, J.D., Hnasko, R.M., Mitloehner, F. 2018. Dosage-dependent effects of Monensin on the rumen microbiota of lactating dairy cattle. Advances in Dairy Research. 8(7):e00783. https://doi.org/10.1002/mbo3.783.
Milczarek, R.R., Liang, P., Wong, T., Augustine, M.P., Smith, J.L., Woods, R., Sedej, I., Olsen, C.W., Vilches, A.M., Haff, R.P., Preece, J.E., Breksa, A.P. 2019. Nondestructive determination of the astringency of pollination-variant persimmons (Diospyros kaki) using near-infrared (NIR) spectroscopy and nuclear magnetic resonance (NMR) relaxometry. Postharvest Biology and Technology. 149:50-57. https://doi.org/10.1016/j.postharvbio.2018.11.006.
Palumbo, J.D., O'Keeffe, T.L., Quejarro, B., Yu, A., Zhao, A. 2019. Comparison of Aspergillus section Nigri species populations in conventional and organic raisin vineyards. Mycotoxin Research. 76(7):848-854. https://doi.org/10.1007/s00284-019-01697-6.
Pennerman, K.K., Gonzalez, J., Chenoweth, L.F., Bennett, J.W., Yin, G., Hua, S.T. 2018. Biocontrol strain Aspergillus flavus WRRL 1519 has differences in chromosomal organization and an increased number of transposon-like elements compared to other strains. Molecular Genetics and Genomics. 293(6):1507-1522. https://doi.org/10.1007/s00438-018-1474-x.
Beck, J.J., Gee, W.S., Cheng, L.W., Higbee, B.S., Wilson, H., Daane, K.M. 2018. Investigating host plant-based semiochemicals for attracting the leaffooted bug (Hemiptera: Coreidae), an insect pest of California agriculture. ACS Symposium Series. 1294:143-165. https://doi.org/10.1021/bk-2018-1294.ch011.
Singh, A., Zhang, D., Tam, C.C., Cheng, L.W., Land, K.M., Kumar, V. 2019. Synthesis and antiprotozoal activity of functionalized 1H-1,2,3-triazole-tethered isatin-ferrocene conjugates. Bioorganic and Medicinal Chemistry Letters. 896:1-4. https://doi.org/10.1016/j.jorganchem.2019.05.025.
Stringer, T., Seldon, R., Liu, N., Warner, D.F., Tam, C.C., Cheng, L.W., Land, K.M., Smith, P.J., Chibale, K., Smith, G.S. 2017. Antimicrobial activity of organometallic isonicotinyl and pyrazinyl ferrocenyl-derived complexes. Dalton Transactions. 46:9875-9885. https://doi.org/10.1039/c7dt01952a.
Noritake, S.M., Liu, J., Kanetake, S., Levin, C.E., Tam, C.C., Cheng, L.W., Land, K.M., Friedman, M. 2017. Phytochemical-rich foods inhibit the growth of pathogenic trichomonads. BMC Complementary and Alternative Medicine. 17(1):461. https://doi.org/10.1186/s12906-017-1967-x.
Chellan, P., Stringer, T., Shokar, A., Au, A., Tam, C.C., Cheng, L.W., Smith, G.S., Land, K.M. 2019. Antiprotozoal activity of palladium (II) salicylaldiminato thiosemicarbazone complexes on metronidazole resistant Trichomonas vaginalis. Inorganic Chemistry. (102):1-4. https://doi.org/10.1016/j.inoche.2019.01.033.
Chellan, P., Avery, V., Duffy, S., Triccas, J.A., Nagalingam, G., Tam, C.C., Cheng, L.W., Liu, J., Land, K.M., Clarkson, G.J., Romero, I., Sadler, P.J. 2018. Organometallic conjugates of the drug sulfadoxine for combatting antimicrobial resistance. Chemistry - A European Journal. 24(40):10078-10090. https://doi.org/10.1002/chem.201801090.
Singh, A., Fong, G., Liu, J., Wu, Y., Chang, K., Park, W., Kim, J., Tam, C.C., Cheng, L.W., Land, K.M., Kumar, V. 2018. Synthesis and preliminary antimicrobial analysis of Isatin-ferrocene and Isatin-ferrocenyl-chalcone conjugates. ACS Omega. 3(5):5808-5813. https://doi.org/10.1021/acsomega.8b00553.
Gumbo, M., Beteck, R.M., Mandizvo, T., Seldon, R., Warner, D.F., Hoppe, H.C., Issacs, M., Laming, D., Tam, C.C., Cheng, L.W., Liu, N., Land, K.M., Khanye, S.D. 2018. Cinnamoyl-oxaborole amides: Synthesis and their in Vitro biological activity. Molecules. 23(8):2038. https://doi.org/10.3390/molecules23082038.
Friedman, M., Huang, V., Quiambao, Q., Noritake, S.S., Liu, J., Kwon, O., Chintalapati, S., Levin, C.E., Tam, C.C., Cheng, L.W., Land, K.M. 2018. Potato peels and their bioactive glycoalkaloids and phenolic compounds inhibit the growth of pathogenic trichomonads. Journal of Agricultural and Food Chemistry. 66(30):7942-7947. https://doi.org/10.1021/acs.jafc.8b01726.
Hua, S.T., Parfitt, D., Sarreal, S.L., Sidhu, G.K. 2019. Dual culture of atoxigenic and toxigenic strains of Aspergillus flavus to gain insight into repression of aflatoxin biosynthesis and fungal interaction. Mycotoxin Research. https://doi.org/10.1007/s12550-019-00364-w.
McGarvey, J.A., Han, R., Tran, T., Hnasko, R.M., Brown, P. 2018. Bacterial population dynamics after foliar fertilization of almond leaves. Journal of Applied Microbiology. 126(3):945-953. https://doi.org/10.1111/jam.14169.