A cyclic lipopeptide from Fusarium graminearum targets plant membranes to promote virulence.

Microbial plant pathogens deploy amphipathic cyclic lipopeptides to reduce surface tension in their environment. While plants can detect these molecules to activate cellular stress responses, the role of these lipopeptides or associated host responses in pathogenesis are not fully clear. The gramillin cyclic lipopeptide is produced by the Fusarium graminearum fungus and is a virulence factor and toxin in maize. Here, we show that gramillin promotes virulence and necrosis in both monocots and dicots by disrupting ion balance across membranes. Gramillin is a cation-conducting ionophore and causes plasma membrane depolarization. This disruption triggers cellular signaling, including a burst of reactive oxygen species (ROS), transcriptional reprogramming, and callose production. Gramillin-induced ROS depends on expression of host ILK1 and RBOHD genes, which promote fungal induction of virulence genes during infection and host susceptibility. We conclude that gramillin's ionophore activity targets plant membranes to coordinate attack by the F. graminearum fungus.

Irrigation Method Matters: Contamination and Die-off Rates of Escherichia coli on Dry Bulb Onions After Overhead and Drip Irrigation in Washington State (2022-2023).

Two U.S. outbreaks of salmonellosis in 2020 and 2021 were epidemiologically linked to red onions. The 2020 outbreak investigation implicated the production of agricultural water as a likely contamination source. Field trials were designed to investigate the prevalence and survival of Escherichia coli (surrogate for Salmonella) on dry bulb onions after the application of contaminated irrigation water at the end of the growing period. Irrigation water was inoculated at 3 log most probable number (MPN)/100 mL (2022 and 2023) or 5 log MPN/100 mL (2023, drip only) with a cocktail of rifampin-resistant E. coli and applied with the final irrigation (0.4 acre-inch/0.4 ha-cm) to onions. Onion bulbs (40 or 80) were sampled immediately after irrigation and throughout field curing (4 weeks) and E. coli was enumerated using an MPN method. For drip irrigation, at 3 log MPN/100 mL E. coli was detected on 13% of onions at 24 h but not detected at 0 h; at 5 log MPN/100 mL for drip irrigation applied to saturated soil, E. coli was detected in 63% of onions at 0 h. Prevalence significantly (P < 0.05), decreased after 7 d of curing with cell densities of 1-1,400 MPN/onion. At the end of field curing in 2023, 1/80 of onions had detectable E. coli (2.04 MPN/onion). E. coli was detected in a significantly smaller percentage of onions (2022: 13%; 2023: 68%) after a contaminated drip irrigation event compared to overhead irrigation (98-100%; P < 0.05). After overhead irrigation, E. coli was detected in onions (1-1,000 MPN/onion) on day 0. Prevalence decreased significantly (P < 0.05) after 7 d of field curing in both years (2022: 15%; 2023: 7%). E. coli was not detected on Calibra onions (80/year) at the end of field curing in either year but was detected at <12 MPN/onion in 2.5-3.75% of onions (n = 80) for other cultivars. These data confirm limited contamination risk associated with drip irrigation water quality and begin to quantify contamination risks associated with overhead irrigation of dry bulb onions.

Behavior of Salmonella During Preparation of a Fermented Cashew Cheese Analog.

Between 2013 and 2021, there were three reported salmonellosis outbreaks in North America linked to the consumption of cashew cheese analogs that were prepared from soaked and fermented cashews. The behavior of Salmonella was evaluated during fermentation of cashews to better understand the risks associated with plant-based fermentations. Single or seven-strain rifampin-resistant Salmonella-inoculated cashews (1-2 log CFU/g) were soaked 1:1 (w/v) in sterile ultrapure water at 4 °C for 24 ± 1 h, drained, and then blended with additional water. Salmonella-inoculated or uninoculated cashews with or without added commercial Lactococcus lactis starter culture (LAB), and with LAB and NaCl (0.8% and 1.6% w/w), citric acid (0.4% w/w), or a combination of NaCl and citric acid, were held at 24 ± 1 °C for up to 72 h. The pH, aerobic plate counts (M17 agar), and Salmonella populations (CHROMagar Salmonella with 50 µg/mL of rifampin) were measured at 0, 24, 48, and 72 h in replicate experiments. When LAB was present, aerobic plate counts increased from ∼8 log CFU/g to ∼9 log CFU/g after 24 h. The pH decreased from an initial pH âˆ¼6 to pH 4.5-5.0 at 24 h in the presence of LAB or at 48 h in the absence of LAB. The presence of LAB significantly (P < 0.0001) impacted populations of Salmonella during the fermentation. There was no significant difference in Salmonella populations between the treatments with LAB alone and the treatments with LAB in combination with added NaCl (P = 0.3484) or citric acid (P = 0.8630). After 24 h, populations of Salmonella increased by 5.3-5.5 log in the absence of LAB and by 0.5-1.7 log in the presence of LAB, with or without added NaCl. These data demonstrate the need to consider a range of control measures for safe preparation of plant-based fermented products.

A comparison between the role of enniatins and deoxynivalenol in Fusarium virulence on different tissues of common wheat.

BACKGROUND: Fusarium graminearum and Fusarium avenaceum are two of the most important causal agents of Fusarium head blight (FHB) of wheat. They can produce mycotoxins that accumulate in infected wheat heads, including deoxynivalenol (DON) and enniatins (ENNs), produced by F. graminearum and F. avenaceum, respectively. While the role of DON as a virulence factor in F. graminearum toward wheat is well known, ENNs in F. avenaceum has been poorly explored. Results obtained to-date indicate that ENNs may confer an advantage to F. avenaceum only on particular hosts. RESULTS: In this study, with the use of ENN-producing and ENN non-producing F. avenaceum strains, the role of ENNs on F. avenaceum virulence was investigated on the root, stem base and head of common wheat, and compared with the role of DON, using DON-producing and DON non-producing F. graminearum strains. The DON-producing F. graminearum strain showed a significantly higher ability to cause symptoms and colonise each of the tested tissues than the non-producing strain. On the other hand, the ability to produce ENNs increased initial symptoms of the disease and fungal biomass accumulation, measured by qPCR, only in wheat heads, and not in roots or stem bases. LC-MS/MS analysis was used to confirm the presence of ENNs and DON in the different strains, and results, both in vitro and in wheat heads, were consistent with the genetics of each strain. CONCLUSION: While the key role of DON on F. graminearum virulence towards three different wheat tissues was noticeable, ENNs seemed to have a role only in influencing F. avenaceum virulence on common wheat heads probably due to an initial delay in the appearance of symptoms.

Survival of Salmonella enterica and Enterococcus faecium on Abiotic Surfaces During Storage at Low Relative Humidity.

Currently, there is limited knowledge on the survival of bacteria on surfaces during postharvest handling of dry products such as onions. Extended survival of microorganisms, coupled with a lack of established and regular, validated cleaning or sanitation methods could enable cross-contamination of these products. The aim of the study was to evaluate the survival of a potential surrogate, Enterococcus faecium, and Salmonella enterica on typical onion handling surfaces, polyurethane (PU), and stainless steel (SS), under low relative humidity. The influence of onion extract on the survival of E. faecium and Salmonella on PU and SS was also investigated. Rifampin-resistant E. faecium NRRL B-2354 and a five-strain cocktail of Salmonella suspended in 0.1% peptone or onion extract were separately inoculated onto PU and SS coupons (2 × 2 cm), at high, moderate, or low (7, 5, or 3 log CFU/cm2) levels. The inoculated surfaces were stored at ∼34% relative humidity and 21°C for up to 84 days. Triplicate samples were enumerated at regular intervals in replicate trials. Samples were enriched when populations fell below the limit of detection by plating (0.48 log CFU/cm2). Scanning electron microscopy was used to observe the cell distribution on the coupons. Reductions of E. faecium of less than ∼2 log were observed on PU and SS over 12 weeks at all inoculum levels and with both inoculum carriers. In 0.1% peptone, Salmonella populations declined by 2 to 3 log over 12 weeks at the high and moderate inoculum levels; at the low inoculum level, Salmonella could not be recovered by enrichment at 84 days. Survival of E. faecium and Salmonella was significantly (P < 0.05) enhanced over 84 days of storage when suspended in onion extract, where cells were covered by a layer of onion extract. E. faecium might have utility as a conservative surrogate for Salmonella when evaluating microbial survival on dry food-contact surfaces.

Phenotypic Characteristics That May Contribute to Persistence of Salmonella Strains in the Pistachio Supply Chain.

Salmonella enterica subsp. enterica strain diversity in California pistachios is limited; some strains have persisted in the pistachio supply chain for ≥10 years. Representative isolates of six persistent strains and three sporadic strains isolated from California pistachios were selected to evaluate copper resistance, growth in pistachio hull slurry, biofilm formation, desiccation tolerance, and survival during subsequent storage. The presence of a copper homeostasis and silver-resistance island sequence in three of the persistent strains was associated with an increase in tolerance to CuSO4 from 7.5 mM to 15 mM under anaerobic but not aerobic conditions; all isolates were resistant to ≥120 mM Cu-EDTA under both anerobic and aerobic conditions. When inoculated into pistachio hull slurry at 2.75 ± 0.04 log CFU/mL and incubated at 30 °C, the populations of Salmonella Enteritidis strain A (sporadic) increased to significantly lower levels than the other strains at 16, 20, 24, and 28 h but not at 40 and 48 h. Maximum populations of 8.70-8.85 log CFU/mL were observed for all strains at ≥40 h of incubation. All nine Salmonella strains produced weak to strong biofilms after 4 days at 25 °C; seven strains, including two sporadic strains, produced moderate biofilms, and Salmonella Liverpool strain A (persistent) produced a strong biofilm. The rdar+ and rdar- morphotypes were observed in both persistent and sporadic Salmonella strains. Population declines of 5.03 log were observed for Salmonella Enteritidis strain A within 18 h of drying on filter paper whereas reductions of 0.50-1.25 log were observed for the other eight Salmonella strains. Population reductions (3.98-5.12 log) of these eight strains were not significantly different after storage at 25 ± 1 °C and 35% relative humidity for 50 days. The phenotypic characteristics evaluated here do not independently account for the persistence of a small number of Salmonella strains associated with the California pistachio production chain.

Reduction Foodborne Pathogens and Surrogate Microorganism on Citrus Fruits after Lab- and Pilot-scale Finishing Wax Application.

After finishing waxes are applied, citrus fruits are typically dried at 32-60°C for 2-3 min before final packing. The survival of Listeria monocytogenes, Salmonella, and Enterococcus faecium NRRL B-2354 was evaluated under laboratory conditions on lemons after applying one of four finishing waxes (F4, F6, F8, and F15) followed by an ambient hold or heated (50 or 60°C) drying step. The reduction of inoculated microorganisms during drying was significantly influenced by wax type and temperature, with greater reductions at higher temperatures. Greater reductions after waxing and drying at 60°C were observed with L. monocytogenes (2.84-4.44 log) than with Salmonella (1.65-3.67 log), and with Salmonella than with E. faecium (0.99-2.93 log). The survival of Salmonella inoculated at 5.8-5.9 log/fruit on lemons and oranges after applying wax F6 and drying at 60°C was evaluated during storage at 4 and 22°C. The reductions of Salmonella after waxing and drying were 1.7 log; additional reductions during storage at 4 or 22°C were 1.40-1.43 or 0.18-0.29 log, respectively, on waxed lemons, and 0.56-1.02 or 0.54-0.57 log, respectively, on waxed oranges. Under pilot-scale packinghouse conditions with wax F4, mean and minimum reductions of E. faecium ranged from 2.15 to 2.89 and 1.64 to 2.12 log, respectively. However, E. faecium was recovered by whole-fruit enrichment (limit of detection: 0.60 log CFU/lemon) but not by plating (LOD: 1.3 log CFU/lemon) from uninoculated lemons run with or after the inoculated lemons. The findings should provide useful information to establish and implement packinghouse food safety plans.

Genetic diversity of Salmonella enterica isolated over 13 years from raw California almonds and from an almond orchard.

A comparative genomic analysis was conducted for 171 Salmonella isolates recovered from raw inshell almonds and raw almond kernels between 2001 and 2013 and for 30 Salmonella Enteritidis phage type (PT) 30 isolates recovered between 2001 and 2006 from a 2001 salmonellosis outbreak-associated almond orchard. Whole genome sequencing was used to measure the genetic distance among isolates by single nucleotide polymorphism (SNP) analyses and to predict the presence of plasmid DNA and of antimicrobial resistance (AMR) and virulence genes. Isolates were classified by serovars with Parsnp, a fast core-genome multi aligner, before being analyzed with the CFSAN SNP Pipeline (U.S. Food and Drug Administration Center for Food Safety and Applied Nutrition). Genetically similar (≤18 SNPs) Salmonella isolates were identified among several serovars isolated years apart. Almond isolates of Salmonella Montevideo (2001 to 2013) and Salmonella Newport (2003 to 2010) differed by ≤9 SNPs. Salmonella Enteritidis PT 30 isolated between 2001 and 2013 from survey, orchard, outbreak, and clinical samples differed by ≤18 SNPs. One to seven plasmids were found in 106 (62%) of the Salmonella isolates. Of the 27 plasmid families that were identified, IncFII and IncFIB plasmids were the most predominant. AMR genes were identified in 16 (9%) of the survey isolates and were plasmid encoded in 11 of 16 cases; 12 isolates (7%) had putative resistance to at least one antibiotic in three or more drug classes. A total of 303 virulence genes were detected among the assembled genomes; a plasmid that harbored a combination of pef, rck, and spv virulence genes was identified in 23% of the isolates. These data provide evidence of long-term survival (years) of Salmonella in agricultural environments.

Characterizing the Genetic Diversity of Salmonella Isolated from U.S. Raw Inshell Pistachios Using Whole Genome Sequencing.

The genetic diversity of 169 Salmonella isolates from pistachios collected from California storage silos during the 2010, 2011, and 2012 harvests (silo survey isolates) was determined by analyzing the whole genome sequence data using the CFSAN SNP pipeline developed by the U.S. Food and Drug Administration's Center for Food Safety and Applied Nutrition. Salmonella isolates clustered by serovars Agona, Enteritidis, Montevideo, Sandiego, Senftenberg, Liverpool, Tennessee, and Worthington in the phylogenetic tree. Within each serovar, isolates grouped into one or two clusters (≤14 SNPs). Two distinct clusters (>14 SNPs; A and B) were identified for Salmonella Enteritidis, Montevideo, and Liverpool for a total of 11 unique strains. Sequences of representative silo survey isolates clustered with sequences of Salmonella strains isolated from U.S. pistachio-associated samples collected between 2008 and 2018 available on the National Center for Biotechnology Information database, and, in all but two cases, not with sequences of Salmonella strains recovered from raw California almonds from 2001 through 2013. The genomic evidence suggests that strains of Salmonella Agona, Liverpool Cluster A, Montevideo Clusters A and B, Senftenberg, and Worthington have persisted in the California pistachio environment for ≥3 years and some of these strains have been reported exclusively in association with pistachios.

Survival of Listeria monocytogenes and Salmonella in citrus storage waxes or on lemons held under common commercial storage conditions.

To prolong cold storage, diluted storage waxes are applied to washed lemons after harvest and before packing, without drying steps, to reduce premature rotting and water loss. The survival of Listeria monocytogenes and Salmonella in undiluted and diluted storage waxes (S1-S4), and on lemon surfaces under common commercial storage were investigated. Populations of L. monocytogenes declined more slowly than Salmonella in undiluted storage waxes over 24 h of storage at 4 or 22 °C. L. monocytogenes (inoculated at ∼6 log CFU/mL) was detected by enrichment in undiluted waxes S2, S3, and S4 after 75-135 days at 4 °C but not after 30, 10, or 105 days, respectively at 22 °C. L. monocytogenes survived better in diluted than in undiluted storage waxes at 22 °C. Populations of L. monocytogenes (∼6 log CFU/lemon) declined by 0.64-1.62 log on lemon surfaces right after waxing. Populations of L. monocytogenes decreased to <1.30 log CFU/lemon after 28 days (1:9 S1) or 75 days (other treatments) at 12 °C and ≥93% RH. Except for 1:9 S1, L. monocytogenes was detected by enrichment in all lemon samples over 87 days of storage. Packinghouses should consider the survival of L. monocytogenes and Salmonella in citrus storage waxes in their food safety programs.

Transfer of Enterococcus faecium and Salmonella enterica during simulated postharvest handling of yellow onions (Allium cepa).

Bacterial transfer during postharvest handling of fresh produce provides a mechanism for spreading pathogens, but risk factors in dry environments are poorly understood. The aim of the study was to investigate factors influencing bacterial transfer between yellow onions (Allium cepa) and polyurethane (PU) or stainless steel (SS) under dry conditions. Rifampin-resistant Enterococcus faecium NRRL B-2354 or a five-strain cocktail of Salmonella was inoculated onto onion skin or PU surfaces at high or moderate levels using peptone, onion extract, or soil water as inoculum carriers. Transfer from inoculated to uninoculated surfaces was conducted using a texture analyzer to control force, time, and number of contacts. Transfer rates (ratio of recipient surface to donor surface populations) of E. faecium (4-5%) were significantly higher than those of Salmonella (0.5-0.6%) at the high (7 log CFU/cm2) but not moderate (5 log CFU/cm2) inoculum levels. Significantly higher populations of E. faecium transferred from onion to PU than from PU to onion. The transfer rates of E. faecium were impacted by inoculum carrier (61% [onion extract], 1.6% [peptone], and 0.31% [soil]) but not by inoculation level or recipient surface (PU versus SS). Bacterial transfer during dry onion handling is significantly dependent on bacterial species, inoculation levels, inoculum carrier, and transfer direction.

U.S. Consumer Practices of Homemade Nut-based Dairy Analogs and Soaked Nuts.

Tree nuts, a low-moisture food, are typically perceived as being a low risk for foodborne illness. In the past five decades, the consumption of tree nuts (dry, soaked, or as nut-based dairy analogs [NBDA]) has increased along with corresponding foodborne illness outbreaks and recalls associated with these products. We developed an online survey to assess tree nut handling practices of U.S. consumers, and to select study participants who have soaked tree nuts and/or made NBDA at home. We distributed our initial survey questions in October 2021 to a convenience sample (n = 12) to test for clarity and comprehension. In January 2022, participants (n = 981) who met the criteria completed the survey. The most popular soaked tree nuts were almonds (54%), followed by cashews (36%), walnuts (32%), and pistachios (22%). Participants soaked tree nuts for direct consumption (67%) and during the preparation of NBDA (80%). Participants soaked tree nuts under refrigerated conditions for 1-24 h (22%), on the countertop at room temperature (est. 65-75°F [18-24°C]) for 1-5 h (21%), or at room temperature for 12 h or more (6%); 16% used a hot or boiling water, short time treatment. Some participants added acid (28%) or salt (25%) to the soaking water. Among those participants who dried their tree nuts after soaking (63%), 89% reported drying at a temperature lower than 46°C (115°F). Some participants (34%) used their tree nuts to make fermented dairy analogs (e.g., "cheese" or "yogurt") by adding "probiotics" (56-86%) or a yogurt starter culture (37-99%), respectively, and then, most frequently, holding at or below 20°C (68°F) for 12 h or less (29%). The safety of many of these practices has not been adequately investigated, but the findings of this study will inform future risk assessment and risk modeling studies on tree nut food safety in home kitchen settings.

Levels and Distribution of Salmonella in Naturally Contaminated Cashews.

Raw materials associated with foodborne illness outbreaks are rarely available for evaluation. The levels and distribution of Salmonella were determined in naturally contaminated raw cashews linked to a salmonellosis outbreak associated with a fermented cashew cheese analog. Two unopened 22.7-kg boxes from a single lot of cashew kernel pieces were each divided into seven approximately equal units, 14 in total. Three 10-g subsamples per unit (n = 21) were evaluated for aerobic plate count (APC), coliform counts, and Escherichia coli counts, and 10 50-g subsamples per unit (n = 70) were enriched for the presence of Salmonella. Presumptive Salmonella-positive colonies were confirmed using CHROMagar Salmonella and real-time PCR (invA) and then serotyped using antigenic methods and genome sequencing prediction tools. APC and coliform counts ranged from 1.81 to 5.47 (mean 2.44 ± 0.63) log CFU/g and 0.60 to 5.20 (mean 1.74 ± 0.80) log CFU/g, respectively. Salmonella was recovered from four units in Box 1 and all seven units in Box 2. One of the 10 subsamples was positive in all but four of the positive units; one (Box 1) and three (Box 2) units had two positive subsamples. The level of Salmonella in the two boxes combined was 0.0023 most probable number/g (95% confidence interval [0.0014, 0.0038]). Salmonella Urbana was isolated from three of five positive subsamples in Box 1 and eight of 10 positive subsamples in Box 2. Salmonella Fresno and Vinohrady were unique to single subsamples from Box 1, and Salmonella Nima was isolated from two subsamples from Box 2. Of the four serovars recovered, Salmonella Urbana and Salmonella Vinohrady were in common with outbreak-associated clinical or product isolates. Understanding the distribution and concentration of Salmonella in naturally contaminated cashews provides important information for hazard analysis and risk assessments for soaked and fermented cashew products.

Impact of irrigation water quality on human norovirus surrogate survival during leafy green production.

Introduction: The impact of water quality on the survival of human norovirus (NoV) was determined in irrigation water field run-off (tail water) and well water from a representative Central Coast vegetable production site in the Salinas Valley, California. Methods: Tail water, well water, and ultrapure water samples were inoculated separately with two surrogate viruses for human NoV-Tulane virus (TV) and murine norovirus (MNV)-to achieve a titer of 1×105 plaque forming units (PFU)/ml. Samples were stored at 11, 19, and 24°C for 28 days. Additionally, inoculated water was applied to soil collected from a vegetable production site in the Salinas Valley or to the surface of growing romaine lettuce leaves, and virus infectivity was evaluated for 28 days in a growth chamber. Results: Virus survival was similar for water stored at 11, 19, and 24°C and there was no difference in infectivity based on water quality. After 28 days, a maximum 1.5 log reduction was observed for both TV and MNV. TV decreased by 1.97-2.26 log and MNV decreased by 1.28- 1.48 logs after 28 days in soil; infectivity was not influenced by water type. Infectious TV and MNV were recovered from lettuce surfaces for up to 7 and 10 days after inoculation, respectively. Across the experiments there was no significant impact of water quality on the stability of the human NoV surrogates. Discussion: Overall, the human NoV surrogates were highly stable in water with a less than 1.5 log reduction over 28 days and no difference observed based on the water quality. In soil, the titer of TV declined by approximately 2 logs over 28 days, while MNV declined by 1 log during the same time interval, suggesting surrogate-specific inactivation dynamics in the soil tested in this study. A 5-log reduction in MNV (day 10 post inoculation) and TV (day 14 post inoculation) was observed on lettuce leaves, and the inactivation kinetics were not significantly impacted by the quality of water used. These results suggest that human NoV would be highly stable in water, and the quality of the water (e.g., nutrient content, salinity, and turbidity) does not significantly impact viral infectivity.

Fate of foodborne pathogens during soaking and drying of walnuts.

Walnuts are among the most popular tree nuts that are soaked at home. Recipes for preparing soaked walnut kernels from online blogs (n = 71) and YouTube videos (n = 29) were reviewed to identify typical consumer handling practices that were then used to determine the fate of foodborne pathogens during soaking and subsequent drying of walnut kernels. Individual five-strain cocktails of rifampin-resistant Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella, grown on agar plates and diluted in water, were inoculated onto walnuts and then dried. Inoculated walnuts were added to sterile water at a ratio of 1:4 (w/v), held at 15, 18, or 22°C for up to 24 h, and then dried at 64°C for up to 24 h (for Salmonella-inoculated walnuts). Pathogen populations during soaking and drying were enumerated on tryptic soy agar with rifampin and on CHROM agar. Initial walnut moisture was ∼4%, increased to ∼30% at 8 and 24 h of soaking and then decreased during drying to ∼4% at 6 h and <1% after 24 h. Initial E. coli, L. monocytogenes, and Salmonella populations were ∼1.0, ∼1.5, and 1.0-2.5 log CFU/g, respectively, after inoculation and drying. No significant (P > 0.05) increase in populations was observed after 24 h at 15 and 18°C or after 12 h at 22°C. Significant increases of 1.9-3.0, 1.2-2.1, and 1.8 log CFU/g for E. coli, L. monocytogenes, and Salmonella, respectively, were observed after 24 h of soaking at 22°C. Growth rates of 0.19, 0.093, and 0.16 log CFU/sample per h, respectively, were observed. Lag times of 8.8 and 11 h at 22°C were determined for E. coli and Salmonella, respectively. Populations of Salmonella declined by 1.04 log CFU/g over 12 h of drying; further significant (P < 0.05) decreases were not observed at 24 h. To limit food safety risks in soaked walnuts, educational materials should emphasize sourcing treated walnuts, kitchen sanitation, hygiene measures, and soaking at cooler temperatures or for shorter times at ambient temperatures.

Water Application Method Influences Survival or Growth of Escherichia coli on Bulb Onions during Field Curing.

ABSTRACT: The impact of water application method on bacterial survival at or after the final irrigation was evaluated in bulb onions during commercially relevant field drying (curing). A three-strain rifampin-resistant cocktail of Escherichia coli was introduced to onions via a single overhead spray application in two separate trials (5.22 [trial 1] or 2.40 [trial 2] log CFU per onion) 2 to 3 days after the final irrigation. Onions were lifted from the soil 8 days after spray inoculation and, in some cases, foliage was removed (topping); onions remained in the field for an additional ca. 2 weeks (total ca. 3 weeks of curing). E. coli populations declined on the onions in the first 4 h after spray inoculation. E. coli was recovered from 38 (48%) or 28 (35%) of 80 whole-onion enrichments at the end of curing in trials 1 or 2, respectively. Topping did not significantly impact the percentage of E. coli-positive onions detected at the end of curing. From 8 h to 21 days, E. coli populations on positive onions ranged from 1 CFU per onion to 7 log CFU per onion in both trials, representing a potential risk of E. coli growth with overhead application of contaminated water at the end of onion production. In trial 2, additional rows of onions were inoculated via a 22-cm subsurface or surface drip irrigation line (1.94 log CFU/mL for 2.5 h). E. coli was detected in 0 (subsurface) and 4 (surface) of 50 whole-onion enrichments 3 h after the initiation of drip irrigation. Positive onions were detected at days 1 (4 of 50) and 7 (1 of 50) with subsurface drip inoculation, and at days 1 (7 of 50), 7 (2 of 50), and 14 (2 of 50) with surface drip inoculation. E. coli was not detected in whole-onion enrichments at the end of curing when inoculated by subsurface (0 of 50) or surface (0 of 50) drip irrigation. Application of contaminated water through drip irrigation, when coupled with field curing, results in low rates of contamination of bulb onions at the time of harvest.

Salmonella enterica subsp. enterica virulence potential can be linked to higher survival within a dynamic in vitro human gastrointestinal model.

Salmonella enterica subsp. enterica is one of the leading causes of human foodborne infections and several outbreaks are now associated with the consumption of fresh fruit and vegetables. This study aims at evaluating whether Salmonella virulence can be linked to an enhanced ability to survive successive digestive environments. Thirteen S. enterica strains were selected according to high and low virulence phenotypes. Lettuce inoculated separately with each S. enterica strain was used as food matrix in the TNO gastrointestinal model (TIM-1) of the human upper gastrointestinal tract. During the passage in the stomach, counts determined using PMA-qPCR were 2-5 logs higher than the cultivable counts for all strains indicating the presence of viable but non-cultivable cells. Bacterial growth was observed in the duodenum compartment after 180 min for all but one strain and growth continued into the ileal compartment. After passage through the simulated gastrointestinal tract, both virulent and avirulent S. enterica strains survived but high virulence strains had a significantly (p = 0.004) better average survival rate (1003 %-3753 %) than low virulence strains (from 25 % to 3730%). The survival rates of S. enterica strains could be linked to the presence of genes associated with acid and bile resistance and their predicted products. The presence of single nucleotide polymorphisms may also impact the function of virulence associated genes and play a role in the resulting phenotype. These data provide an understanding of the relationship between measured virulence potential and survival of S. enterica during dynamic simulated gastrointestinal transit.

Naturally Occurring Fusarium Species and Mycotoxins in Oat Grains from Manitoba, Canada.

Fusarium head blight (FHB) can lead to dramatic yield losses and mycotoxin contamination in small grain cereals in Canada. To assess the extent and severity of FHB in oat, samples collected from 168 commercial oat fields in the province of Manitoba, Canada, during 2016-2018 were analyzed for the occurrence of Fusarium head blight and associated mycotoxins. Through morphological and molecular analysis, F. poae was found to be the predominant Fusarium species affecting oat, followed by F. graminearum, F. sporotrichioides, F. avenaceum, and F. culmorum. Deoxynivalenol (DON) and nivalenol (NIV), type B trichothecenes, were the two most abundant Fusarium mycotoxins detected in oat. Beauvericin (BEA) was also frequently detected, though at lower concentrations. Close clustering of F. poae and NIV/BEA, F. graminearum and DON, and F. sporotrichioides and HT2/T2 (type A trichothecenes) was detected in the principal component analysis. Sampling location and crop rotation significantly impacted the concentrations of Fusarium mycotoxins in oat. A phylogenetic analysis of 95 F. poae strains from Manitoba was conducted using the concatenated nucleotide sequences of Tef-1α, Tri1, and Tri8 genes. The results indicated that all F. poae strains belong to a monophyletic lineage. Four subgroups of F. poae strains were identified; however, no correlations were observed between the grouping of F. poae strains and sample locations/crop rotations.

Apicidin biosynthesis is linked to accessory chromosomes in Fusarium poae isolates.

BACKGROUND: Fusarium head blight is a disease of global concern that reduces crop yields and renders grains unfit for consumption due to mycotoxin contamination. Fusarium poae is frequently associated with cereal crops showing symptoms of Fusarium head blight. While previous studies have shown F. poae isolates produce a range of known mycotoxins, including type A and B trichothecenes, fusarins and beauvericin, genomic analysis suggests that this species may have lineage-specific accessory chromosomes with secondary metabolite biosynthetic gene clusters awaiting description. METHODS: We examined the biosynthetic potential of 38 F. poae isolates from Eastern Canada using a combination of long-read and short-read genome sequencing and untargeted, high resolution mass spectrometry metabolome analysis of extracts from isolates cultured in multiple media conditions. RESULTS: A high-quality assembly of isolate DAOMC 252244 (Fp157) contained four core chromosomes as well as seven additional contigs with traits associated with accessory chromosomes. One of the predicted accessory contigs harbours a functional biosynthetic gene cluster containing homologs of all genes associated with the production of apicidins. Metabolomic and genomic analyses confirm apicidins are produced in 4 of the 38 isolates investigated and genomic PCR screening detected the apicidin synthetase gene APS1 in approximately 7% of Eastern Canadian isolates surveyed. CONCLUSIONS: Apicidin biosynthesis is linked to isolate-specific putative accessory chromosomes in F. poae. The data produced here are an important resource for furthering our understanding of accessory chromosome evolution and the biosynthetic potential of F. poae.

Reduction of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella on Whole Yellow Onions (Allium cepa) Exposed to Hot Water.

ABSTRACT: In-home or food service antimicrobial treatment options for fresh produce are limited. Hot water treatments for whole (unpeeled) produce have been proposed, but data to support this practice for onions are not available. Separate cocktails of rifampin-resistant Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella were cultured on agar and suspended in sterile water. The outer papery skin at the equator or root or stem ends of the whole yellow onions was spot inoculated at 6 log CFU per onion. After drying for 30 min and, in some cases, storage at 4°C for 6 days, onions were immersed in water at ca. 100°C for 5 s or 85°C for 10 to 180 s. No significant difference (P > 0.05) in the mean decline of Salmonella was found on onions that were exposed to hot water after drying the inoculum for 30 min or after storage at 4°C for 6 days. Exposure of whole onions at 100°C for 5 s reduced E. coli O157:H7 and L. monocytogenes populations by >5 log CFU per onion at all inoculum sites and Salmonella populations by >5 log CFU per onion at the stem end and equator but not consistently at the root end. Mean root-end reductions of ≥5 log CFU per onion of E. coli O157:H7, L. monocytogenes, and Salmonella were achieved consistently when the root end was fully immersed in 85°C hot water for 45 or 60 s except in a small number of cases (4 of 57; 7%) when the root end was oriented upward and above the water line during treatment. When onions were held at 85°C for 180 s with the root end above the water line in an uncovered water bath, no significant declines in Salmonella populations were observed; significant mean declines in Salmonella were achieved (mean, 5 log CFU per onion; range, 3.49 to 6.25 log CFU per onion) when the water bath was covered. Short exposure to hot water can significantly reduce pathogens on the surface of whole onions. Reductions are more consistent when the root end is submerged and when the water bath is covered.