Bacterial community shifts of commercial apples, oranges, and peaches at different harvest points across multiple growing seasons.

Assessing the microbes present on tree fruit carpospheres as the fruit enters postharvest processing could have useful applications, as these microbes could have a major influence on spoilage, food safety, verification of packing process controls, or other aspects of processing. The goal of this study was to establish a baseline profile of bacterial communities associated with apple (pome fruit), peach (stone fruit), and Navel orange (citrus fruit) at harvest. We found that commercial peaches had the greatest bacterial richness followed by oranges then apples. Time of harvest significantly changed bacterial diversity in oranges and peaches, but not apples. Shifts in diversity varied by fruit type, where 70% of the variability in beta diversity on the apple carposphere was driven by the gain and loss of species (i.e., nestedness). The peach and orange carposphere bacterial community shifts were driven by nearly an even split between turnover (species replacement) and nestedness. We identified a small core microbiome for apples across and between growing seasons that included only Methylobacteriaceae and Sphingomonadaceae among the samples, while peaches had a larger core microbiome composed of five bacterial families: Bacillaceae, Geodermtophilaceae, Nocardioidaceae, Micrococcaeceae, and Trueperaceae. There was a relatively diverse core microbiome for oranges that shared all the families present on apples and peaches, except for Trueperaceae, but also included an additional nine bacterial families not shared including Oxalobacteraceae, Cytophagaceae, and Comamonadaceae. Overall, our findings illustrate the important temporal dynamics of bacterial communities found on major commercial tree fruit, but also the core bacterial families that constantly remain with both implications being important entering postharvest packing and processing.

Weather stressors correlate with Escherichia coli and Salmonella enterica persister formation rates in the phyllosphere: a mathematical modeling study.

Enteric pathogens can enter a persister state in which they survive exposure to antibiotics and physicochemical stresses. Subpopulations of such phenotypic dormant variants have been detected in vivo and in planta in the laboratory, but their formation in the natural environment remains largely unexplored. We applied a mathematical model predicting the switch rate to persister cell in the phyllosphere to identify weather-related stressors associated with E. coli and S. enterica persister formation on plants based on their population dynamics in published field studies from the USA and Spain. Model outputs accurately depicted the bi-phasic decay of bacterial population sizes measured in the lettuce and spinach phyllosphere in these studies. Predicted E. coli persister switch rate on leaves was positively and negatively correlated with solar radiation intensity and wind velocity, respectively. Likewise, predicted S. enterica persister switch rate correlated positively with solar radiation intensity; however, a negative correlation was observed with air temperature, relative humidity, and dew point, factors involved in water deposition onto the phylloplane. These findings suggest that specific environmental factors may enrich for dormant bacterial cells on plants. Our model quantifiably links persister cell subpopulations in the plant habitat with broader physical conditions, spanning processes at different granular scales.

Verification of peroxyacetic acid treatment against L. monocytogenes on fresh apples using E. faecium NRRL B-2354 as a surrogate in commercial spray-bar operations.

Peroxyacetic acid (PAA) is a commonly used antimicrobial in apple spray bar interventions during post-harvest packing. However, limited information is available about its efficacy against foodborne pathogens on fresh apples under commercial packing conditions. In this study, the practical efficacies of PAA against Listeria monocytogenes on fresh apples during spray bar operation at ambient and elevated temperature were validated in three commercial packing facilities using Enterococcus faecium NRRL B-2354 as a surrogate strain. Apples were inoculated with E. faecium at ~6.5 Log10 CFU/apple and subjected to PAA spray bar interventions per commercial packing line practice. At each temperature and contact time intervention combination, 20-24 inoculated apples were processed together with 72-80 non-inoculated apples. Applying 80 ppm PAA at ambient temperature (17-21 °C) achieved a similar log reduction (P > 0.05) of E. faecium on Granny Smith apples (GSA) in three apple packing facilities, which caused 1.12-1.23 and 1.18-1.32 Log10 CFU/apple reductions of E. faecium on GSA for 30-sec and 60-sec intervention, respectively. Increasing the temperature of the PAA solution to 43-45 °C enhanced its bactericidal effect against E. faecium, causing 1.45, 1.86 and 2.19 Log10 CFU/apple reductions in three packing facilities for a 30-sec contact, and 1.50, 2.24, and 2.29 Log10 CFU/apple reductions for a 60-sec contact, respectively. Similar efficacies (P > 0.05) of PAA at both ambient and elevated temperature were also observed on Fuji apples. Spraying PAA on apples at ambient or elevated temperature reduced the level of E. faecium cross-contamination from inoculated apples to non-inoculated apples but could not eliminate cross-contamination. Data from this study provides valuable technical information and a reference point for the apple industry in controlling L. monocytogenes and verifying the effectiveness of their practices.

Effect of Weather on the Die-Off of Escherichia coli and Attenuated Salmonella enterica Serovar Typhimurium on Preharvest Leafy Greens following Irrigation with Contaminated Water.

The Food Safety Modernization Act (FSMA) includes a time-to-harvest interval following the application of noncompliant water to preharvest produce to allow for microbial die-off. However, additional scientific evidence is needed to support this rule. This study aimed to determine the impact of weather on the die-off rate of Escherichia coli and Salmonella on spinach and lettuce under field conditions. Standardized, replicated field trials were conducted in California, New York, and Spain over 2 years. Baby spinach and lettuce were grown and inoculated with an ∼104-CFU/ml cocktail of E. coli and attenuated Salmonella Leaf samples were collected at 7 time points (0 to 96 h) following inoculation; E. coli and Salmonella were enumerated. The associations of die-off with study design factors (location, produce type, and bacteria) and weather were assessed using log-linear and biphasic segmented log-linear regression. A segmented log-linear model best fit die-off on inoculated leaves in most cases, with a greater variation in the segment 1 die-off rate across trials (-0.46 [95% confidence interval {95% CI}, -0.52, -0.41] to -6.99 [95% CI, -7.38, -6.59] log10 die-off/day) than in the segment 2 die-off rate (0.28 [95% CI, -0.20, 0.77] to -1.00 [95% CI, -1.16, -0.85] log10 die-off/day). A lower relative humidity was associated with a faster segment 1 die-off and an earlier breakpoint (the time when segment 1 die-off rate switches to the segment 2 rate). Relative humidity was also found to be associated with whether die-off would comply with FSMA's specified die-off rate of -0.5 log10 die-off/day.IMPORTANCE The log-linear die-off rate proposed by FSMA is not always appropriate, as the die-off rates of foodborne bacterial pathogens and specified agricultural water quality indicator organisms appear to commonly follow a biphasic pattern with an initial rapid decline followed by a period of tailing. While we observed substantial variation in the net culturable population levels of Salmonella and E. coli at each time point, die-off rate and FSMA compliance (i.e., at least a 2 log10 die-off over 4 days) appear to be impacted by produce type, bacteria, and weather; die-off on lettuce tended to be faster than that on spinach, die-off of E. coli tended to be faster than that of attenuated Salmonella, and die-off tended to become faster as relative humidity decreased. Thus, the use of a single die-off rate for estimating time-to-harvest intervals across different weather conditions, produce types, and bacteria should be revised.

Enhanced Efficacy of Peroxyacetic Acid Against Listeria monocytogenes on Fresh Apples at Elevated Temperature.

Peroxyacetic acid (PAA) is the most commonly used antimicrobial in spray bar antimicrobial treatment during fresh apple packing and processing. However, there are limited data regarding its practical efficacy against Listeria monocytogenes on fresh apples. This study evaluated the antimicrobial activity of PAA against L. monocytogenes on fresh apples applicable to current industry practice, and further examined practical parameters impacting its efficacy to maximize the biocidal effects. Apples were inoculated with a three-strain L. monocytogenes cocktail at ~6.0 Log10 CFU/apple and then subjected to comparative antimicrobial treatments after 48 h post-inoculation. An 80 ppm PAA treatment, at 30-s and 2-min exposure, reduced L. monocytogenes on fresh apples by ~1.3 or 1.7 Log10 CFU/apple, respectively. The anti-Listeria efficacy of PAA was not affected by the water hardness and pH of PAA solution, while it improved dramatically when applied at elevated temperature. A 2-min exposure of 80 ppm PAA at 43 and 46°C resulted in a 2.3 and 2.6 Log10 CFU/apple reduction, respectively. A 30-s contact time of 80 ppm PAA at 43-46°C reduced L. monocytogenes on apples by 2.2-2.4 Log10 CFU/apple. Similarly, PAA intervention at elevated temperatures significantly strengthened its effectiveness against naturally occurring apple microbiota. PAA treatment at 43-46°C can provide a vital method to improve antimicrobial efficacy against both L. monocytogenes and indigenous microbiota on fresh apples. Our data provide valuable information and reference points for the apple industry to further validate or verify process controls.

Occurrence of Escherichia coli O157:H7 in Pest Flies Captured in Leafy Greens Plots Grown Near a Beef Cattle Feedlot.

Leafy greens are leading vehicles for Escherichia coli O157:H7 foodborne illness. Pest flies can harbor this pathogen and may disseminate it to produce. We determined the occurrence of E. coli O157:H7-positive flies in leafy greens planted up to 180 m from a cattle feedlot and assessed their relative risk to transmit this pathogen to leafy greens. The primary fly groups captured on sticky traps at the feedlot and leafy greens plots included house flies (Musca domestica L.), face flies (Musca autumnalis L.), stable flies (Stomoxys calcitrans L.), flesh flies (family Sarcophagidae), and blow flies (family Calliphoridae). E. coli O157:H7 carriage rates of house, face, flesh, and blow flies were similar (P > 0.05), ranging from 22.3 to 29.0 flies per 1,000 flies. In contrast, the carriage rate of stable flies was lower at 1.1 flies per 1,000 flies (P < 0.05). Differences in carriage rates are likely due to the uses of fresh bovine feces and manure by these different pest fly groups. E. coli O157:H7 carriage rates of total flies did not differ (P > 0.05) by distance (ranging from 0 to 180 m) from the feedlot. Most fly isolates were the same predominant pulsed-field gel electrophoresis types found in feedlot surface manure and leafy greens, suggesting a possible role for flies in transmitting E. coli O157:H7 to the leafy greens. However, further research is needed to clarify this role and to determine set-back distances between cattle production facilities and produce crops that will reduce the risk for pathogen contamination by challenging mechanisms like flies.

Evaluation of post-contamination survival and persistence of applied attenuated E. coli O157:H7 and naturally-contaminating E. coli O157:H7 on spinach under field conditions and following postharvest handling.

This study determined the variability in population uniformity of an applied mixture of attenuated E. coli O157:H7 (attEcO157) on spinach leaves as impacted by sampling mass and detection technique over spatial and temporal conditions. Opportunistically, the survival and distribution of naturally contaminating pathogenic E. coli O157:H7 (EcO157), in a single packaged lot following commercial postharvest handling and washing, was also evaluated. From the main study outcomes, differences in the applied inoculum dose of 100-fold, resulted in indistinguishable population densities of approximately Log 1.1 CFU g-1 by 14 days post-inoculation (DPI). Composite leaf samples of 150 g and the inclusion of the spinach petiole resulted in the greatest numerical sensitivity of detection of attEcO157 when compared to 25 and 150 g samples without petioles (P < 0.05). Differences in population density and protected-site survival and potential leaf internalization were observed between growing seasons and locations in California (P < 0.05). A Double Weibull model best described and identified two distinct populations with different inactivation rates of the inoculated attEcO157. Linear die-off rates varied between 0.14 and 0.29 Log/Day irrespective of location. Detection of EcO157- stx1-negative and stx2-positive, resulting from a natural contamination event, was observed in 11 of 26 quarantined commercial units of washed spinach by applying the 150 g sample mass protocol. The capacity to detect EcO157 varied between commercial test kits and non-commercial qPCR. Our findings suggest the need for modifications to routine pathogen sampling protocols employed for lot acceptance of spinach and other leafy greens.

Evaluation of JC9450 and Neutral Electrolyzed Water in Controlling Listeria monocytogenes on Fresh Apples and Preventing Cross-Contamination.

Recent multistate outbreaks and recalls of fresh apples due to Listeria monocytogenes contamination have increased consumer concerns regarding fresh and processed apple safety. This study aimed to evaluate the antimicrobial efficacy of two sanitizers, mineral oxychloride (JC9450) and neutral electrolyzed water (NEW), for inactivation of L. monocytogenes on fresh apples. A 2-min treatment of 0.125% (v/v) JC9450 with 100 ppm free available chlorine (FAC) or NEW with 110 ppm FAC caused 0.9-1.2 log10 CFU/apple reduction of L. monocytogenes on both Granny Smith and Fuji apples 24 h post-inoculation. Increasing JC9450 concentration to 0.25 and 0.50% significantly improved its bactericidal effect and reduced L. monocytogenes on Granny Smith apples by ~2.0 and 3.8 log10 CFU/apple, respectively, after a contact time of 2 min. At a shorter contact time of 30 sec, the inactivation efficacy of chlorine and 0.25-0.50% JC9450 against L. monocytogenes on apples was significantly reduced compared with the respective 2-min wash. Furthermore, no L. monocytogenes was recovered in deionized water prepared antimicrobial wash solution or on non-inoculated apples post-NEW with 110 ppm FAC or 0.125-0.5% JC9450 washes, indicating their ability to prevent cross-contamination. In addition, a 2-min exposure to NEW with 110 ppm FAC and 0.50% JC9450 reduced apple native microbiota including total plate count by 0.14 and 0.65 log10 CFU/apple, respectively, and yeast and mold counts by 0.55 and 1.63 log10 CFU/apple, respectively. In summary, L. monocytogenes attached on apples was difficult to eliminate. JC9450 and NEW demonstrated a dose-dependent reduction in L. monocytogenes on apples and successfully prevented cross-contamination, indicating their application potential in post-harvest washes of apples.

Impact of chlorine dioxide disinfection of irrigation water on the epiphytic bacterial community of baby spinach and underlying soil.

The contamination of pathogenic bacteria through irrigation water is a recognized risk factor for fresh produce. Irrigation water disinfection is an intervention strategy that could be applied to reduce the probability of microbiological contamination of crops. Disinfection treatments should be applied ensuring minimum effective doses, which are efficient in inhibiting the microbial contamination while avoiding formation and accumulation of chemical residues. Among disinfection technologies available for growers, chlorine dioxide (ClO2) represents, after sodium hypochlorite, an alternative disinfection treatment, which is commercially applied by growers in the USA and Spain. However, in most of the cases, the suitability of this treatment has been tested against pathogenic bacteria and low attention have been given to the impact of chemical residues on the bacterial community of the vegetable tissue. The aim of this study was to (i) to evaluate the continual application of chlorine dioxide (ClO2) as a water disinfection treatment of irrigation water during baby spinach growth in commercial production open fields, and (ii) to determine the subsequent impact of these treatments on the bacterial communities in water, soil, and baby spinach. To gain insight into the changes in the bacterial community elicited by ClO2, samples of treated and untreated irrigation water as well as the irrigated soil and baby spinach were analyzed using Miseq® Illumina sequencing platform. Next generation sequencing and multivariate statistical analysis revealed that ClO2 treatment of irrigation water did not affect the diversity of the bacterial community of water, soil and crop, but significant differences were observed in the relative abundance of specific bacterial genera. This demonstrates the different susceptibility of the bacteria genera to the ClO2 treatment. Based on the obtained results it can be concluded that the phyllosphere bacterial community of baby spinach was more influenced by the soil bacteria community rather than that of irrigation water. In the case of baby spinach, the use of low residual ClO2 concentrations (approx. 0.25 mg/L) to treat irrigation water decreased the relative abundance of Pseudomonaceae (2.28-fold) and Enterobacteriaceae (2.5-fold) when comparing treated versus untreated baby spinach. Members of these two bacterial families are responsible for food spoilage and foodborne illnesses. Therefore, a reduction of these bacterial families might be beneficial for the crop and for food safety. In general it can be concluded that the constant application of ClO2 as a disinfection treatment for irrigation water only caused changes in two bacterial families of the baby spinach and soil microbiota, without affecting the major phyla and classes. The significance of these changes in the bacterial community should be further evaluated.

High-Level Culturability of Epiphytic Bacteria and Frequency of Biosurfactant Producers on Leaves.

UNLABELLED: To better characterize the bacterial community members capable of biosurfactant production on leaves, we distinguished culturable biosurfactant-producing bacteria from nonproducers and used community sequencing to compare the composition of these distinct cultured populations with that from DNA directly recovered from leaves. Communities on spinach, romaine, and head lettuce leaves were compared with communities from adjacent samples of soil and irrigation source water. Soil communities were poorly described by culturing, with recovery of cultured representatives from only 21% of the prevalent operational taxonomic units (OTUs) (>0.2% reads) identified. The dominant biosurfactant producers cultured from soil included bacilli and pseudomonads. In contrast, the cultured communities from leaves are highly representative of the culture-independent communities, with over 85% of the prevalent OTUs recovered. The dominant taxa of surfactant producers from leaves were pseudomonads as well as members of the infrequently studied genus Chryseobacterium The proportions of bacteria cultured from head lettuce and romaine leaves that produce biosurfactants were directly correlated with the culture-independent proportion of pseudomonads in a given sample, whereas spinach harbored a wider diversity of biosurfactant producers. A subset of the culturable bacteria in irrigation water also became enriched on romaine leaves that were irrigated overhead. Although our study was designed to identify surfactant producers on plants, we also provide evidence that most bacteria in some habitats, such as agronomic plant surfaces, are culturable, and these communities can be readily investigated and described by more classical culturing methods. IMPORTANCE: The importance of biosurfactant production to the bacteria that live on waxy leaf surfaces as well as their ability to be accurately assessed using culture-based methodologies was determined by interrogating epiphytic populations by both culture-dependent and culture-independent methods. Biosurfactant production was much more frequently observed in cultured communities on leaves than in other nearby habitats, such as soil and water, suggesting that this trait is important to life on a leaf by altering either the leaf itself or the interaction of bacteria with water. While pseudomonads were the most common biosurfactant producers isolated, this habitat also selects for taxa, such as Chryseobacterium, for which this trait was previously unrecognized. The finding that most epiphytic bacterial taxa were culturable validates strategies using more classical culturing methodologies for their study in this habitat.

Effect of proximity to a cattle feedlot on Escherichia coli O157:H7 contamination of leafy greens and evaluation of the potential for airborne transmission.

The impact of proximity to a beef cattle feedlot on Escherichia coli O157:H7 contamination of leafy greens was examined. In each of 2 years, leafy greens were planted in nine plots located 60, 120, and 180 m from a cattle feedlot (3 plots at each distance). Leafy greens (270) and feedlot manure samples (100) were collected six different times from June to September in each year. Both E. coli O157:H7 and total E. coli bacteria were recovered from leafy greens at all plot distances. E. coli O157:H7 was recovered from 3.5% of leafy green samples per plot at 60 m, which was higher (P < 0.05) than the 1.8% of positive samples per plot at 180 m, indicating a decrease in contamination as distance from the feedlot was increased. Although E. coli O157:H7 was not recovered from air samples at any distance, total E. coli was recovered from air samples at the feedlot edge and all plot distances, indicating that airborne transport of the pathogen can occur. Results suggest that risk for airborne transport of E. coli O157:H7 from cattle production is increased when cattle pen surfaces are very dry and when this situation is combined with cattle management or cattle behaviors that generate airborne dust. Current leafy green field distance guidelines of 120 m (400 feet) may not be adequate to limit the transmission of E. coli O157:H7 to produce crops planted near concentrated animal feeding operations. Additional research is needed to determine safe set-back distances between cattle feedlots and crop production that will reduce fresh produce contamination.

Pre- and postharvest preventive measures and intervention strategies to control microbial food safety hazards of fresh leafy vegetables.

This review includes an overview of the most important preventive measures along the farm to fork chain to prevent microbial contamination of leafy greens. It also includes the technological and managerial interventions related to primary production, postharvest handling, processing practices, distribution, and consumer handling to eliminate pathogens in leafy greens. When the microbiological risk is already present, preventive measures to limit actual contamination events or pathogen survival are considered intervention strategies. In codes of practice the focus is mainly put on explaining preventive measures. However, it is also important to establish more focused intervention strategies. This review is centered mainly on leafy vegetables as the commodity identified as the highest priority in terms of fresh produce microbial safety from a global perspective. There is no unique preventive measure or intervention strategy that could be applied at one point of the food chain. We should encourage growers of leafy greens to establish procedures based on the HACCP principles at the level of primary production. The traceability of leafy vegetables along the chain is an essential element in ensuring food safety. Thus, in dealing with the food safety issues associated with fresh produce it is clear that a multidisciplinary farm to fork strategy is required.

Colorimetric paper-based detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes of agricultural water.

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of agricultural waters. Here, water is filtered through sterile Modified Moore Swabs (MMS), which consist of a simple gauze filter enclosed in a plastic cartridge, to concentrate bacteria. Following filtration, non-selective or selective enrichments for the target bacteria are performed in the MMS. For colorimetric detection of the target bacteria, the enrichments are then assayed using paper-based analytical devices (µPADs) embedded with bacteria-indicative substrates. Each substrate reacts with target-indicative bacterial enzymes, generating colored products that can be detected visually (qualitative detection) on the µPAD. Alternatively, digital images of the reacted µPADs can be generated with common scanning or photographic devices and analyzed using ImageJ software, allowing for more objective and standardized interpretation of results. Although the biochemical screening procedures are designed to identify the aforementioned bacterial pathogens, in some cases enzymes produced by background microbiota or the degradation of the colorimetric substrates may produce a false positive. Therefore, confirmation using a more discriminatory diagnostic is needed. Nonetheless, this bacterial concentration and detection platform is inexpensive, sensitive (0.1 CFU/ml detection limit), easy to perform, and rapid (concentration, enrichment, and detection are performed within approximately 24 hr), justifying its use as an initial screening method for the microbiological quality of agricultural water.

Adhesion of and to soil in runoff as influenced by polyacrylamide.

Polyacrylamide (PAM) is used in agriculture to reduce soil erosion and has been reported to reduce turbidity, nutrients, and pollutants in surface runoff water. The objective of this work was to determine the effect of PAM on the concentration of enteric bacteria in surface runoff by comparing four enteric bacteria representing phenotypically different motility and hydrophobicity from three soils. Results demonstrated that bacterial surface runoff was differentially influenced by the PAM treatment. Polyacrylamide treatment increased surface runoff for adhered and planktonic cells from a clay soil; significantly decreased surface runoff of adhered bacteria, while no difference was observed for planktonic bacteria from the sandy loam; and significantly decreased the surface runoff of planktonic cells, while no difference was observed for adhered bacteria from the clay loam. Comparing strains from a final water sample collected after 48 h showed a greater loss of while serovar Poona was almost not detected. Thus, (i) the PAM efficiency in reducing the concentration of enteric bacteria in surface runoff was influenced by soil type and (ii) variation in the loss of enteric bacteria highlights the importance of strain-specific properties that may not be captured with general fecal indicator bacteria.

A framework for developing research protocols for evaluation of microbial hazards and controls during production that pertain to the application of untreated soil amendments of animal origin on land used to grow produce that may be consumed raw.

Application of manure or soil amendments of animal origin (untreated soil amendments; UTSAs) to agricultural land has been a long-standing practice to maintain or improve soil quality through addition of organic matter, nitrogen, and phosphorus. Much smaller quantities of these types of UTSAs are applied to land used for food crops than to land used for animal grain and forage. UTSAs can harbor zoonotic enteric pathogens that may survive for extended periods after application. Additional studies are needed to enhance our understanding of preharvest microbial food safety hazards and control measures pertaining to the application of UTSAs especially for land used to grow produce that may be consumed raw. This document is intended to provide an approach to study design and a framework for defining the scope and type of data required. This document also provides a tool for evaluating the strength of existing data and thus can aid the produce industry and regulatory authorities in identifying additional research needs. Ultimately, this framework provides a means by which researchers can increase consistency among and between studies and facilitates direct comparison of hazards and efficacy of controls applied to different regions, conditions, and practices.

Resident bacteria on leaves enhance survival of immigrant cells of Salmonella enterica.

Although Salmonella enterica apparently has comparatively low epiphytic fitness on plants, external factors that would influence its ability to survive on plants after contamination would be of significance in the epidemiology of human diseases caused by this human pathogen. Viable population sizes of S. enterica applied to plants preinoculated with Pseudomonas syringae or either of two Erwinia herbicola strains was ≥10-fold higher than that on control plants that were not precolonized by such indigenous bacteria when assessed 24 to 72 h after the imposition of desiccation stress. The protective effect of P. fluorescens, which exhibited antibiosis toward S. enterica in vitro, was only ≈50% that conferred by other bacterial strains. Although S. enterica could produce small cellular aggregates after incubation on wet leaves for several days, and the cells in such aggregates were less susceptible to death upon acute dehydration than solitary cells (as determined by propidium iodide staining), most Salmonella cells were found as isolated cells when it was applied to leaves previously colonized by other bacterial species. The proportion of solitary cells of S. enterica coincident with aggregates of cells of preexisting epiphytic species that subsequently were judged as nonviable by viability staining on dry leaves was as much as 10-fold less than those that had landed on uncolonized portions of the leaf. Thus, survival of immigrant cells of S. enterica on plants appears to be strongly context dependent, and the presence of common epiphytic bacteria on plants can protect such immigrants from at least one key stress (i.e., desiccation) encountered on leaf surfaces.

Texture, composition and anatomy of spinach leaves in relation to nitrogen fertilization.

BACKGROUND: The postharvest quality and shelf life of spinach are greatly influenced by cultural practices. Reduced spinach shelf life is a common quandary in the Salinas Valley, California, where current agronomic practices depend on high nitrogen (N) rates. This study aimed to describe the postharvest fracture properties of spinach leaves in relation to N fertilization, leaf age and spinach cultivar. RESULTS: Force-displacement curves, generated by a puncture test, showed a negative correlation between N fertilization and the toughness, stiffness and strength of spinach leaves (P > 0.05). Younger leaves (leaves 12 and 16) from all N treatments were tougher than older leaves (leaves 6 and 8) (P > 0.05). Leaves from the 50 and 75 ppm total N treatments irrespective of spinach cultivar had higher fracture properties and nutritional quality than leaves from other N treatments (P > 0.05). Total alcohol-insoluble residues (AIR) and pectins were present at higher concentrations in low-N grown plants. These plants also had smaller cells and intercellular spaces than high-N grown leaves (P > 0.05). CONCLUSION: Observed changes in physicochemical and mechanical properties of spinach leaves due to excess nitrogen fertilization were significantly associated with greater postharvest leaf fragility and lower nutritional quality.

A framework for developing research protocols for evaluation of microbial hazards and controls during production that pertain to the quality of agricultural water contacting fresh produce that may be consumed raw.

Agricultural water may contact fresh produce during irrigation and/or when crop protection sprays (e.g., cooling to prevent sunburn, frost protection, and agrochemical mixtures) are applied. This document provides a framework for designing research studies that would add to our understanding of preharvest microbial food safety hazards and control measures pertaining to agricultural water. Researchers will be able to use this document to design studies, to anticipate the scope and detail of data required, and to evaluate previously published work. This document should also be useful for evaluating the strength of existing data and thus should aid in identifying future research needs. Use of this document by the research community may lead to greater consistency or comparability than currently exists among research studies, which may ultimately facilitate direct comparison of hazards and efficacy of controls among different commodities, conditions, and practices.

Assessment of root uptake and systemic vine-transport of Salmonella enterica sv. Typhimurium by melon (Cucumis melo) during field production.

Among melons, cantaloupes are most frequently implicated in outbreaks and surveillance-based recalls due to Salmonella enterica. There is limited but compelling evidence that associates irrigation water quality as a significant risk of preharvest contamination of melons. However, the potential for root uptake from water and soil and subsequent systemic transport of Salmonella into melon fruit is uncharacterized. The aim of this work was to determine whether root uptake of S. enterica results in systemic transport to fruit at high doses of applied inoculum through sub-surface drip and furrow irrigation during field production of melons. Cantaloupe and honeydew were grown under field conditions, in a silt clay loam soil using standard agronomic practices for California. An attenuated S. enterica sv. Typhimurium strain was applied during furrow irrigation and, in separate plots, buried drip-emitter lines delivered the inoculum directly into the established root zone. Contamination of the water resulted in soil contamination within furrows however Salmonella was not detected on top of the beds or around melon roots of furrow-irrigated rows demonstrating absence of detectable lateral transfer across the soil profile. In contrast, positive detection of the applied isolate occurred in soil and the rhizosphere in drip injected plots; survival of Salmonella was at least 41 days. Despite high populations of the applied bacteria in the rhizosphere, after surface disinfection, internalized Salmonella was not detected in mature melon fruit (n=485). Contamination of the applied Salmonella was detected on the rind surface of melons if fruit developed in contact with soil on the sides of the inoculated furrows. Following an unusual and heavy rain event during fruit maturation, melons collected from the central area of the beds, were shown to harbor the furrow-applied Salmonella. Delivery of Salmonella directly into the peduncle, after minor puncture wounding, resulted in detection of applied Salmonella in the sub-rind tissue below the fruit abscission zone. Results indicate that Salmonella internalization from soil and vascular systemic transport to fruit is unlikely to occur from irrigation water in CA production regions, even if substantially above normal presumptive levels of contamination. Although contaminated irrigation water and subsequently soil in contact with fruit remains a concern for contamination of the external rind, results suggest an acceptable microbial indicator threshold and critical limit for the presence of Salmonella in applied water may be possible by defining appropriate microbiological standards for melon irrigation in California and regions with similar climate, soil texture, and crop management practices.

Leaf microbiota in an agroecosystem: spatiotemporal variation in bacterial community composition on field-grown lettuce.

The presence, size and importance of bacterial communities on plant leaf surfaces are widely appreciated. However, information is scarce regarding their composition and how it changes along geographical and seasonal scales. We collected 106 samples of field-grown Romaine lettuce from commercial production regions in California and Arizona during the 2009-2010 crop cycle. Total bacterial populations averaged between 10(5) and 10(6) per gram of tissue, whereas counts of culturable bacteria were on average one (summer season) or two (winter season) orders of magnitude lower. Pyrosequencing of 16S rRNA gene amplicons from 88 samples revealed that Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria were the most abundantly represented phyla. At the genus level, Pseudomonas, Bacillus, Massilia, Arthrobacter and Pantoea were the most consistently found across samples, suggesting that they form the bacterial 'core' phyllosphere microbiota on lettuce. The foliar presence of Xanthomonas campestris pv. vitians, which is the causal agent of bacterial leaf spot of lettuce, correlated positively with the relative representation of bacteria from the genus Alkanindiges, but negatively with Bacillus, Erwinia and Pantoea. Summer samples showed an overrepresentation of Enterobacteriaceae sequences and culturable coliforms compared with winter samples. The distance between fields or the timing of a dust storm, but not Romaine cultivar, explained differences in bacterial community composition between several of the fields sampled. As one of the largest surveys of leaf surface microbiology, this study offers new insights into the extent and underlying causes of variability in bacterial community composition on plant leaves as a function of time, space and environment.