Understanding Pre-Harvest Routes of Fresh Produce Contamination in Soils

Non-Technical Summary: Soil, irrigation water and land applied manures can harbor problematic bacteria but the transfer from these materials onto produce (e.g., vegetables) has not been well established. Questions remain as to whether a direct transfer is possible, growth on the surface or if combination of two is needed. While data suggests that human pathogens can occur in the food supply, this project is interested in seeing to what degree common agricultural practices and contacts between produce and contamination sources will alter the potential loading of cells from the environmental sources to the plant surface. That is, how many bacteria are required to induce a negative situation in a commercial production system and what conditions are required for soil or other environmental sources to supply the bacteria Secondly, it is not clear when in the processing chain produce is most susceptible to contamination. Therefore, this project is interested in how bacteria are transferred from environmental sources to plant surfaces and if this transfer leads to an effective colonization of the product, or if growth on the product can occur or is required to occur to induce a condition where a health effects can result. In order to better understand the different factors that are involved in the contamination of vegetables by human pathogens, this project will grow plants in two types of systems. In the first system, vegetables (lettuce, spinach, tomato and melon) will be grown in test tubes containing water agar supplemented with all the nutrients required for their growth. By using this system, we will follow and monitor the number and distribution of two kinds of human pathogens (E. coli and Salmonella) on the vegetables. The bacteria are tagged and can easily be tracked along the different plant parts once they are introduced. We will introduce these bacteria into the plant system in a number of ways including by contaminating the intact or damaged seeds before germination or by contaminating the growth medium. We will then take plant samples (e.g., roots, leaves) and examine the occurrence of and quantify the bacteria in the different plant parts, including the inside the plant tissues. In the second system, we will grow the vegetables in soils, which have been contaminated under different scenarios which are common in the field (e.g., transplanting contaminated seedlings, use of contaminated seeds, use of contaminated irrigation water or land application of animal manure). We will then investigate which of the contamination scenarios mentioned are more risky in terms of allowing the survival and transmission of the bacteria on the vegetables so that prevention techniques are tailored towards the contamination scenarios which are more risky. The expected outcomes include better understanding of the role of rhizosphere in supporting contamination bacteria in a plant system and identification of the role of a number of potential preharvest contamination routes on the survival, transferability and spread of pathogens on vegetables so that producers can prioritize on strategies targeting the preharvest routes of contamination that pose meaningful risk. <P> Approach: In order to investigate the extent to which the rhizosphere conditions can support or increase the number of contamination bacteria in a plant system and to differentiate the response and to track the key contamination locations, plants will be grown in test tubes containing water agar supplemented with Hoagland nutrient solution. This system is particularly useful for real time tracking of the distribution of human pathogens on plant surfaces. The project will use E. coli O157:H7 and Salmonella spp marked with gfp or antibiotic resistance to track and monitor the movement and distribution of these bacteria on vegetables under a number of contamination scenarios (e.g. seed vs media contamination; intact vs damaged seeds). Lettuce, spinach, tomato and melon seeds will be contaminated with the bacteria (10, 102 105 or 107 CFU ml-1) and put under 16/8 light/dark cycle in a growth chamber after germination in the dark at room temperature. The seedling will be monitored for bacteria distribution and number in different locations (rhizosphere vs phyllosphere) with time by taking samples destructively periodically. They will also be investigated for occurrence of internalization (penetration into the plant's vascular system) of the bacteria in the early growth stages of the seedlings especially in the systems developed around damaged seeds. To investigate the different preharvest routes of vegetable contamination in soils and their effect on the survival, transferability and spread of pathogens on vegetables, two contrasting soil types (sandy and clay) will be used. The following preharvest practices will be considered as potential contamination routes: transplanting contaminated seedlings, use of contaminated seeds, use of contaminated irrigation water or land application of animal manure. The potential of rainfall splash in transporting pathogens from soils to plant surfaces after germination will also be investigated. All contaminated entities will have bacteria at 102 104 and 106 CFU g-1 or mL-1. The vegetables will be grown in soils that will be put in wooden beds in a controlled walk-in growth chamber. Antibiotic resistant strains of E. coli 2+ and Salmonella spp, which facilitate their retrieval from the soil environment, will be used. In each bed, representing each contamination scenarios, lettuce, spinach and tomato will be planted. Plant samples will be taken three times during the growing season (during thinning, half way before harvest, and at harvest) while three soil core samples will be taken from each beds biweekly until harvest. All samples will be analyzed for the bacteria according to established methods. All the experiments will be replicated four times. All data will be summarized into descriptive statistics. Analysis of variance will also be carried out on data to investigate the statistical significance of the different modes of contamination (e.g., seed vs medium) on the concentration of the bacteria on different plant parts well as the effect of the different treatments (e.g., routes of contamination) on the transferability and survival of the bacteria on the plant materials and soils with SAS at p = 0.05.