Recipient Organization
VIRGINIA POLYTECHNIC INSTITUTE
(N/A)
BLACKSBURG,VA 24061
Performing Department
SPES
Non Technical Summary
Alternative irrigation water sources are key to managing water supplies, but often contain greater salt concentrations that might increase food safety risks. The salinity of traditional irrigation waters is rising in many locations and producers are turning to alternative irrigation sources, which also have moderate salinity levels. This is true for drier regions like California and Arizona, but also in more temperate climates like Virginia. For example, the Eastern Shore of Virginia, is a large vegetable producing region, where Virginia ranks in the top ten of fresh-market tomato production in the US. Producers on Delmarva often capture rainwater and hold it in retention ponds to supplement or replace traditional groundwater irrigation. Irrigation water and soil are reservoirs of bacteria that can contaminate vegetable crops. At least five salmonellosis outbreaks have been linked to Delmarva since 2002, with at least one outbreak associated with use of contaminated irrigation pond water. Likewise, outbreaks of pathogenic E. coli have been tracked to irrigation canals in production regions where the salinity of soils and water tends to be high. Whether or not the salinity directly played a role in creating these environmental reservoirs is unknown.Salinity changes the survival of fecal indicator bacteria and pathogens, as well as the overall community structure of microbiota present in water and soils. Very high salt concentrations preserves food because it killspathogens. However, the effects of small increases in salinity on pathogen persistence in water and soil at the field end of the farm-to-fork continuum have not been tested. Recent work by our team shows small increases in salinity and specific salt types can actually increase the persistence of fecal indicator bacteria in water. Non-pathogenic E. coli is the current fecal indicator bacteria for agricultural water under the new Food Safety Modernization Act's Produce Safety Rule. If salinity also increases pathogen persistence, the use of alternative irrigation water sources with higher salinities, such as brackish, return flow, or recycled waters, or salinization of traditional sources, may increases risks to food safety.The research objective of this proposed work is to determine how salinity and salt type in alternative irrigation water alters the fate of FIB and foodborne pathogens in food production. Based on preliminary data, the central hypothesis is that moderate salinity levels (0.35 - 1.0 dS m-1 increase the persistence and concentration of FIB and foodborne pathogens in water, soil, and plant fruits and leaves, but the magnitude of the effects depends on salt type. The rationale is this work will provide: 1) knowledge necessary to determine whether alternative irrigation sources increase the persistence of FIB and foodborne pathogens, and risk of contamination; and 2) informed recommendations for alternative and salinizing irrigation waters, which will ultimately improve management of alternative water sources in vegetable production. We will test the central hypothesis bycollectingirrigation water samples from sources in Virginia, measure the in-situ concentrations of FIB and the water chemistry, and in the greenhouse,inoculatingand measure the persistence of Salmonella, E. coli O157:H7, and FIB in water, soil and plantmesocosms with differing salinities and salt types.Through our project activities we will develop recommendations to mitigate potential food safety risks from pathogen contamination of vegetables via irrigation waters by identifying salinity ranges and types of irrigation waters that are safe, and those that require additional management. This application supports thegoal of providing science-based, credible and reliable benchmarks for nontraditional water used to irrigate food crops, by addressing risks of biological contamination due to salinization. Broadly, we will help US agriculture maintain food safety and human health while reducing the freshwater demand for irrigation by substituting the use of nontraditional water sources.
Animal Health Component
25%
Research Effort Categories
Basic
75%
Applied
25%
Developmental
0%
Goals / Objectives
The goal of this project is to provide: 1) knowledge necessary to determine whether alternative irrigation sources increase the persistence of FIB and foodborne pathogens, and risk of contamination; and 2) informed recommendations for alternative and salinizing irrigation waters, which will ultimately improve management of alternative water sources in vegetable production. The research objectiveis to determine how salinity and salt type in alternative irrigation water alters the fate of FIB and foodborne pathogens in food production. The central hypothesis is that moderate salinity levels (0.35 - 1.0 dS m-1) increase the persistence and concentration of FIB and foodborne pathogens in water, soil, and plant fruits and leaves, but the magnitude of the effects depends on salt type. We will achieve themajor goals via the following specific objectives:Specific Objective 1: Survey salinity, salt type, and FIB concentrations of alternative and traditional irrigation water sources used in the Commonwealth of Virginia. Specific hypothesis 1: Compared to traditional sources, alternative irrigation water will have a higher average salinity and greater concentrations of FIB. Approach 1: We will collect irrigation water samples from sources in Virginia, measure the in-situ concentrations of FIB and the water chemistry.Specific Objective 2: Experimentally determine the effects of salinity and salt type on the persistence of FIB and foodborne pathogens in water. Specific hypothesis 2: salinity, and specifically Mg2+, will increase the persistence of pathogenic bacteria and FIB in water. Approach 2: We will inoculate and measure the persistence of Salmonella, E. coli O157:H7, and FIB in water mesocosms with differing salinities and salt types. Specific Objective 3: Determine the effects of irrigation salinity and salt type on the persistence of FIB and foodborne pathogens in soil and on growing tomato and romaine lettuce plants. Specific hypothesis 3: increasing salinity alters the persistence of pathogenic bacteria in the soil, on tomato fruits and lettuce leaves. Approach 3: We will grow tomatoes and romaine lettuce in the greenhouse using irrigation water with different salinities and salt types, then measure the persistence of Salmonella, and E. coli O157:H7 in the soil and on plant fruit and leaves after inoculations.
Project Methods
Specific Objective 1:Irrigation water survey: We will identify and sample alternative and traditional water sources in Virginia. Producersmay have more than one water source (both traditional and alternative). Each water source will be collected three times during the growing season. At each sampling, we will collect 1 L of water using acid-washed, autoclaved HPDE bottles, identifying the source type and location.Biological and Chemical Analyses:Anions will be measured on a Dionex ion chromatograph (Dionex Corp. Sunnyvale, CA). Cations will be measured by the Virginia Tech Soil Testing Laboratory using inductively coupled argon plasma atomic emission spectroscopy (Spectro AROCOS ICP Model FHS16, Spectro Analytical Instruments, Inc., Mahwah, NJ, USA). Total carbon and nitrogen will be measured as non-purgeable organics using a high-temperature, Pt-catalyzed combustion Shimadzu TOC-V (Shimadzu Corp. Houston, TX, USA). For FIB, concentrations of culturable total coliforms and E. coli will be measured via the Colilert method (IDEXX Laboratories, Inc., Westbrook, ME, USA) using the Quanti-Tray/2000 for enumeration.Data Analysis: General correlations between all log-transformed FIB concentrations and water chemistry measurements will be analyzed using a Pearson's correlation. In addition, a multivariate approach will be used to determine sets of factors that account for highest concentrations of FIB, specific test used depend on sample size and data structure. Types of traditional and alternative irrigation waters will be compared using ANOVA with post hoc comparisons. An Optimized Hot Spot analysis will be performed to identify areas within the region of higher and lower concentrations of salts, types and FIB.Specific Objective 2:Mesocosms:Mesocosms will be set up in a Biological Safety Level 2 (BSL 2) greenhouse on the main campus of Virginia Tech in Blacksburg, Virginia. After incubation, each mesocosm receive carbon, nitrogen and phosphorus at 5 mg L-1 at the Redfield ratio of 106: 16: 1 (C: N: P), so not to stress the bacteria for lack of other nutrients.The experimental design will be a fully replicated factorial in a randomized complete block. We will test three different chloride salts - sodium, calcium, and magnesium- at five conductivities: 0.05 dS m-1, 0.25 dS m-1, 0.50 dS m-1, 1.00 dS m-1, and 2.00 dS m-1. Each treatment combination will be replicated three times for a total of 45 mesocosms. We will inoculate with standard strains of Salmonella, E. coli O157:H7, and non-pathogenic E. coli. E. coli O157:H7 isolated from produce outbreaks will be used. Non-pathogenic E. coli isolated from environmental samples from produce growing regions. Pathogen and FIB concentrations will be measured five times over two weeks, an interval based on the survival rates observed in preliminary observations and literature.Indicator bacteria enumeration: FIB concentrations will be determined as described in Objective 1.E. coli O157:H7 enrichment and isolation: Enrichments will be subjected to immunomagnetic separation (IMS) to concentrate E. coli O157:H7 cells as previously described (Grimont et al., 2007). Washed IMS beads (50 μl) will be plated onto two selective and differential media: modified sorbitol-MacConkey agar (mSMAC; Becton Dickinson, Franklin Lakes, NJ) supplemented with 20 mg/L of novobiocin and 2.5 mg/L of potassium tellurite (Sigma-Aldrich, St. Louis, MO) and CHROMagar O157 agar (CHROMagar, Paris, France). CHROMagar O157 and mSMAC plates will be incubated at 37±2°C for 24 and 48 h, respectively. Up to ten presumptive E. coli O157:H7 colonies will be sub-streaked onto BHI and incubated at 37±2°C for 24 h. Presumptive E. coli O157:H7 colonies will be confirmed using a multiplex PCR assay. Salmonella detection and isolation will be performed using a modified version of the procedures outlined in the Food and Drug Administration's Bacteriological Analytical Manual (FDA, 2019). A 1.0- and 0.1-mL aliquot of non-selective pTSB enrichment will be transferred to 9 and 9.9 mL of tetrathionate (TT; Oxoid) and Rappaport Vassiliadis (RV; Oxoid; Fisher; Acros Organic, Belgium), respectively. These selective enrichment cultures will be incubated in a shaking water bath at 42±2°C for 24 h. A 50 μl aliquot selective enrichment will be plated onto xylose lysine deoxycholate agar (XLD; Neogen, Lansing, MI) and CHROMagar Salmonella (CHROMagar) agar, and incubated at 35 and 37±2°C for 24 and 48 h, respectively. Up to 20 presumptive Salmonella colonies will be sub-streaked to BHI and incubated at 37±2°C for 24 h. Presumptive Salmonella colonies will be confirmed using a previously described PCR assay that detects invA.Data Analysis: Persistence will be measured as K values. K values are the slope of the linear regression model of log-transformed counts. K values of different salt treatments and bacterial type (pathogen vs. indicator) will be compared using ANOVA. K values will be empirically modelled with salinity using AIC regression.Specific Objective 3. ?Greenhouse experiment: To minimize risks, we will use the same BSL-2 greenhouse complex at Virginia Tech in Blacksburg, VA.For tomatoes, we will use 5-gallon containers filled with a homogenized soil mix of sand, vermiculite, and top soil mix. Soil will be tested for presence of FIB prior to planting. Romaine lettuce will be grown in commercial top soil, prepared by mixing top soil (Sun Gro Metro-mix 510; Bellevue, WA) with Marathon 1% Granular Insecticide (OHP, Inc., Mainland, PA) and slow-release fertilizer (Osmocote, Scotts Miracle-Gro, Marysville, OH). Plants will be grown in planter trays.We will irrigate all plants with an overhead system with waters of differing salinity and salt types.We will test two chloride salts of sodium, calcium, or magnesium, at five conductivities: 0.05 dS m-1, 0.25 dS m-1, 0.50 dS m-1, 1.00 dS m-1, and 2.00 dS m-1.We will use a replicated factorial in a randomized complete block design. Treatment combinations will be replicated three times for a total of 45 mesocosms for tomato and lettuce. When tomatoes and lettuce are in production stage, we will inoculate the plants and soil with indicator E. coli and either Salmonella (tomato) or E. coli O157:H7 (lettuce) strains. We will spray fruits of tomato and leaves of lettuce until runoff with a bacterial suspension in irrigation water. Although bacteria concentration in solution will be determined through turbidity, the actual number of bacteria on tomato fruits and lettuce leaves will be determined by sampling just after inoculation and the CFU measurement will be time = 0. We will sample four more times over ten days. After inoculation, pots will be watered carefully by hand only applying water to the soil, so not to wash off bacteria from the tomatoes. At each sampling, 5 mature red fruits or 5 lettuce heads will randomly be collected from each container. Plant parts will be placed into a sanitized pre-labeled harvesting bin. A total of 45 samples (5 fruits/pot x 3 replications x 5 time points) will collected for each treatment for pathogen detection. All the bins with harvested plant parts will be stored in a walk-in cold storage room at 12-15°C prior to microbial analysis. The methods described in Objective 2 will be used to quantify pathogens.Data analysis: Persistence will be measured as K values, where concentrations just after inoculation provide the zero-time point, and decrease of concentrations in subsequent samplings represent the decay rate. K values are the slope of the linear regression model of log-transformed counts. K values of different salt treatments and bacterial type (pathogen vs. indicator) will be compared using ANOVA.