1a. Objectives (from AD-416):
The overall objective of this project is to develop commercially viable methods for the control of pathogens in tree nuts (almonds, pistachios and walnuts) and raisins which lead to foodborne illness in humans and animals. This includes both the control of fungal pathogens (mycotoxins) as well as bacterial pathogens such as E. coli and Salmonella. One approach will be the development of biocontrol agents using bacteria, yeast and non-toxigenic Aspergillus carbonarius. In addition, a better understanding of organic and conventional farming systems will provide new insights on mycotoxin control. The specific objectives for the period covered by this project plan are as follows: Objective 1: Define the critical control points for pathogen contamination during the production stream. Place particular emphasis on agricultural water sources including dairy waste water. Using both cultural and non-cultural based methods we will identify the points in the developmental process, as well as the processing cycle where tree nuts are most likely to become contaminated with human pathogenic bacteria and mycotoxigenic fungi. Objective 2: Evaluate the microbial ecology of organic v. conventional practices. Although several surveys have reported that consumers equate organically grown food stuffs with higher levels of food safety, little is known about how these practices affect the microbial population structure or mycotoxin levels in tree nuts. We will address the influence of phyllosphere microbial community on the population diversity of A. flavus in tree nut orchards, and A. carbonarius on grape surfaces in both farming systems. Objective 3: Delineate the factors affecting cross-contamination during processing and develop a potential intervention strategy during storage. A number of experiments point to a strong possibility that cross-contamination of toxigenic fungi is possible during processing and storage, although no research appears to have been done on the transfer in actual (not laboratory) processing and storage conditions. Development of novel approaches to prevent the growth of storage fungi and production of harmful toxins is a high priority in the almond industry. Edible films and coatings (EFC) containing antimicrobial natural compounds will be tested for their efficiency to reduce mycotoxin in stored almonds. Objective 4: Develop biological-control/intervention technologies using competitive or antagonistic microorganisms such as yeasts or bacteria that can be mass-produced and effectively utilized in a variety of pre- or post-harvest environments. Bacterial and yeast biocontrol agents will be tested in almond orchards and vineyards to control A. flavus and A. carbonarius, as well as human pathogenic bacteria, E. coli and Salmonella. EPA registration of the patented yeast, Pichia anomala will be pursued for commercial application. Methods to enhance the biocontrol efficacy will be developed. The flowchart shown in Figure 1 illustrates the integration of the four objectives to achieve the ultimate goal of eliminating mycotoxins and human bacterial pathogen contamination (i.e. Salmonella, E. coli O157:H7, etc.) in tree nuts and raisins.
1b. Approach (from AD-416):
Develop mentods to control insect pests and toxic fungi of tree nuts. Insects include naval orangeworm, codling moth and peach twig borer. Feeding damage by these insects leads to infection by aflatoxigenic aspergilli. Control methods for insects are to be environmentally benign and employ semiochemicals to disrupt insect behavior. Control of toxic fungi focuses on biological control using competitive or antagonistic microorganisms. These microorganisms include either yeasts or bacteria that can be mass-produced and effectively utilized in a variety of pre- or post harvest environments.
3. Progress Report:
Fungal populations from raisins produced under different management practices have been isolated and characterized. Analysis of ochratoxin and fumonisin production by Aspergillus isolates has been performed. Molecular analysis of fumonisin biosynthesis genes in fumonisin-nonproducing strains has been performed. We have examined almonds grown in 3 conventional and 3 organic orchards at 4 different time points: 1. When the nut first forms; 2. When the nut is set; 3. When the nut is mature; and, 4. When the hull splits, using small subunit ribosomal sequence analysis of DNA extracted from the nut surfaces. Our results indicate that there are significant differences in the types of bacteria present on the surfaces of almonds grown under conventional and organic methodologies. We did not observe any sequences representative of Salmonella enterica in the organically grown almonds at any point in the maturation of the nut. However we did observe 2 sequences representative of Salmonella enterica in the nuts grown under conventional methodologies. Mixing and collection of samples for analysis for sub-objectives 3.1 and 3.2 are complete. Aflatoxin analysis is ongoing and nearing completion. A visual screening method for anti-fungal activity of essential oil edible films has been developed to screen for effective edible films. Oregano oil was shown to inhibit both the growth and aflatoxin production of A. flavus. Allspice oil and thyme oil were demonstrated to be effective as well. These edible films are being tested on mini-bins to prevent fungal growth in stored almond nuts. Production of antibiotic compounds and fungal cell wall-degrading enzymes by candidate bacterial biocontrol agents has been demonstrated in solid and liquid culture. Small-scale field trials to determine efficacy of bacterial biocontrol agents on corn for reducing aflatoxin contamination showed that the bacterial strains that performed best in laboratory assays did not produce measurable reduction of aflatoxin on corn. Large-scale field trials were not initiated. Methods of application of biocontrol agents (to soil, for example) are currently being re-evaluated. Stimulation of Pichia anomala growth and cell yield by Aspergillus flavus was observed in a dual culture system. Medium composition influenced the increases. Cell yield determined by colony forming unit (CFU) showed that yeast grown in the presence of A. flavus had 30% higher CFUs than the control without A. flavus.
1. Ochratoxin and fumonisin production in conventional vs. organic orchards. Black Aspergillus isolates have been collected and identified from vineyard samples of raisins. These isolates have been screened for ochratoxin and fumonisin production. Initial analysis showed no significant differences in population diversity of black Aspergillus species between conventional and organic vineyards. This result supports the hypothesis that organic farming practices do not lead to greater risk of ochratoxin or fumonisin contamination.
2. Genetic basis of fumonisin production. Genetic analysis of fumonisin-nonproducing Aspergillus Niger and Aspergillus awamori strains showed that in A. awamori strains, a larger section of the fumonisin bysynthetic gene cluster is deleted. This deletion in the gene cluster resulted in a loss of fumonisin production. In A. Niger, fumonisin-nonproducing strains contain the entire biosynthetic gene cluster, indicating that the nonproduction is due to other genetic factors. This research increases the fundamental understanding of the genetic basis of fumonisin production in these species.
3. Repression of mycotoxin genes in Aspergillus flavus. The biocontrol yeast, Pichia anomala WRL-076 has been demonstrated to repress the expression of aflatoxin and cyclopiazonic acid biosynthetic genes from Aspergillus flavus by quantitative reverse transcriptase PCR ( qRT PCR ). Genes demonstrated to be repressed in aflatoxin biosynthesis are: aflR (coding for transcription activator), aflJ (coding for transcription enhancer), omtB (coding for 0-methyltranferase B) and pksA (coding for polyketide synthase). Genes demonstrated to be repressed in cyclopianzonic (CPA) acid biosynthesis are: pks-npls1 (coding for polyketide synthase and nonribosomal peptide synthase), hydA (coding for CPA amidohydrolase and ctfR1 (coding for CPA C6-type transcription factor). The molecular data validated that the patented P. anomala is a suitable biocontrol agent for reducing both aflatoxin and cyclopiazonic acid in plant food products.Du, W., Avena Bustillos, R.D., Hua, S.T., Mchugh, T.H. 2011. Antimicrobial volatile essential oils in edible films for food safety. In: Science against Microbial Pathogens: Communicating Current Research and Technological Advances. Badajoh, Spain: Formatex. p. 1124-1134.