Final Report for GNE14-086
Cantaloupes serve as one of the major crops in the state of Delaware accounting for approximately $850,000 in estimated annual sales. However; a recent turning point for the cantaloupe industry occurred in 2011 when the Jensen brothers received criminal charges for growing produce that was contaminated with Listeria monocytogenes and was associated with an outbreak resulting in the deaths of 34 individuals. The purpose of this project was to evaluate the use of a plant growth promoting rhizobacteria, Bacillus subtilis UD1022, to reduce the contamination of cantaloupes by Listeria monocytogenes in the pre-harvest environment as well as in the packinghouse environment. Preliminary data has demontrated the ability of UD1022 to help increase crop yield through complex plant-microbe interactions that increase plant defenses toward plant pathogens. Preliminary data has also shown that UD1022 can directly inhibit growth of Listeria monocytogenes, in culture as well as indirectly on leaves of Romaine lettuce plants when UD1022 is inoculated onto the roots of plants. The objectives of this study were to determine the effects of a novel biocontrol agent, B. subtilis UD1022, to inhibit the growth of L. monocytogenes throughout the cantaloupe production chain including in soil, on seeds, cantaloupe rind and food production surfaces. Cantaloupe seeds and soil were inoculated with L. monocytogenes then either left untreated, treated with UD1022 commercial spores, or lyohpilized P. fluorescence-1. Samples were collected over a 30 day period. UD1022 was able to significantly inhibit the growth of L. monocytogenes on cantaloupe seeds after 20 (p=0.04/p=0.03) and 30 days (p=0.19/p=0.005) and in cantaloupe soil post inoculation compared to the untreated control/P. fluorescens-1 treated controls. Similarly, cantaloupe rind pieces were inoculated with L. monocytogenes and dipped into either sterile water or UD1022 supernatant. Pieces were then incubated for 8 h or 24 h at 4, 22 or 37°C. Inhibition was significant at room temperature (22°C) (p=0.0024) on pieces that were treated with UD1022. Lastly, UD1022 biofilms were allowed to form on stainless steel coupons, then coupons were rinsed and placed into fresh media and inoculated with L. monocytogenes. While biofilm formation of UD1022 did reduce the ability of L. monocytogenes to attach to stainless steel surfaces there was no significant difference in the attachment of L. monocytogenes to stainless steel surfaces that were not pretreated with UD1022 biofilms (p=0.22). These results indicate that UD1022 may be used as a natural biocontrol agent to reduce the risk of contamination by L. monocytogenes through UD1022 treatment of cantaloupe seeds, as a soil amendment as well as on cantaloupe rind during times of temperature abuse in the packing house, during storage or transport. Further optimization is required for treatment of cantaloupe production surfaces with UD1022 to reduce contamination by L. monocytogenes in packing houses and production facilities.
The purpose of this project was to determine if Bacillus subtilis UD1022 was able to help reduce the contamination of cantaloupes (Cucumis melo spp. Melo var. cantaloupensis) by Listeria monocytogenes in the pre-harvest environment as well as the packing house environment. Our preliminary data showed evidence that this biocontrol agent can not only help increase crop yield by helping to increase plant defenses toward plant pathogens, but it can also help decrease crop contamination by human pathogens, specifically L. monocytogenes. The use of UD1022 as a biocontrol agent will help provide a sustainable means for growers, including those raising organic crops, to reduce environmental and human health risks associated with growing cantaloupes as well as improving productivity and crop yield thus; reducing cost of production and increasing net farm income. Biocontrol and manipulation of pathogens on plants can reduce the initial contamination that may be unavoidable, at times, and spread by cross-contamination during washing or during packaging. Microbial contamination can originate from countless areas along the farm to fork continuum. At greatest risk are those aspects of contamination that can occur within the pre-harvest environment, whereby microbial contamination can come in contact with plant tissues from water, soil amendments, wind, birds, insects, animals, and other fomites (Beuchat, 2002). While a wide array of research has been conducted on ways to reduce microbial contamination through better sanitation and use of safe irrigation water and soil amendments, contamination events still occur. This proposed project focuses on an important aspect of microbial contamination which has been previously overlooked but is extremely important for addressing new methods to increase sustainability methods for increasing food safety in the pre-harvest environment.
Recently the Centers for Disease Control and Prevention attributed 46% of illnesses to fresh produce (Painter et al., 2013), indicating that greater and more sophisticated efforts are needed to prevent contamination on these commodities that cause foodborne illness. Plants grow in close association with large communities of microbes, yet comparatively little is known about the diversity of microbes that associate with plants, and their interactions and effects on performance, crop yields and plant protection. Previous work has shown for the first time an existence of an intraplant root-to-shoot signal that regulates stomatal functions in plants (Kumar et al., 2012). In the proposed work we intend to determine the timing and application of this beneficial bacterium UD1022 (previously FB17) to reduce contamination of cantaloupes after a contamination event. We have preliminary data to suggest that application of UD1022 can reduce growth of Listeria monocytogenes in culture as well as on cantaloupe rinds. The successful outcome of our proposed research will provide fundamental insight into the role of diverse microbial communities on plant health enabling farmers to grow safer crops of larger quantity.
Recent field trials conducted at the University of Delaware and elsewhere provide evidence that rainfall significantly contributes to bacterial contamination of leaves (Park et al., 2014; Spanninger et al. 2013). Likewise aerosol contamination from airborne transmission of pathogens in applied manure or other soil amendments has great potential for contamination (Millner et al., 2009). These are examples of contamination that cannot necessarily be explained or controlled; however, a biocontrol agent like the one proposed in this study would be useful under these conditions to reduce contamination at the field. In higher-risk crops like cantaloupe, contamination is believed to occur in the field or in the packing house along distribution. The FDA is currently performing unannounced visits to cantaloupe packing houses across the US as a means of risk mitigation to identify risk-based behaviors. These facts serve as evidence for the need for a control measure for conventional and organic growers to reduce the chance of contamination of cantaloupe. Preliminary data indicates that the cell free lysate (CFL) of UD1022 can serve as a biocontrol agent for pathogenic bacteria like Listeria for use on plants through root application or through application to cantaloupe surfaces. This novel application is quite useful to safeguard cantaloupe which is one of the most popular fruits sold in the United States, and fresh-cut cantaloupe is a major value-added product made from the peeled fruit (Fang et al., 2013).
The first objective, according to the original proposal, was to investigate the ability of UD1022 to prevent Listeria monocytogenes growth and biofilm formation on cantaloupe rind at various storage temperatures such as those that occur in the packinghouse environment. This experiment has been completed and results can be found in the impacts/outcomes section. The final objective was to evaluate the effects of UD1022 treated cantaloupe seeds on seed germination and persistence of Listeria monocytogenes during plant growth in the pre-harvest environment. We were able to complete trials investigating the use of UD1022 during periods of cantaloupe seed storage where results are recorded in the results section. Principal investigators also investigated the use of UD1022 as a soil amendment in cantaloupe fields to reduce risk of L. monocytogenes contamination in the pre-harvest environment. Results can also be found in the results section. Principal investigators chose to also investigate the use of UD1022 biofilms to reduce potential contamination by L. monocytognes on food production equipment. Although 2 complete trials of this experiment were completed, both trials showed significantly different results. Further explanation can be found in the results section.
- Application on Cantaloupe Rind: Cantaloupe rind was cut into 2x2 cm pieces and inoculated by a paint brush with 100 µL of monocytogenes (5.57 log CFU/ml). After drying, the cantaloupe rind pieces were then dipped in either sterile water or UD1022 supernatant directly following inoculation or 8 h after incubation. Following the dipping process, the pieces were incubated at either 37, 22 or 4° C. Serial dilutions were performed on each sample and plated on Brilliance Listeria Agar.
- Application on Cantaloupe Seeds: Cantaloupe seed samples (1 g of orange sherbet variety) were treated with 1 g of B. subtilis UD1022 commercial spores, 1 g of lyophilized Pseudomonas fluorescens -1 (control), or not treated at all (control). Following treatment seeds were inoculated with 1 ml of L. monocytogenes 390-1 (environmental isolate) containing 10^8 cells. Seeds were allowed to dry in a biosafety cabinet overnight. Samples were collected on days 0, 5, 10, 15, 20 and 30. For enumeration of L. monocytogenes, 10 ml of BPW was added to each sample and samples were vortexed for 1 min. Serial dilutions were performed in distilled deionized sterile water before being plated on Brilliance Listeria Agar.
- Application in soil: Soil was collected from cantaloupe fields in Georgetown, DE. Soil samples (50g) were inoculated with 1 ml of L. monocytogenes 390-1 (environmental isolate) containing 10^7 cells and vortexed well. Soil samples were then treated with 1g of B. subtilis UD1022 commercial spores, 1 g of lyophilized Pseudomonas fluorescence -1 (control), or not treated at all (control). Samples were collected on days 0, 5, 10, 15, 20 and 30. For enumeration of L. monocytogenes, 30 ml of BPW was added to each sample and samples were vortexed for 1 min. Serial dilutions were performed in distilled deionized sterile water before being plated on Brilliance Listeria Agar.
- Application on production surfaces: Sterile stainless steel coupons (SSC) were placed into 50 ml sterile tubes (n=4) containing 30 ml of 10% LB broth. 3 ml of UD1022 was added to each tube and incubated at ambient temperature for 48 h to allow for biofilm formation. SSC were removed from each tube. Each side of the coupon was rinsed with 10 ml sterile water to remove unattached cells. Coupons were incubated with 3 ml monocytogenes in 10% LB broth at ambient temperature for 48 h. SSC were placed into new tubes containing 30 ml BPW. 1 g of sterile glass beads were added to each tube and vortexed for 1 min to remove any attached L. monocytogenes cells. Listeria on SCC not pretreated with UD1022 served as a negative control. Listeria was enumerated by performing serial dilutions followed by plating on Brilliance Listeria Agar.
The first objective of the proposal was completed in October 2014. The data for these experiments has been analyzed. We determined that efficacy of UD1022 application to cantaloupe rind was best at ambient temperature (Figures 1-3). In the future we would like to perform experiments to extend the storage period for studies performed at 4°C. The principal investigators hypothesize that a 24 h experiment was not enough time to determine the efficacy of UD1022 given the slow growth of both organisms at this temperature. However; results indicated that application of UD1022 to cantaloupe rind could potentially reduce L. moncytogenes persistence during periods of temperature abuse. It was an interesting finding that application of UD1022 to cantaloupe seeds and the soil of cantaloupe plants could significantly reduce the persistence of L. monocytogenes after only 20 days (Figure 4 and Figure 6) . In one of the seed storage trials, there was a spike in the concentration of L. monocytogenes seeds treated with UD1022 that was attributed to a spike in temperature on sampling day 10. Results from biofilm formation studies differed in trial 1 and trial 2, which was disappointing (Figure 5). However; in both trials L. monocytognes growth was lower on stainless steel coupons pre-treated with UD1022 biofilm indicating the potential for optimization.
The results of these experiments have demonstrated that Bacillus subtilis UD1022 has the potential to reduce the risk of contamination by L. monocytogenes on cantaloupes during temperature abuse such as that may occur in the packing house, during storage or transport. The use of UD1022 may provide a sustainable method for cantaloupe growers to protect their crops from plant pathogens as well as human pathogens in the pre-harvest environment as well as the post-harvest environment. An additional benefit to increasing the safety of cantaloupes is that UD1022 has been demonstrated to increase crop yields. This work was presented at the Northeast Region of the American Society for Horticulture Science 2015 Annual Meeting in Newark, DE on January 6th and 7th as well as the International Association for Food Protection Annual Meeting in Portland OR on July 25th – 28th.
Education & Outreach Activities and Participation Summary
This work was presented at the Northeast Region of the American Society for Horticulture Science 2015 Annual Meeting in Newark, DE on January 6th and 7th as well as the International Association for Food Protection Annual Meeting in Portland OR on July 25th – 28th. The data was well received by both audiences and there was a significant amount of overall interest
The economic impact of the use of UD1022 on cantaloupes was not determined in this study. However; as stated previously the ability of UD1022 to increase plant help and crop yield could be a potential advantage for farmers.
The use of the studied methods have not yet been applied in a commercial farm setting. Adoption of these protocols by a cantaloupe grower could potentially lead to increased crop yields and subsequently higher profits. Application of UD1022 to cantaloupe seeds as well as a soil amendment could potentially help prevent contamination by L. monocytogenes in the pre-harvest environment. Application to the cantaloupe rind post-harvest may help prevent growth of L. monocytogenes during times of temperature abuse.
Areas needing additional study
Additional work should be completed to optimize the use of UD1022 application on cantaloupe rind at 4°C to determine its efficacy during storage in the cantaloupe packing house. Additionally further studies should assess the use of UD1022 biofilms to reduce contamination of production equipment by L. monocytognes.