Steven L. Rideout

Baseline survey of the anatomical microbial ecology of an important food plant: Solanum lycopersicum (tomato).

BackgroundResearch to understand and control microbiological risks associated with the consumption of fresh fruits and vegetables has examined many environments in the farm to fork continuum. An important data gap however, that remains poorly studied is the baseline description of microflora that may be associated with plant anatomy either endemically or in response to environmental pressures. Specific anatomical niches of plants may contribute to persistence of human pathogens in agricultural environments in ways we have yet to describe. Tomatoes have been implicated in outbreaks of Salmonella at least 17 times during the years spanning 1990 to 2010. Our research seeks to provide a baseline description of the tomato microbiome and possibly identify whether or not there is something distinctive about tomatoes or their growing ecology that contributes to persistence of Salmonella in this important food crop.ResultsDNA was recovered from washes of epiphytic surfaces of tomato anatomical organs; leaves, stems, roots, flowers and fruits of Solanum lycopersicum (BHN602), grown at a site in close proximity to commercial farms previously implicated in tomato-Salmonella outbreaks. DNA was amplified for targeted 16S and 18S rRNA genes and sheared for shotgun metagenomic sequencing. Amplicons and metagenomes were used to describe “native” bacterial microflora for diverse anatomical parts of Virginia-grown tomatoes.ConclusionsDistinct groupings of microbial communities were associated with different tomato plant organs and a gradient of compositional similarity could be correlated to the distance of a given plant part from the soil. Unique bacterial phylotypes (at 95% identity) were associated with fruits and flowers of tomato plants. These include Microvirga, Pseudomonas, Sphingomonas, Brachybacterium, Rhizobiales, Paracocccus, Chryseomonas and Microbacterium. The most frequently observed bacterial taxa across aerial plant regions were Pseudomonas and Xanthomonas. Dominant fungal taxa that could be identified to genus with 18S amplicons included Hypocrea, Aureobasidium and Cryptococcus. No definitive presence of Salmonella could be confirmed in any of the plant samples, although 16S sequences suggested that closely related genera were present on leaves, fruits and roots.

Co-Enriching Microflora Associated with Culture Based Methods to Detect Salmonella from Tomato Phyllosphere

The ability to detect a specific organism from a complex environment is vitally important to many fields of public health, including food safety. For example, tomatoes have been implicated numerous times as vehicles of foodborne outbreaks due to strains of Salmonella but few studies have ever recovered Salmonella from a tomato phyllosphere environment. Precision of culturing techniques that target agents associated with outbreaks depend on numerous factors. One important factor to better understand is which species co-enrich during enrichment procedures and how microbial dynamics may impede or enhance detection of target pathogens. We used a shotgun sequence approach to describe taxa associated with samples pre-enrichment and throughout the enrichment steps of the Bacteriological Analytical Manuals (BAM) protocol for detection of Salmonella from environmental tomato samples. Recent work has shown that during efforts to enrich Salmonella (Proteobacteria) from tomato field samples, Firmicute genera are also co-enriched and at least one co-enriching Firmicute genus (Paenibacillus sp.) can inhibit and even kills strains of Salmonella. Here we provide a baseline description of microflora that co-culture during detection efforts and the utility of a bioinformatic approach to detect specific taxa from metagenomic sequence data. We observed that uncultured samples clustered together with distinct taxonomic profiles relative to the three cultured treatments (Universal Pre-enrichment broth (UPB), Tetrathionate (TT), and Rappaport-Vassiliadis (RV)). There was little consistency among samples exposed to the same culturing medias, suggesting significant microbial differences in starting matrices or stochasticity associated with enrichment processes. Interestingly, Paenibacillus sp. (Salmonella inhibitor) was significantly enriched from uncultured to cultured (UPB) samples. Also of interest was the sequence based identification of a number of sequences as Salmonella despite indication by all media, that samples were culture negative for Salmonella. Our results substantiate the nascent utility of metagenomic methods to improve both biological and bioinformatic pathogen detection methods.

Ecological prevalence, genetic diversity, and epidemiological aspects of Salmonella isolated from tomato agricultural regions of the Virginia Eastern Shore.

Virginia is the third largest producer of fresh-market tomatoes in the United States. Tomatoes grown along the eastern shore of Virginia are implicated almost yearly in Salmonella illnesses. Traceback implicates contamination occurring in the pre-harvest environment. To get a better understanding of the ecological niches of Salmonella in the tomato agricultural environment, a 2-year study was undertaken at a regional agricultural research farm in Virginia. Environmental samples, including tomato (fruit, blossoms, and leaves), irrigation water, surface water and sediment, were collected over the growing season. These samples were analyzed for the presence of Salmonella using modified FDA-BAM methods. Molecular assays were used to screen the samples. Over 1500 samples were tested. Seventy-five samples tested positive for Salmonella yielding over 230 isolates. The most commonly isolated serovars were S. Newport and S. Javiana with pulsed-field gel electrophoresis yielding 39 different patterns. Genetic diversity was further underscored among many other serotypes, which showed multiple PFGE subtypes. Whole genome sequencing (WGS) of several S. Newport isolates collected in 2010 compared to clinical isolates associated with tomato consumption showed very few single nucleotide differences between environmental isolates and clinical isolates suggesting a source link to Salmonella contaminated tomatoes. Nearly all isolates collected during two growing seasons of surveillance were obtained from surface water and sediment sources pointing to these sites as long-term reservoirs for persistent and endemic contamination of this environment.

In Situ Evaluation of Paenibacillus alvei in Reducing Carriage of Salmonella enterica Serovar Newport on Whole Tomato Plants

ABSTRACT Recently, tomatoes have been implicated as a primary vehicle in food-borne outbreaks of Salmonella enterica serovar Newport and other Salmonella serovars. Long-term intervention measures to reduce Salmonella prevalence on tomatoes remain elusive for growing and postharvest environments. A naturally occurring bacterium identified by 16S rRNA gene sequencing as Paenibacillus alvei was isolated epiphytically from plants native to the Virginia Eastern Shore tomato-growing region. After initial antimicrobial activity screening against Salmonella and 10 other bacterial pathogens associated with the human food supply, strain TS-15 was further used to challenge an attenuated strain of S. Newport on inoculated fruits, leaves, and blossoms of tomato plants in an insect-screened high tunnel with a split-plot design. Survival of Salmonella after inoculation was measured for groups with and those without the antagonist at days 0, 1, 2, and 3 and either day 5 for blossoms or day 6 for fruits and leaves. Strain TS-15 exhibited broad-range antimicrobial activity against both major food-borne pathogens and major bacterial phytopathogens of tomato. After P. alvei strain TS-15 was applied onto the fruits, leaves, and blossoms of tomato plants, the concentration of S. Newport declined significantly (P ≤ 0.05) compared with controls. Astonishingly, >90% of the plants had no detectable levels of Salmonella by day 5 for blossoms. The naturally occurring antagonist strain TS-15 is highly effective in reducing the carriage of Salmonella Newport on whole tomato plants. The application of P. alvei strain TS-15 is a promising approach for reducing the risk of Salmonella contamination during tomato production.

The impact of systemic and copper pesticide applications on the phyllosphere microflora of tomatoes

Background Contamination of tomatoes by Salmonella can occur in agricultural settings. Little is currently understood about how agricultural inputs such as pesticide applications may impact epiphytic crop microflora and potentially play a role in contamination events. We examined the impact of two materials commonly used in Virginia tomato agriculture: acibenzolar-S-methyl (crop protectant) and copper oxychloride (pesticide) to identify the effects these materials may exert on baseline tomato microflora and on the incidence of three specific genera; Salmonella, Xanthomonas and Paenibacillus. Results Approximately 186 441 16S rRNA gene and 39 381 18S rRNA gene sequences per independent replicate were used to analyze the impact of the pesticide applications on tomato microflora. An average of 3 346 677 (634 892 974 bases) shotgun sequences per replicate were used for metagenomic analyses. Conclusion A significant decrease in the presence of Gammaproteobacteria was observed between controls and copper-treated plants, suggesting that copper is effective at suppressing growth of certain taxa in this class. A higher mean abundance of Salmonella and Paenibacillus in control samples compared to treatments may suggest that both systemic and copper applications diminish the presence of these genera in the phyllosphere; however, owing to the lack of statistical significance, this could also be due to other factors. The most distinctive separation of shared membership was observed in shotgun data between the two different sampling time-points (not between treatments), potentially supporting the hypothesis that environmental pressures may exert more selective pressures on epiphytic microflora than do certain agricultural management practices.

Potential Interactions between Salmonella enterica and Ralstonia solanacearum in tomato plants.

Over the past decade, the Eastern Shore of Virginia (ESV) has been implicated in at least four outbreaks of salmonellosis associated with tomato, all originating from the same serovar, Salmonella enterica serovar Newport. In addition to Salmonella Newport contamination, the devastating plant disease bacterial wilt, caused by the phytopathogen Ralstonia solanacearum, threatens the sustainability of ESV tomato production. Bacterial wilt is present in most ESV tomato fields and causes devastating yield losses each year. Although the connection between bacterial wilt and tomato-related salmonellosis outbreaks in ESV is of interest, the relationship between the two pathogens has never been investigated. In this study, tomato plants were root dip inoculated with one of four treatments: (i) 8 log CFU of Salmonella Newport per ml, (ii) 5 log CFU of R. solanacearum per ml, (iii) a coinoculation of 8 log CFU of Salmonella Newport per ml plus 5 log CFU of R. solanacearum per ml, and (iv) sterile water as control. Leaf, stem, and fruit samples were collected at the early-green-fruit stage, and S. enterica contamination in the internal tissues was detected. S. enterica was recovered in 1.4 and 2.9% of leaf samples from plants inoculated with Salmonella Newport only and from plants coinoculated with Salmonella Newport plus R. solanacearum, respectively. S. enterica was recovered from 1.7 and 3.5% of fruit samples from plants inoculated with Salmonella Newport only and from plants coinoculated with Salmonella Newport plus R. solanacearum, respectively. There were significantly more stem samples from plants coinoculated with Salmonella Newport plus R. solanacearum that were positive for S. enterica (18.6%) than stem samples collected from plants inoculated with Salmonella Newport only (5.7%). Results suggested that R. solanacearum could influence S. enterica survival and transportation throughout the internal tissues of tomato plants.

Effects of pesticides on the reduction of plant and human pathogenic bacteria in application water.

Overhead spray applications of in-field tomato treatments dissolved in aqueous solutions have specific pest targets (fungal, bacterial, insect, or other). Any organism present in the solution or on treated plant surfaces that is not a specific target of the application is unlikely inactivated and can instead be spread through the phyllosphere. In this laboratory study, commercially labeled pesticides (including Actigard 50WG, Bravo Weather Stik 6F, Cabrio 20EG, Kasumin, Kocide 3000 46WG, Oxidate 27L, Penncozeb 75DF, ProPhyt 54.5L, Stimplex 100L, Firewall, 22.4WP, and Tanos 50DF) in common use in commercial tomato production fields of the Eastern Shore of Virginia were investigated for activity against in vitro bacterial contamination of pesticide application waters. Pesticides of interest were tank mixed individually with one of the plant pathogens Ralstonia solanacearum, Xanthomonas campestris pv. vesicatoria, Pseudomonas syringae pv. tomato, Erwinia carotovora subsp. carotovora, or one of two serovars (Newport and Montevideo) of the human pathogen Salmonella enterica to assess reduction values during the average time between mixing and initial application. Observations suggested that while some treatments had a noticeable effect on population levels, only the oxidizer, peroxyacetic acid, showed significant and consistent levels of suppression against all bacteria investigated, at levels that could have practical implications.

Nitrogen Fertilizer and Growth Regulator Impacts on Tuber Deformity, Rot, and Yield for Russet Potatoes

Potatoes (Solanum tuberosum) are an important high-value commodity for producers in the Mid-Atlantic Region. Current production recommendations were based on white potatoes, and practices for Russet potatoes have not been researched in this region. The objective of this study was to test impacts of N rate (0, 67, 134, 201, and 268 kg N ha−1), N application timing (100% applied with planter, 2-way split (30% with planter and 70% band applied approximately 30 days after planting at dragoff), and three-way split (30% with planter, 50% band applied prior to drag-off, and 20% band applied at first sight of bloom)), and additions of the growth regulator maleic hydrazide (MH-30). We tested “Goldrush” and “Norkotah” Russet potato varieties on marketability, total yield, tuber deformity, and tuber soft rot incidence for sandy loam soils in the Mid-Atlantic. Overall, year variations were significant with substantial rots (up to 86.5%) occurring in year 3. Maleic hydrazide and N application timing had little consistent effect on any tested parameter. Nitrogen rate and variety factors had the greatest impacts on deformity, tuber rots, and yields for Russet potatoes in the Mid-Atlantic Region with 134 kg N ha−1 producing the highest total yields in 2009 and 2010. If tuber rots can be controlled, both “Goldrush” and “Norkotah” are acceptable varieties under the Mid-Atlantic production practices.

Microbial Quality of Agricultural Water Used in Produce Preharvest Production on the Eastern Shore of Virginia

Several produce-borne outbreaks have been associated with the use of contaminated water during preharvest applications. Salmonella has been implicated in a number of these outbreaks. The purpose of this study was to evaluate the microbial quality of agricultural surface water used in preharvest production on the Eastern Shore of Virginia in accordance with the Food Safety Modernization Acts Produce Safety Rule water standards. The study also examined the prevalence, concentration, and diversity of Salmonella in those water sources. Water samples (1 L) from 20 agricultural ponds were collected during the 2015 and 2016 growing seasons ( n = 400). Total aerobic bacteria, total coliforms, and Escherichia coli were enumerated for each sample. Population levels of each microorganism were calculated per 100-mL sample and log transformed, when necessary. Samples (250 mL) were also enriched for Salmonella. Presumptive Salmonella isolates were confirmed by PCR ( invA gene) and were serotyped. In 2016, the concentration of Salmonella in each sample was also estimated by most probable number (MPN). Indicator bacteria and environmental and meteorological factors were analyzed for their association with the detection of a Salmonella-positive water sample by using logistic regression analysis. Seventeen of the 20 ponds met the Food Safety Modernization Acts Produce Safety Rule standards for production agricultural water. Three ponds did not meet the standards because the statistical threshold value exceeded the limit. Salmonella was detected in 19% of water samples in each year (38 of 200 in 2015 and 38 of 200 in 2016). Of the 118 Salmonella isolates serotyped, 14 serotypes were identified with the most prevalent being Salmonella Newport. E. coli concentration, farm, and total aerobic bacteria concentration were significantly associated with the likelihood of detecting a Salmonella-positive sample The average concentration of Salmonella in all samples was 4.44 MPN/100 mL, with the limit of detection being 3.00 MPN/100 mL. The highest concentration of Salmonella was 93.0 MPN/100 mL. These data will assist in a better understanding of the risks that production water poses to produce contamination events.

Agricultural Practices Influence Salmonella Contamination and Survival in Pre-harvest Tomato Production

Between 2000 and 2010 the Eastern Shore of Virginia was implicated in four Salmonella outbreaks associated with tomato. Therefore, a multi-year study (2012-2015) was performed to investigate presumptive factors associated with the contamination of Salmonella within tomato fields at Virginia Techs Eastern Shore Agricultural Research and Extension Center. Factors including irrigation water sources (pond and well), type of soil amendment: fresh poultry litter (PL), PL ash, and a conventional fertilizer (triple superphosphate - TSP), and production practices: staked with plastic mulch (SP), staked without plastic mulch (SW), and non-staked without plastic mulch (NW), were evaluated by split-plot or complete-block design. All field experiments relied on naturally occurring Salmonella contamination, except one follow up experiment (worst-case scenario) which examined the potential for contamination in tomato fruits when Salmonella was applied through drip irrigation. Samples were collected from pond and well water; PL, PL ash, and TSP; and the rhizosphere, leaves, and fruits of tomato plants. Salmonella was quantified using a most probable number method and contamination ratios were calculated for each treatment. Salmonella serovar was determined by molecular serotyping. Salmonella populations varied significantly by year; however, similar trends were evident each year. Findings showed use of untreated pond water and raw PL amendment increased the likelihood of Salmonella detection in tomato plots. Salmonella Newport and Typhimurium were the most frequently detected serovars in pond water and PL amendment samples, respectively. Interestingly, while these factors increased the likelihood of Salmonella detection in tomato plots (rhizosphere and leaves), all tomato fruits sampled (n = 4800) from these plots were Salmonella negative. Contamination of tomato fruits was extremely low (< 1%) even when tomato plots were artificially inoculated with an attenuated Salmonella Newport strain (104 CFU/mL). Furthermore, Salmonella was not detected in tomato plots irrigated using well water and amended with PL ash or TSP. Production practices also influenced the likelihood of Salmonella detection in tomato plots. Salmonella detection was higher in tomato leaf samples for NW plots, compared to SP and SW plots. This study provides evidence that attention to agricultural inputs and production practices may help reduce the likelihood of Salmonella contamination in tomato fields.