Progress 06/15/13 to 02/14/14
Outputs
Target Audience: Our target audiences initially were the leafy green vegetable growers in California, who have sustained great economic hardship from food born contamination. There are two areas of the state of California (Imperial Valley and Central Valley) that are responsible, in 2012, for approximately 80% of California’s 6.8 billion in vegetable crops gross revenue. We felt this would be a natural starting point for our search for collaborating partners. The FDA has put out a proposal, which at this moment is under review and open for comment. This proposal would require all agricultural operations that irrigate food products for human consumption to test their water on a weekly basis for possible E-coli contamination. The consequences, for operations that are found to have E-coli contaminations, as of this moment are yet undetermined. Most growers that we have talked to feel they could have very serious consequences for their business. The thought that there may be an irrigation method that could reduce and/or eliminate E-coli contamination could be huge. We have contacted and discussed our plans to move forward with a more thorough study with field trials and were looking for partners to collaborate with. We spoke with Kay Pricola, the manager of the Imperial Valley Vegetable Growers Association in Imperial Valley, and she expressed interest in collaboration on field trial research. We have also made contact and discussed with individual growers (1) Larry Fox of Fox Farms Vegetables in Brawley and (2) Ralph Strahm a large farmer in El Centro. A couple of the reason we contacted individuals in the Imperial Valley area where, that their proximity to our facility and because in this area there is very high Hispanic minority population as their labor force working for minimum wage. From the success of our Phase I trials, where we achieved such a high pathogen reduction with our irrigation method, we have expanded our projected target market scope to include all agricultural operation that irrigate food products for human consumption. This includes not only California but all of the United States from, the individual grower and the growers associations, to the FDA and the federally funded state crop advisors. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest? We have contacted and discussed our plans to move forward with a more thorough study with field trials and were looking for partners to collaborate with. We spoke with Kay Pricola, the manager of the Imperial Valley Vegetable Growers Association in Imperial Valley, we have also made contact and discussed with individual growers (1) Larry Fox of Fox Farms Vegetables in Brawley and (2) Ralph Strahm a large farmer in El Centro. Growers and managers expressed interest in collaboration on field trial research during the Phase II. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Lettuce seeds of a standard commercial variety were used in the study. Soil and hydroponic systems were researched. In soil system 160 pots were split into four equal groups. Additionally each group was divided into two subgroups “Healthy” and “Damaged” plants. Spray (over-head) method of irrigation was used for soil system. Pots were watered one time a day for 10 min at flow 1 gpm. Four levels of dissolved ozone in irrigation water were applied: (a). Control (no ozone); (b). Low Ozone (0.5 ppm); (c). Middle Ozone (1 ppm); (d). High ozone (1.5 ppm). In hydroponic system 160 pots were arranged in hydroponic channels. Flood and Drain method of irrigation was used. Hydroponic solution from tank flooded the hydroponic channels. After 20 min. solution was drained back in the tank. To compensate the lost of water by plant’s uptake and evaporation the drip irrigation method was used. Drip irrigation method applied two levels of dissolved ozone in water: (a). Control (no ozone); (b). Middle Ozone (1 ppm). Similar to soil system, plants were divided on two equal groups – damaged and healthy plants. In both systems plants were mechanically damaged (root decapitation and/or leaf’s cuts) before each inoculation. Plants grown in soil and hydroponic systems were inoculated by E. coli O157:H7 (ATCC 25922) strain through soil and surface (soil system) and media (hydroponic system) at level 107 CFU/ml. Plant contamination in soil system occurred during the entire growth cycle. Starting on day 14 after sowing, the bacteria inocula were introduce into soil and plant surface on a weekly basis. After inoculation, lettuce plant samples were collected on the next day. The harvest scheme in hydroponic system was different from the soil system. It was conducted three separate experiments during the entire growth cycle (after 1st , 2nd and 3rd month). Duration of each experiment was one week. The group of “Damaged Plants” had mechanical injures to the root tips. Plant samples were collected on 1st, 3rd and 5th day after inoculation. Among the 144 lettuce leaf samples (soil system) exanimated for surface contamination, 62.5% plants gave positive results. The highest incidence was observed in control group (no ozone) – 80.5%. In the low ozone group (0.5 ppm) the percentage of contaminated plants was 66.7%, in middle ozone group (1.0 ppm) – 58.3% and in high ozone group (1.5 ppm) – 44.4%. The average external contamination level was reduced in 22 times from 224.2 CFU/cm2 (control group) to 10.2 CFU/cm2 (high ozone group) when ozone was applied if taken into account of the sample surface. Most important results were obtained regarding of internal contamination. Obtained data have shown that incidents of internal contamination were dramatically reduced in groups where ozone was applied. No internal contamination (0/54) was found in “Healthy group” at all three levels of ozone in irrigation water (0.5 ppm, 1.0 ppm and 1.5 ppm). At the same time among 18 healthy plants in control group (no ozone), internal E.coli contamination was determined in 5 plant tissues. Number of incidents of internal contamination in “Damaged Plants” group dropped from 77.8% (control group) to 38.9% (high ozone group) when ozone was applied. The actual internal contamination level was reduced in 30 times from 15,264.8 CFU/g to 527.5 CFU/g. It was found that internal contamination had a direct relationship with external pathogen concentrations for damaged plants (correlation coefficient 0.98). It was allowed to suggest that the enteric pathogen internalization relates to damaged plant tissue which can be used by bacteria to entry into the vasculature. The lack of an internal contamination in groups of plants where ozone was applied can be explained by plant defense mechanism that is triggered by ozone. Defense responses involving phenylpropanoids include synthesis of isoflavonoid phytoalexins and reinforcement of cell walls by deposition of lignin and related wall-bound phenolics. Continuous use of ozonated water develop conditions when plants react on ozone as on microbial contamination and plant defense mechanism constantly protect them from internal contamination even when pathogens are not a threat. Very few reports have examined the effect of plant age on the internal contamination by enteric pathogens. Our results showed that incidents of internalization were observed more often for older plants. Similar results we obtained in soil and hydroponic systems. If the number of incidents in the soil system increased from 10 to 25 during the growth cycle than the number of incidents in hydroponic system increased from 0 to 9. It was very important finding from this study that ozone effects on E. coli O157:H7 survival in soil. This finding can be critical for green produce safety. As was found that even low ozone concentration (0.5 ppm) effects on pathogen’s survival and quickly reduces their level from 105 CFU/g to zero. Thus, irrigation by ozonated water can dramatically reduces the chance of probability of internalization through the root zone. Obviously the reduction of pathogen microorganisms in the growth media is the key of any type of mechanisms of internalization. Manure amended soil is one of the most probable scenario of microbiological soil contamination and green produce internalization. Obtained results show that the use of ozonated water for irrigation purpose can reduce the probability of food-born outbreaks and improve produce safety. Experiments conducted in hydroponic system have showed that among 90 lettuce samples examined for internal E.coli contamination in control group (no ozone), nine yielded a positive result. The actual internal contamination level was 258 CFU/g in group of damaged plants and 27 CFU/g (the detection limit of direct plating) in group of healthy plants. The group of plants that were irrigated by ozonated water at concentration 1 ppm showed only one positive result (damaged plant) on internal contamination. Microbiological analysis of hydroponic solution has shown that pathogen concentration in group of plants where ozone was applied was reduced from 107 CFU/ml to 103 CFU/ml compare to control group (from 107 CFU/ml to 105 CFU/ml). Total amount of incidents obtained in soil system was much higher that in hydroponic system (49/72 and 12/180, accordingly). It could be concluded that lettuce grown in hydroponic conditions had much less probability to be contaminated compare to plants grown in soil systems. Obtained results have showed that ozonated water dramatically reduces the risk of internal contamination in both soil and hydroponic systems because it not only reduces the total count of pathogen microorganisms on the plant surface, in soil and hydroponic solution, but also triggers the defense plant mechanism that typically occurs in response to microbial infection. Comparison of control and ozonated plants groups has showed significant reduction of incidents and actual contamination level (CFU/g) in group of ozonated plants. If among healthy plants the internal contamination occurred 18 times in control group then number of incidents in groups of ozonated plants was not found at all (0/54 in soil system and 0/90 in hydroponic system). In group of damaged plants the actual internal level of contamination was reduced in 30 times for soil system and in 7 times for hydroponic system. Obtained results allow us to assume that a new irrigation technique in which ozonated water is used for irrigation can increase the safety of green fresh produce, reduce the risk of internal contamination and eliminate the foodborne outbreaks country wide.
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