Screening of biocontrol organisms for the management of phytopathogenic fungi and foodborne pathogens on produce

The multibillion dollar agricultural industry is an important part of the United States economy, and the management of factors that affect crop and human health is imperative to maintaining this economic sector. The fungi Botrytis cinerea, Fusarium pallidoroseum, and Fusarium moniliforme are the causative agents of several plant diseases and can cause significant crop loss both before and after harvest in commodities such as strawberries, lettuce, citrus, and grains. Fungicides are employed to control these phytopathogens, but the use of these chemicals has led to an increase in fungicide resistance and may negatively affect the environment and human health. In addition to plant pathogens, foodborne pathogens also have a substantial impact on the agricultural industry. Foodborne disease outbreaks involving Listeria monocytogenes, Salmonella, and Escherichia coli O157:H7 not only cause considerable economic losses, but can also result in devastating health problems for consumers. The increase in fungicide resistance and number of produce-related foodborne disease outbreaks warrants investigation into additional methods of microbial control for use in the agricultural industry. Many bacterial species, including Lactic Acid Bacteria (LAB) and Bacillus species, produce antifungal and antimicrobial compounds, thus the use of biological control agents pre- and postharvest could augment current methods of pathogen management. The purpose of this study was to screen 22 bacterial isolates for inhibitory activity against the fungal phytopathogens Botrytis cinerea, Fusarium pallidoroseum, and Fusarium moniliforme and the foodborne pathogens Listeria monocytogenes, Salmonella, and Escherichia coli O157:H7 in vitro, then evaluate antimicrobial efficacy of select isolates against the foodborne pathogens on fresh produce. To evaluate antifungal activity, the bacterial isolates were individually spot-inoculated onto Tryptic Soy Agar, Potato Dextrose Agar, or MRS agar, depending on isolate growth requirements and then a plug of fungal-colonized agar was placed onto the center of the isolate-inoculated plate. Plates were incubated at 24°C for 10 days; fungal growth was evaluated daily, beginning on Day 3. Nine of the 22 isolates screened inhibited all three fungi; inhibition by these isolates ranged from 51-62% for B. cinerea, 60-68% for F. pallidoroseum, and 40-61% for F. moniliforme. Isolates were also screened for biosurfactant activity using the drop-collapse test. Biosurfactant production was detected in seven of the nine isolates. Bacillus megaterium, Bacillus coagulans, Bacillus thuringiensis BT2 and three Bacillus amyloliquefaciens isolates demonstrated strong biosurfactant activity and suppression of all three fungi, and therefore are recommended for further study. Antimicrobial activity of the isolates was assessed using two methods: LAB isolates were screened using a seeded-overlay method and all other isolates were evaluated by spot inoculating the isolate on pathogen-seeded TSA. Three LAB isolates and six Bacillus isolates suppressed L. monocytogenes, Salmonella, and E. coli O157:H7 in vitro. Based on the results of the screening, three LAB isolates—Lactobacillus plantarum, Pediococcus acidilactici, and Pediococcus pentosaceus—were selected for further evaluation and use in challenge studies on fresh produce. The role of organic acids in pathogen inhibition was evaluated by incubating L. monocytogenes, Salmonella, and E. coli O157:H7 cultures in the cell-free supernatant (CFS; pH 3.81-4.27) or the neutralized cell-free supernatant (pH adjusted to 6.5 -7.0) of each isolate. When neutralized, the antimicrobial activity of the CFS of the three LAB isolates was greatly diminished, illustrating the role of lactic acid in the inhibition of pathogen growth. To assess antimicrobial efficacy on Iceberg lettuce, a cocktail of the three LAB isolates (7-8 log CFU/g) was sprayed onto lettuce spot-inoculated with L. monocytogenes (2-3 log CFU/g); lettuce was incubated at 10°C for 14 d. L. monocytogenes levels were 1.84 log lower on LAB-treated lettuce than on untreated lettuce at the end of incubation. Because the LAB cocktail suppressed the growth of L. monocytogenes on lettuce, testing on fresh produce continued using DF1, which was a powdered product comprised of the three LAB isolates and media components. Because DF1 caused substantial browning of Iceberg lettuce after 2 d, Gala apples were chosen to evaluate the antimicrobial activity of DF1 against L. monocytogenes, Salmonella, and E. coli O157:H7. The effect of DF1 on L. monocytogenes, Salmonella, and E. coli O157:H7 on Gala apples was determined by spraying a Gala apple spot-inoculated with pathogen (6-7 log CFU/plug) with approximately 3 mL of a 20% DF1 solution, then incubating at 20°C for 5 d. After 5 d incubation, L. monocytogenes, Salmonella, and E. coli O157:H7 levels on DF1-treated apples were approximately 4, 2, and 2 log higher than the control, respectively. Based on the results of these experiments, DF1 is not the optimal formulation for the biocontrol of foodborne pathogens on fresh produce. This study identified several bacterial isolates with potential for use in the biocontrol of plant and foodborne pathogens. Further investigation is required to assess possible use in the agricultural industry, including characterization of bioactive compounds, optimization of biocontrol product formulation, and evaluation of the commercial viability of the biocontrol produc