Testing wetting agents for soil drag and bootie swabs and validating them in varied agricultural soils

Practical and powerful soil sampling methods can improve microbial quality and safety in produce production. Aggregative sampling methods, such as drag and bootie swabs, have been studied in animal and produce production but have not been fully optimized for produce. This project optimizes the wetting agent for drag and bootie swabs for use in different agricultural soils. We will first test increasingly practical wetting agents buffered peptone water, neutralizing buffer, and phosphate-buffered saline and compare them with reference methods. We use evaporated milk as a reference for the optimization of wetting agents and soil composite grabs as a reference for the validation of aggregation. The optimal wetting agent should be shelf stable, not animal sourced, not allergenic and should show at least as good, if not better, results for bacteria recovery than reference methods. In addition, this project will validate the best wetting agent testing in varying soils created by various stages of ground preparation for different commodities, using bacteria recovery and diversity as metrics. This project will determine the best wetting agent for aggregative soil sampling across multiple produce-relevant soil types.

Aggregative sampling methods, such as drag and bootie swabs, have been studied in animal and produce production but have not been fully optimized for produce – for example the typical wetting agent is evaporated milk, which is a potential allergen, animal sourced, and not shelf stable. Thus, there is a need to optimize wetting agent composition for drag and bootie swabs of produce soil. In this proposal, we use soil composite grabs as a reference to ensure the aggregation is valid and evaporated milk as a reference to ensure the optimization for wetting agents is valid. Three sample schemes will be used to collect samples, paired steps and scoops, scoping at some steps, and walking a serpentine pattern. In objective 1, we will compare evaporated milk, buffered peptone water, neutralizing buffer, and phosphate-buffered saline wetting agents for organism recovery from soil sprayed with untreated-manure, over up to 6 months of for die-off. Aerobic count recovery, total coliforms, and generic E. coli will be tested for microbial quality and safety purposes. Compared to reference methods, the drag and bootie swabs hydrated with optimal wetting agent could be (i) more practical if it was clean label, easier to use, faster to collect samples, and less labor; and (ii) more powerful if it would show at least as good, if not better, results for bacteria recovery abilities. In objective 2, drag and bootie swabs will be hydrated by the optimal wetting agent and tested on three commodities in sustainable student farms and two commodities in specialty crop farms. For each commodity, we will collect samples on tilled soil, immediately or 1-2 days post-irrigation, and after opportunity events like rainfall or animal intrusion. The bacteria recovery ability will be analyzed by plate counts and enrichment. 16s rRNA sequencing will be used to test bacteria diversity. We will keep track of the soil parameters, such as texture, pH, soil organic carbon, and microbial biomass, every visit for both objectives 1 and 2. The goal of this project is to enable evidence-based changes in produce soil sampling through industry and academia to ultimately allow producers to better protect public health by evaluating food quality and safety indicators.

Objective 1. Test increasingly practical wetting agents for drag and bootie swabs. 

Objective 2. Validate optimal wetting agent for the drag and bootie swabs on varying soil created by various stages of ground preparation for different commodities.

This work focuses on optimizing wetting agents for drags and booties used in collecting soil samples to improve preharvest produce safety management.

First, to identify practical wetting agents for drag and bootie swabs, five increasingly practical wetting agents were evaluated: skim milk, tryptic soy broth, buffered peptone water, phosphate buffered saline, and deionized water. A total of 484 samples were collected from fallow, row-crop fields where untreated animal manure was applied, and samples tested for aerobic bacteria, total coliforms, and generic Escherichia coli. Applying untreated animal manure was necessary to ensure recoverable levels of typically rare fecal indicator organisms. The results suggested that variations between wetting agents or sampling methods were small and biologically not meaningful (although some statistically significant differences were observed). Therefore, the choice of wetting agent can be guided by practicality, where simple, stable wetting agents like phosphate buffered saline may be preferred.

The next phase was to validate promising wetting agents for collecting soil samples from produce fields. The most promising wetting agents were buffered peptone water and phosphate buffered saline. A total of 240 samples (96 drags, 96 booties, and 48 soil grabs) were collected from a commercial melon farm, a small mixed produce farm (leafy greens, peppers, beets), and an apple orchard. Aerobic bacteria, total coliforms, and E. coli were enumerated, and samples were also enriched for E. coli. Overall results show little biologically meaningful variation between the performance of buffered peptone water and phosphate buffered saline for detecting any of the indicator microorganisms. Further, in some fields bootie and drag swabs, regardless of wetting agent, recovered higher levels of indicator bacteria than composite soil grabs, and, overall, more booties and drags than soil grabs tested positive for E. coli. Microbiome analysis revealed no major differences in diversity between samples collected with the two different hydration buffers.

These data confirm that bootie and drag swabs are a promising alternative to composite soil grab sampling, and that protocols can use whatever wetting agents are most practical.