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 Sub-objective 1A, researchers obtained pistachio mummies (unharvested nuts) and classified them on the level of hull degradation, the presence of a kernel, and whether visible mold was observed. Volatile emissions from these groups were identified and compared with the same mummies in an atmosphere of increased humidity that allowed for the production of microbial volatiles. A group of volatiles unique to the mummies was identified and categorized as originating from pistachio tissues with or without fungal damage. The ability of these volatiles to attract navel orange worm (NOW) adults is being evaluated. Researchers tested various insect traps in commercial orchards to determine the most effective design for use in monitoring the leaf-footed bug (LFB). A colony of LFBs was established from a wild population in a commercial pomegranate orchard and two additional populations from laboratory-reared colonies were maintained. A selection of volatiles common to the host plants of the LFB (i.e. almond, pistachio, pomegranate, citrus, and tomato) was compiled. The sensitivity of LFB antennae to detect each of these host plant volatiles was compared for male and female LFB from both the wild and lab-reared colonies. The most promising host plant volatiles were combined into blends and tested for their ability to lure LFBs to orchard traps. Field trials for the efficacy of these lures in almond, pistachio, and pomegranate orchards are ongoing. Under Sub-objective 1B, researchers have developed a portable x-ray irradiation unit for use in pistachio orchards to sterilize or kill Navel Orangeworm larvae overwintering in pistachio mummies on the orchard floor. A new approach using high pressure steam has also been developed and is being tested for the ability to kill the overwintering larvae, eliminating the need for using irradiation. A novel rearing system has been developed substantially increasing egg production for use in the sterile insect technique (SIT). Irradiation experiments testing the effect of adding certain natural compounds to insect diet, to reduce the required x-ray dose for sterilization, and/or increase their fitness following irradiation, have been completed. Irradiation experiments testing the effect of modified atmospheres on insect radio sensitivity have also been completed, potentially reducing dose requirements for insect sterilization for SIT. Under Sub-objective 2A, researchers have isolated multiple bacteria capable of inhibiting the growth of Salmonella in an in vitro fluorescence assay. Because of California Department of Food and Agriculture regulations restricting the use of experimental bio-pesticides in field trials, the model for growth and persistence on produce was changed from almond drupes to cantaloupe melons, which are available year-round and are suitable for growth in the greenhouse. These bacteria have been shown to grow and persist in cantaloupes, and in addition, inhibit the growth of Salmonella. The genomes of these bacteria have been sequenced and possible antimicrobial compounds have been identified. Two of these organisms have been patented and two different companies have expressed interest in using them in field trials. Under Sub-objective 2B, researchers in collaborative studies with researchers at the University of California, Davis, are in the third year of a project to determine the safety of applying composted cow manure to almond orchards. Samples have been taken every year and analyzed for E. coli O157:H7, Salmonella enterica and Listeria monocytogenes presence by culture methods. Samples are also being analyzed by 16S rRNA gene sequence analysis to describe changes in the microbial population structure in the soils. Under Sub-objective 2C, researchers have isolated genomic DNA from a biocontrol Aspergillus flavus strain followed by DNA sequencing. DNA fragments were assembled to construct a genomic library. Total RNA from this strain was also extracted and converted to cDNA and sequenced to obtain a transcriptomic library. Both sets of data were deposited into National Center for Biotechnology Information (NCBI). In-depth analysis of the genes involved in aflatoxin and cyclopiazonic acid (CPA) synthesis, as well as the genes associated with biocontrol efficacy are in progress. Researchers paired atoxigenic strains of Aspergillus flavus with toxigenic strains and grew them in dual cultures. Fungal material was collected and used to extract RNA for evaluation of the biosynthesis of aflatoxin of toxigenic strains (the toxigenic strain is the cause of aflatoxin contamination in almond, pistachio, peanut and corn). The results demonstrated that the regulatory gene, aflR for the toxin biosynthetic pathway, was inhibited by the presence of the atoxigenic strain. High-performance liquid chromatography analysis of the dual cultures showed significant reduction of aflatoxin presence by more than 90%. Under Sub-objective 2D, researchers described the ecology of black-spored toxigenic Aspergilli. Soil and fruit samples were collected from conventionally and organically farmed raisin grape vineyards in California for the second consecutive year. Samples were taken after berry formation, early in fruit ripening, at full ripeness, and following sun-drying into raisins. DNA from microorganisms present in soil and in washes of fruit surfaces were isolated and used in quantitative polymerase chain reaction (PCR) experiments to determine the relative amounts of the four predominant black-spored Aspergillus species. Results indicated that populations of fungi within vineyards fluctuate during the growing season. However, the relative population sizes and proportions of species (most importantly of ochratoxin-producing A. carbonarius) in soil and on fruit were not significantly different between conventional and organic vineyards, and were similar from year to year. These data suggest that biocontrol interventions would act similarly in conventional and organic vineyards. Also under Sub-objective 2D, researchers initiated studies to identify bacterial populations physically associated with toxigenic and nontoxigenic Aspergillus species in soil. Methods for isolating microbiome components of fungi from soil are in development, and metagenomic analysis of the bacterial species will be performed using next-generation sequencing of 16s ribosomal DNA fragments amplified from fungus-associated bacterial populations. These studies will provide information regarding differences in bacteria associated with toxigenic and non-toxigenic populations of the same Aspergillus species. Bacterial species identified in these studies will be screened for antifungal phenotypes that could be used in biocontrol applications. Under Objective 3, researchers have identified cinnamic acid derivatives that disrupt the cell wall integrity systems of fungi. While disruption of the fungal cell wall is an effective intervention strategy, certain commercial cell wall disruptants cannot completely inhibit the growth of filamentous fungi, including mycotoxin-producing Aspergillus. Four cinnamic acids identified possessed the highest cell wall-disrupting activity. In addition, the efficacy of commercial cell wall disruptants, such as caspofungin, could also be augmented by the co-application of cinnamic acids. Cinnamic acids further overcame resistance to antifungal agents such as fludioxonil. Thus, cinnamic acids can be developed as target-based (namely, disruption of cell wall and/or antioxidant system) intervention catalysts for the inhibition of toxigenic Aspergillus.
1. Mass rearing of navel orangeworm. Mass rearing (i.e. millions of moths per day) is one of the critical elements of an efficient sterile insect technology (SIT) program. The current SIT program operated by Animal and Plant Health Inspection Service (APHIS) in Phoenix, Arizona, utilizes glass jars under simulated daylight for mass rearing. ARS scientists in Albany, California, developed a new rearing system in which the larvae develop without light, resulting in faster insect development and substantially higher egg count. Results indicate that this method significantly increases moth production with minimal added effort or cost.
2. Isolation and characterization of biocontrol agents to reduce the growth of Salmonella on produce. ARS Scientists in Albany, California, created a library of bacteria normally associated with produce. This library was screened to identify bacteria capable of inhibiting the growth of Salmonella. Two isolates inhibited the growth of Salmonella on cantaloupe melons. The genomes of these bacteria were sequenced, and have revealed clues as to the mechanisms of Salmonella growth inhibition. Patents have been filed for these organisms, which could be used as biocontrol agents for farmers interested in inhibiting growth of bacterial pathogens in produce and farm environments.
3. Identification of an atoxigenic strain of Aspergillus flavus for use in the biocontrol of toxigenic fungal species. Aflatoxins, which can be present in nuts, are widely recognized as a major health problem. ARS scientists in Albany, California, identified an effective biocontrol agent that inhibited aflatoxin production by toxigenic Aspergillus flavus. The DNA and RNA of this strain was extracted, sequenced and assembled into two databases for gene discovery and expression studies. The libraries are useful to elucidate the genetic bases of effective biocontrol agents and for the identification of the relevant metabolic capabilities of atoxigenic A. flavus strains that promote biocontrol-related management of toxin contamination. These studies help define the most effective mycotoxin biocontrol agents for nut and farm crops.
4. Identification of natural cinnamic derivatives that interfere with fungal cell wall system. Due to increasing concerns about the safety of certain antifungal drugs that have been in wide use, and the impact of repeated exposure to these compounds on health and fungal resistance, there are constant demands for new antifungals or drug potentiators with improved health and safety profiles. To that end, ARS researchers in Albany, California, identified natural cinnamic derivatives that prevent fungal growth by disrupting the cell wall integrity of pathogens. The efficacy of caspofungin or octyl gallate, commercial cell wall disrupting agents, can be augmented by the co-application of cinnamic acid-1 (CA-1). Cinnamic derivatives can also overcome fludioxonil (fungicide) tolerance of Aspergillus antioxidant mutants. Collectively, natural cinnamic derivatives can be used to enhance current anti-pathogenic fungi treatments, allowing the reduced use of toxic antifungal agents or fungicides, thus leading to better human health and environmental impacts.
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