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.

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.

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.

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.

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.

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.

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.