Investigation of E.coli survival on contaminated crop residue.

Our research team has conducted several in-field studies in the Salinas Valley and found that if leafy green crops were exposed to E. coli either prior to emergence from soil or early in their growth cycle, the plants at harvest were rarely contaminated and E. coli was rarely recovered from soil or water. However, we found that if a mature spinach crop was contaminated with E. coli and then disked into the field, we could recover E. coli from soil for a much longer period of time (over 85 days). We propose to investigate the dynamics of E. coli survival on lettuce residue incorporated into soil. We will establish experimental plots in commercial field settings and inoculate plants prior to disking. As treatments, we will employ commercial ground preparation practices (multiple diskings, addition of irrigation water) and evaluate their effects on survival and decline of E. coli in soil. Such experiments should provide the grower with practical information on the duration of bacterial survival in soil and the role of post-harvest field practices in reducing bacterial carry over. Because the research will be conducted in the Salinas Valley, our results should reflect real world dynamics of the production environment in coastal California.

Fresh market leafy green vegetables are periodically contaminated with foodborne human pathogens such as E. coli. As extension researchers stationed in the Salinas Valley, our approach has been to conduct field studies under commercial situations in order to study the ecology of E. coli and how this bacterium might survive in the field. Our in-field studies in the Salinas Valley indicate that if lettuce or spinach crops were exposed to E. coli either prior to emergence from soil or early in their growth cycle, the plants at harvest were rarely contaminated and E. coli was rarely recovered from soil or water. However, if a mature spinach crop was contaminated with E. coli and then disked into the field, we could recover E. coli from soil for over 85 days. (Note that for these plots, spinach was inoculated with E. coli, disked into the soil, and left undisturbed for the 85+ days.) Because of the extended soil survival as observed in this previous field trial, we propose to investigate further the dynamics of E. coli survival on crop residue incorporated into soil. We will establish experimental plots in commercial field settings and inoculate lettuce with generic, rifampicin-resistant E. coli strains prior to disking and incorporating the crop residue. Treatments will consist of farm practices, such as disking the soil one or more times and irrigating plots, used in preparing the soil for leafy green production. We will evaluate the effects of these practices on survival and decline of E. coli in the soil by using direct plating and enrichment recovery methods. Such experiments should provide the grower with practical information on the duration of bacterial survival in soil and the role of post-harvest field practices in reducing bacterial carry over. Information generated from such experiments could guide the grower if faced with positive test results that would prevent harvest and use of the crop. Because the research will be conducted in the Salinas Valley, our results should reflect real world dynamics of the production environment in coastal California. In addition, our research team is very familiar with field production aspects of leafy vegetables in coastal California. Our experience as field researchers and extension educators provides us with strong industry connections, and our project will therefore have a prominent extension component as we work directly with growers and other industry personnel.

The overall objective is to further document survival of E. coli and Salmonella strains in soil when a contaminated leafy-green crop (lettuce) is incorporated into soil and the site is prepared for subsequent plantings. Within this objective, the cultivation practices are varied to evaluate several different production scenarios. 

Objective 1: Evaluate impact of different pre-incorporation practices, including mowing and ring-rolling of a contaminated crop, on bacterial survival. 

Objective 2: Evaluate impact of additional irrigations on bacterial survival in contaminated soil. 

Objective 3: Evaluate whether environmentally-adapted E. coli and Salmonella bacteria will transfer to a leafy-green crop re-planted in a previously contaminated soil.

There were several differences between the inoculation procedures in this trial as compared with previous years. First, a lower concentration of inoculum was applied: log 6.0 cfu/ml this year as compared to log 8 cfu/ml in previous years. Also, for this year only half the volume of inoculum was applied per square foot as compared with previous years. Finally, the crop was left intact for a minimum of 48 hours after inoculation before being incorporated into the soil whereas in previous trials the crop had been immediately incorporated after inoculation. All of these factors likely contributed to the lower rate of recovery of the inoculated strains once began. The decision to decrease the volume was based primarily on practical considerations of the size of the experiment and the application methods available. The decision to decrease the concentration was intended, in part, to bring the experiment closer to a possible real-world contamination scenario. Despite the decreases in inoculum concentration and volume, a very large amount of bacteria was applied to the trial crops, an amount exponentially greater than what would be anticipated in a likely irrigation water contamination event. For practical and resource considerations, we did not test the difference between incorporating an inoculated crop immediately versus waiting 48 hours before incorporation. It is likely that the exposure to the elements during the 48 hour hold period in this experiment, as has been shown in our previous studies, had a great impact on the frequency of recovery of the inoculated strains. Salmonella appears to have been more persistent in the environment than E. coli, with a greater number of samples testing positive for Salmonella than for E. coli across the experiment. Salmonella also appears to have thrived (or competitors that interfere with detection declined) as it adapted to the environment, as greater numbers of overall samples tested positive for Salmonella as time progressed in the sampling season. Treatments 2, 4, 6, and 8 returned at least one positive replication of Salmonella for every sampling date. All other treatments had at least one sampling date with no positive replications. While treatments 2, 4, and 6 received an additional irrigation one week after incorporation, treatment 8 did not share this characteristic. Genetic confirmation is still pending for the presumptive positive results reported here. This project builds upon data from previous years in examining the survival of generic strains of E. coli when inoculated into leafy-green production systems. The purpose was to provide documented information on the ecology and survival of E. coli and Salmonella under field conditions and to address practical questions facing growers. If a mature, presumably contaminated crop is not harvested and is disked down, what is the fate and survival of the bacteria on crop residue and in the soil? Such information, strengthened by multiple years of data, could be useful in addressing regulatory and metric concerns faced by growers in California.