1a. Objectives (from AD-416):
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.
1b. Approach (from AD-416):
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
3. Progress Report:
Under Objective 1A, ARS researchers from Albany, California performed research on the volatile profiles of stored almond and pistachio products as they await processing. Humidity (moisture) played a large role in the activation of fungi on both almonds and pistachios. Distinct volatile biomarkers can be identified based on humidity levels and could be used as signals for early detection of fungal contamination. In order to identify new semiochemicals for attracting insect pests in pistachios, ARS researchers from Albany, California identified and compared the volatile emissions from several categories of developing pistachios. During the growing season, pistachios are susceptible to navel orangeworm (NOW) infestation when the hull splits open. Early split pistachios open when the fruit is immature, while regular split and tattered hull pistachios occur as part of the normal ripening process, close to the time of harvest. All of the pistachio types allow for insect access through the hull and are at risk for NOW infestation. The volatile profiles of the split and unsplit pistachios consisted almost exclusively of terpenes. Insect assays showed that NOW adult females responded to these terpenes; however, blends of these compounds were ineffective as lures in pistachio orchards. Because these plant-derived volatiles were ineffective as field lures in pistachio orchards, a new effort was undertaken to identify microbe-derived volatiles as navel orangeworm attractants. These microbes (fungi) are most abundant in almond stocks; thus, stockpiled almonds were sorted to identify samples with field-generated mold contamination. Moldy almonds were incubated under elevated humidity to allow for the production of fungal volatiles, but not additional fungal growth. The presence of mold contamination increased the complexity and level of volatiles from the almond tissues, and resulted in the production of many compounds that tested well in NOW evaluations. These compounds will be used to produce new blends to test as NOW lures under field conditions. Another insect pest that vectors pathogenic fungi is the leaffooted bug (LFB), which has a wide range of host plants. In order to identify semiochemicals for use in pest lures, volatile extracts were prepared from a variety of fruits that are susceptible to LFB damage such as ripe pomegranates, moldy and split pomegranates, oil stock pistachios, oil stock almonds, and ripe oranges. Volatile profiles (either from pure compounds or blends) of these extracts were evaluated for LFB attraction in the laboratory. 30 different blends of these volatiles were tested for field attractiveness to LFB. Unfortunately, the field results could not be adequately evaluated due to the lack of effective traps for LFB (unlike for NOW, LFB adults escaped traps easily). Research is ongoing to develop an effective field test and better insect traps to evaluate volatile blends as lures for LFB. Under Objective 1B, a portable x-ray irradiation unit for use in pistachio fields has been completed, but due to equipment failure, implementation has been temporarily delayed. A novel rearing system has been developed in which NOW are “trained” to form their cocoons on flat sheets of paper, which are then attached to a cylinder that rotates below an x-ray source. This allows a high throughput of irradiated NOW pupae and overcomes the problem of non-uniformity of absorbed dose when irradiating insects in containers. New methodologies for rearing NOW have been developed to increase insect production with less required labor in anticipation of the need to provide sufficient quantities of sterile insects for sterile insect technique (SIT) implementation. Dosimetry methodologies have been improved and upgraded allowing more precise determination of absorbed dose. Under Objective 2A, a produce-associated bacterial library, containing over 80,000 isolates, was screened for the ability to inhibit Salmonella enterica growth using our recently developed in vitro fluorescence assay. Researchers identified 30 isolates that inhibited the growth of S. enterica between 36- and 164-fold after 48 hours, as compared to a phosphate buffered saline negative control. The isolates were members of the phyla Firmicutes (genera: Aerococcus, Bacillus, Carnobacterium, Enterobacter, Lactococcus and Weissella), and Proteobacteria (genera: Citrbacter, Hafnia, Klebsiella, Pantoea, Pseudomonas and Serratia). The entire library (80,000 isolates) was screened for the ability to inhibit the growth of E. coli O157:H7 and Listeria monocytogenes. Isolates that can inhibit these pathogens were identified and are currently being characterized. S. enterica growth inhibiting isolates are also being tested to determine if they can grow, persist and inhibit the growth of S. enterica on fresh produce. Under Objective 2B, researchers studied whether manure application affected pathogenic bacterial populations. Manure was applied to an orchard floor for two years and the microbial population structure was examined. Soil and composted manure were tested for the presence of the pathogens E. coli O157:H7, Salmonella enterica, and Listeria monocytogenes by culture methods. Increased microbial diversity was observed in the soils receiving manure and pathogens have not been idenfitied in any of the soils tested. Samples will be collected again at the end of this year and manure will be reapplied one more time. Under Objective 2C, in order to develop new Aspergilli biocontrol agents, ten atoxigenic Aspergillus flavus isolates, with deletions in both aflatoxin and cyclopiazonic biosynthesis genes were isolated and characterized. The efficacy of the atoxigenic strains to reduce aflatoxin production were evaluated in a dual cultural system. Different ratios of atoxigenic and toxigenic A. flavus strains were co-cultured for five days and the amount of toxin produced was determined. Several mixtures of atoxigenic and toxigenic A. flavus produced significantly lower amounts of aflatoxin, some by as much as 90%. Under Objective 2D, to determine the ecology of black-spored toxigenic Aspergilli, soil and fruit samples were taken from conventionally and organically farmed raisin grape vineyards in California at four stages during the growing season: after berries formed, at the early stages of fruit ripening, at fruit harvest, and following sun-drying into raisins. DNA from soil and from microorganisms adhering to fruit surfaces was isolated and used in quantitative polymerase chain reaction (PCR) experiments to determine fungal population size and structure (relative amounts of four predominant black-spored Aspergillus species) in soil and on fruit. Results from the first year of this study indicate that there are no significant differences in the size or composition of Aspergillus populations between conventional and organic vineyards, especially with regard to ochratoxin A (OTA)-producing A. carbonarius populations. This suggests that biocontrol interventions should act the same on these vineyards regardless of the farming practice. A second year of sampling of these vineyards to determine year-to-year variation in fungal populations is ongoing. The same vineyard samples were used to isolate bacterial strains, to test for antifungal activity against A. carbonarius under lab conditions. Researchers developed assays to identify bacteria that inhibit fungal growth on agar plates (diffusion of antifungal compounds), in liquid media (diffusion of antifungal compounds or direct cell-cell interactions), and via production of volatile compounds (gas-phase antifungal compounds). Analysis of antifungal activities and identification of the antifungal compounds and the bacteria that produce them are ongoing. Under Objective 3, natural products have been identified that inhibited fungal growth (by disrupting fungal metabolism). One of the compounds identified acts as a natural fumigant and can enhance the potency of commercial fungicides (e.g. fludioxonil). This chemical possesses both antifungal and herbicidal activities; thus, the compound not only eliminates pathogens from raw tree nuts, but also controls the growth of weeds in orchards (a reservoir for pathogenic fungi). The development of a fungal pathogen control methodology for use in orchards using nutshell particles as a delivery matrix for these newly identified volatile compounds is underway.
1. New natural fumigant that inhibits pathogenic fungal growth identified. ARS researchers at Albany, California, identified benzaldehyde-1 (BA-1) as a natural fumigant that can effectively prevent fungal growth in tree nuts. Co-application of BA-1 with a conventional fungicide fludioxonil (FD) inhibited growth of FD resistant strains of Aspergillus sp. and Penicillium sp. Benzaldehydes prevented fungal growth by disrupting metal chelation in the metabolism processes. BA-1 also inhibited the germination of weed seeds, a natural reservoir for fungal pathogens. Thus, natural products such as BA-1, when used alone or in combination with existing fungicides, could serve as potent antifungals by controlling the growth of pathogenic fungi and their reservoirs. This research benefits crop industries by identifying safe and natural antifungals that can replace or reduce the use of existing fungicides.
2. Improved X-ray irradiation technique for generating sterile Navel Orangeworm (NOW). Insects reared for SIT (sterile insect techniques) programs are generally irradiated in relatively large containers, resulting in a non-uniform distribution of absorbed dose (i.e. insects closer to the edge of the container receive a higher dose than those at the center). Since it is essential to sterilize all insects in the container, the applied dose tends to over radiate some proportion of the insects, resulting in reduced fitness. ARS scientists at Albany, California, have developed a rearing-irradiating system in which NOW larvae are manipulated to form their cocoons on flat sheets of paper, which are subsequently attached to a rotating cylinder below an x-ray source. Since the insects are thus presented to the x-ray source in a single plane, the distribution of absorbed dose is much more uniform than that achieved using traditional irradiation techniques. This provides the means for producing sterile insects with higher overall fitness. This technique contributes toward controlling NOW, a major agricultural pest of fruits and nuts.
3. Development of a host plant volatile blend that attracts navel orangeworm in almonds. Damage of fruit nuts by navel orangeworm (NOW) has been associated with increases in Aspergillus infection, a risk factor for aflatoxin contamination. ARS researchers in Albany, California, developed and patented (U.S. Patent No. 9,655,366) a new blend of host plant volatiles that attract the navel orangeworm to almonds. The efficacy of this blend in attracting both male and female NOW was demonstrated in orchards and used in the monitoring of NOW infestation and as an aid in pest management. The NOW blend is being developed as a product for commercialization. This new technology will help farmers monitor and reduce NOW infestation in orchards and thus eliminate aflatoxin contamination.
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