Cold-adapted Exiguobacterium sibiricum K1 as a potential bioinoculant in cold regions: Physiological and genomic elucidation of biocontrol and plant growth promotion.

The scarcity of soil nutrient availability under cold conditions of Himalayan regions needs a sustainable approach for better crop yields. The cold-adapted bacteria, Exiguobacterium sibiricum K1, with the potential to produce several plant growth-promoting (PGP) attributes, nitrogen fixation, indole acetic acid production, phosphate and potassium solubilization at 10 °C can provide an opportunity to promote crop yield improvement in an eco-friendly way under cold conditions. The bacterium also exhibited biocontrol activity against two phytopathogens and produced siderophore (53.0 ± 0.5 % psu). The strain's PGP properties were investigated using a spinach-based bioassay under controlled conditions. The bacterized seeds showed a notable increase in germination rate (23.2 %), shoot length (65.3 %), root length (56.6 %), leaf area (73.7 %), number of leaflets (65.2 %), and dry matter (65.2 %). Additionally, the leaf analysis indicated elevated chlorophyll pigments, i.e., chlorophyll a (55.5 %), chlorophyll b (42.8 %), carotenoids (35.2 %), percentage radical scavenging activity (47.4 %), and leaf nutrient uptake such as nitrogen (23.4 %), calcium (60.8 %), potassium (62.3 %), and magnesium (28.9 %). Moreover, the whole-genome sequencing and genome mining endorsed various biofertilisation-related genes, including genes for potassium and phosphate solubilization, iron and nitrogen acquisition, carbon dioxide fixation, and biocontrol ability of Exiguobacterium sibiricum K1. Overall, this study highlights the role of Exiguobacterium sibiricum K1 as a potential bioinoculant for improving crop yield under cold environments.

Strain-specific Growth Parameters are Important to Accurately Model Bacterial Growth on Baby Spinach in Simulation Models.

Digital tools to predict produce shelf life have the potential to reduce food waste and improve consumer satisfaction. To address this need, we (i) performed an observational study on the microbial quality of baby spinach, (ii) completed growth experiments of bacteria that are representative of the baby spinach microbiota, and (iii) developed an initial simulation model of bacterial growth on baby spinach. Our observational data showed that the predominant genera found on baby spinach were Pseudomonas, Pantoea and Exiguobacterium. Rifampicin-resistant mutants (rifR mutants) of representative bacterial subtypes were subsequently generated to obtain strain-specific growth parameters on baby spinach. These experiments showed that: (i) it is difficult to select rifR mutants that do not have fitness costs affecting growth (9 of 15 rifR mutants showed substantial differences in growth, compared to their corresponding wild-type strain) and (ii) based on estimates from primary growth models, the mean (geometric) maximum population of rifR mutants on baby spinach (7.6 log10 CFU/g, at 6°C) appears lower than that of the spinach microbiota (9.6 log10 CFU/g, at 6°C), even if rifR mutants did not have substantial growth-related fitness costs. Thus, a simulation model, parameterized with the data obtained here as well as literature data on home refrigeration temperatures, underestimated bacterial growth on baby spinach. The root mean square error of the simulation's output, compared against data from the observational study, was 1.11 log10 CFU/g. Sensitivity analysis was used to identify key parameters (e.g., strain maximum population) that impact the simulation model's output, allowing for prioritization of future data collection to improve the simulation model. Overall, this study provides a roadmap for the development of models to predict bacterial growth on leafy vegetables with strain-specific parameters and suggests that additional data are required to improve these models.

Fates of attached E. coli o157:h7 on intact leaf surfaces revealed leafy green susceptibility.

Leafy greens, especially lettuce, are repeatedly linked to foodborne outbreaks. This paper studied the susceptibility of different leafy greens to human pathogens. Five commonly consumed leafy greens, including romaine lettuce, green-leaf lettuce, baby spinach, kale, and collard, were selected by their outbreak frequencies. The behavior of E. coli O157:H7 87-23 on intact leaf surfaces and in their lysates was investigated. Bacterial attachment was positively correlated with leaf surface roughness and affected by the epicuticular wax composition. At room temperature, E. coli O157:H7 had the best growth potentials on romaine and green-leaf lettuce surfaces. The bacterial growth was positively correlated with stomata size and affected by epicuticular wax compositions. At 37 °C, E. coli O157:H7 87-23 was largely inhibited by spinach and collard lysates, and it became undetectable in kale lysate after 24 h of incubation. Kale and collard lysates also delayed or partially inhibited the bacterial growth in TSB and lettuce lysate at 37 °C, and they sharply reduced the E. coli O157:H7 population on green leaf lettuce at 4 °C. In summary, the susceptibility of leafy greens to E. coli O157:H7 is determined by a produce-specific combination of physiochemical properties and temperature.

Lytic Escherichia phage OSYSP acts additively and synergistically with gaseous ozone against Escherichia coli O157:H7 on spinach leaves.

Bacteriophage and gaseous ozone are evolving as meritorious alternatives to conventional sanitizers in food postharvest applications. Here, we investigated the efficacy of sequential treatments of a lytic bacteriophage and gaseous ozone, during vacuum cooling of fresh produce, against Escherichia coli O157:H7. Spinach leaves were spot-inoculated with 105-107 CFU g-1 E. coli O157:H7 B6-914 and treated with Escherichia phage OSYSP spray (109 PFU g-1), gaseous ozone, or their combination. Vacuum cooling, which preceded or followed phage application but ran concomitantly with ozone treatment, was performed in a custom-made vessel at the following process sequence: vacuum to 28.5 in. Hg, vessel pressurization to 10 psig with gas containing 1.5 g ozone/kg gas-mix, holding for 30 min, and vessel depressurization to ambient pressure. Bacteriophage or gaseous ozone inactivated E. coli O157:H7, applied at different initial populations on spinach leaves, by 1.7-2.0 or 1.8-3.5 log CFU g-1, respectively. At the high inoculum levels tested (7.1 log CFU g-1), sequential treatments of phage and ozone reduced E. coli O157:H7 population by 4.0 log CFU g-1, but when treatment order was reversed (i.e., ozone followed by bacteriophage), the combination synergistically decreased pathogen's population on spinach leaves by 5.2 log CFU g-1. Regardless the antibacterial application order, E. coli O157:H7 populations, applied initially at ~ 105 CFU g-1, were reduced below the enumeration method's detection level (i.e., < 101 CFU g-1). The study proved that bacteriophage-ozone combination, applied in conjunction with vacuum cooling, is a potent pathogen intervention strategy in fresh produce post-harvest applications.

Disinfection of Clostridioides difficile on spinach with epigallocatechin-based antimicrobial solutions and sodium hypochlorite.

The removal of C. difficile inoculated on fresh spinach leaves washed with antimicrobial solutions was investigated. In addition, the effect of washing solutions on the total aerobic mesophilic bacteria (TAMB) and Enterobacteriaceae in the fresh spinach was examined. The fresh spinach was washed through immersion in different concentrations (MIC, 2xMIC, and 4xMIC) of the natural disinfectant solution (NDS) consisting of EDTA, borax, and epigallocatechin gallate (EGCG) content developed in our laboratory and green tea extract-acetic acid (GTE-AA) for varying contact times (5 and 15 min). Different concentrations (50, 100, and 200 ppm) of sodium hypochlorite (NaOCl) and tap water as the control group were used to compare the effectiveness of the NDS. In addition, the effects of washing on the color, texture, and total phenol content of the spinach were determined. No statistical difference was observed in the washing of the spinach leaves with NDS prepared at 2xMIC and 4xMIC concentrations, while inhibition of C. difficile ranged between 2.11 and 2.32 logs. The highest inhibition was observed in the application of 50 ppm NaOCl for 15 min with a decrease of 2.88 logs in C. difficile spores. The GTE-AA and NDS decreased the number of TAMB by 2.27-3.08 log and, 3.21-3.66 log, respectively. Washing spinach leaves with natural disinfectant for 5 min caused a decrease of 2.58 logs in Enterobacteriaceae load, while washing with 50 ppm NaOCl for 15 min reduced Enterobacteriaceae load by 4 logs. Tap water was ineffective in reducing any microbial load. No difference was detected in the color parameters of the spinach through all washes. Although all antimicrobial washes made a difference in the texture of the spinach, the greatest loss in firmness was observed in the spinach washed with NaOCl. Washing spinach with epigallocatechin-based wash solutions can remove C. difficile in possible C. difficile contamination, thereby reducing the environmental load of C. difficile. Epigallocatechin-based disinfectants can be an alternative to chlorine-based disinfectants in improving the microbial quality of vegetables.

Pre-harvest biocontrol of Listeria and Escherichia coli O157 on lettuce and spinach by lactic acid bacteria.

Recent outbreaks linked to contaminated leafy greens underline the need for identifying effective natural approaches to improve produce safety at pre-harvest level. Lactic acid bacteria (LAB) have been evaluated as biocontrol agents in food products. In this study, the efficacy of a cocktail of LAB including Lactococcus lactis, Lactiplantibacillus plantarum, Lactobacillus johnsonii, and Lactobacillus acidophilus as pre-harvest biocontrol agents against Listeria and Escherichia coli O157 on lettuce and spinach was investigated. Bacterial pathogens L. monocytogenes and E. coli O157:H7 and the non-pathogenic surrogates L. innocua and E. coli O157:H12 were used to spray-inoculate cultivars of lettuce and spinach grown in growth chamber and in field, respectively. Inoculated plants were spray-treated with water or a cocktail of LAB. On day 0, 3, and 5 post-inoculation, four samples from each group were collected and bacterial populations were determined by serial dilution and spiral plating on selective agars. LAB treatment exhibited an immediate antimicrobial efficacy against L. monocytogenes and E. coli O157:H7 on "Green Star" lettuce by ~2 and ~ 1 log reductions under growth chamber conditions, respectively (P < 0.05). The effect of LAB against E. coli O157:H7 on "New Red Fire" lettuce remained effective during the 5-day period in growth chamber (P < 0.05). Treatment of LAB delivered an effective bactericidal effect against E. coli O157:H12 immediately after application on the field-grown lettuce plants (P < 0.05). Approximately 1 log L. innocua reduction was observed on "Matador" and "Palco" spinach on day 5 (P < 0.05). Results of this study support that LAB could potentially be applied as biocontrol agents for controlling Listeria and E. coli O157 contamination on leafy greens at the pre-harvest level.

Pre-heated blades for harvesting baby-leaves reduce the risk of Escherichia coli internalization in leaves.

BACKGROUND: Pathogenic enterobacteria can travel through the plant vascular bundles by penetrating from cuts and persisting into ready-to-eat leafy greens. Because the cutting site is the main point of entrance and uptake, we tested how different cutting strategies can reduce bacterial internalization in leaves. Horizontal cuts at the base of the leaves were performed with two different types of tools: the first with a scalpel (by pulling the blade) and the second with a scissor-action that has blades that cuts by gliding against a thicker blade. Scissor-action generally makes closer border cuts. Blades of both types of tools have worked at 25 °C and 200 °C. The present study aimed to determine how these different types of cuts and temperatures affected bacterial uptake in leaves. Experiments were repeated on different plant genotypes and at different wilting stages. RESULTS: Our findings showed that cutting baby-leaves with a scissor action at 200 °C significantly reduced the bacterial uptake compared to the not heated (which simulates a mechanized lettuce harvester). The most effective cutting treatments for reducing bacterial uptake were in the order: scissor 200 °C > scissor 25 °C > scalpel 200 °C > scalpel 25 °C. The scissor heated at 200 °C also prevented bacterial uptake on wilted baby-leaves. CONCLUSION: The findings of the present study could provide a further contribution in terms of safety during harvest and suggest that a pre-heated blade supports safety during harvest of leafy greens. © 2022 Society of Chemical Industry.

trans-Cinnamaldehyde-encapsulated zeolitic imidazolate framework-8 nanoparticle complex solutions to inactivate Escherichia coli O157:H7 on fresh spinach leaves.

This study synthesized and characterized ZIF-8 nanoparticles encapsulated with trans-cinnamaldehyde oil (TC) and evaluated their antimicrobial effectiveness against Escherichia coli O157:H7 on fresh spinach leaves. The antimicrobial activity of different mass ratios of TC-encapsulated ZIF-8 against E. coli O157:H7 (ATCC 43895) strain was assessed and the best mass ratio of 1:2 TC to ZIF-8 identified. Spinach leaves were treated with (1) 0.5TC@ZIF-8_PL nanoparticle complexes solution, (2) 200 ppm chlorine, (3) free TC, and (4) sterilized distilled water (control). All sample groups were rinsed for 1 min, dried in a biosafety cabinet, weighted, and packed in sterilized Whirl-pkTM Stand-Up sampling bags, and stored at 4°C for 15 days for shelf life studies. Samples were dipped into a solution of nanoparticles and another group was sprayed. The quality of spinach samples was assessed by monitoring changes in moisture content (MC), water activity (Aw), color, pH, texture (firmness and work), vitamin C content, total carotenoid, and chlorophyll content. Spinach leaves treated with 0.5TC@ZIF-8_PL had less (p < 0.05) water, total chlorophyll, and total carotenoid losses, with minimal changes in pH. However, treatment did not prevent the color degradation (p > 0.05) and adversely affected spinach firmness. The spinach samples treated with 200 ppm chlorine and free TC had higher (p < 0.05) total chlorophyll degradation than the samples treated with the nanoparticles. The mass ratio of TC-encapsulated ZIF-8 must be readjusted to reduce potential toxicity issues while maintaining the antimicrobial properties. PRACTICAL APPLICATION: Zeolitic imidazolate framework-8 (ZIF-8) nanoparticle complex can be used to encapsulate natural antimicrobials to inhibit growth of pathogens on fresh produce. A 2-log reduction in populations of Escherichia coli O157:H7 on fresh spinach leaves was achieved using trans-cinnamaldehyde at low concentrations. The results can be used to embed the compounds into polymeric films for antimicrobial packaging applications.

Rapid detection of Salmonella enterica in leafy greens by a novel DNA microarray-based PathogenDx system.

The diverse matrices pose great challenges for rapid detection of low Salmonella level (<10 CFU) in fresh produce. The applicability of microarray-based PathogenDx system for detecting low contamination of Salmonella Newport from leafy greens was evaluated. A pre-PCR preparation protocol including enrichment in universal pre-enrichment broth for 3 h followed by sample concentration using an InnovaPrep bio-concentrator or 6 h enrichment without a concentration step was used for detecting S. Newport from leafy greens with initial inoculum level at ∼6 CFU/25 g. Among 205 samples tested, 98%, 93%, 76%, and 60% of Romaine lettuce, Iceberg lettuce, kale, and spinach samples were tested positive after 3 h of enrichment with sample concentration. After 6 h of enrichment, 100%, 98%, 90%, and 82% of Romaine lettuce, Iceberg lettuce, kale, and spinach samples were positive. The samples were parallelly tested by the FDA bacterial analytical manual (BAM) method and 100% of spiked produce samples were tested positive. The overall analysis time of this methodology was between 8 and 11 h, including all pre-enrichment and concentration steps, in contrast to 4-5 days required for BAM method. The system correctly differentiated all 108 Salmonella strains and 35 non-Salmonella strains used in the study. This novel microarray approach provides a rapid method for detecting Salmonella in leafy greens.

Metagenetic characterization of bacterial communities associated with ready-to-eat leafy vegetables and study of temperature effect on their composition during storage.

Ready-to-eat (RTE) and fresh-cut vegetables meet the current needs for healthy and easy-to-prepare food. However, raw vegetables are widely known to harbor large and diverse bacterial communities promoting spoilage and reducing their shelf-life. A better understanding of their bacterial community and the impact of various environmental factors on its composition is essential to ensure the production of high-quality fresh-cut produce. Therefore, a metagenetic amplicon approach, based on gyrB sequencing, was applied for deciphering the bacterial communities associated with the spoilage of RTE rocket and baby spinach and monitoring the changes occurring in their composition during storage at different temperatures. Our results indicated that Pseudomonas genus was the main spoilage group for both leafy vegetables. Specifically, Pseudomonas viridiflava was dominant in most samples of rocket, while a new Pseudomonas species as well as, Pseudomonas fluorescens and/or Pseudomonas fragi were highly abundant in baby spinach. A significant variability on bacterial species composition among different batches of each vegetable type was observed. In the case of baby spinach, the impact of temperature and/or storage time on bacterial microbiota was not explicitly revealed at batch-level. Concerning rocket, the storage time was the most influential factor resulting in the reduction of Pseudomonas species' abundances and the parallel increase of lactic acid bacteria abundances. The results suggest that a large-scale sampling and further investigation of the various environmental factors shaping the microbiota are needed for gaining deeper knowledge of the diverse bacterial communities on RTE leafy vegetables and thus, enhance the quality of these products.

Seed inoculation with antagonistic bacteria limits occurrence of E. coli O157:H7gfp + on baby spinach leaves.

BACKROUND: During the last decades, outbreaks of foodborne illnesses have increasingly been linked to fresh and/or minimally processed fruit and vegetables. Enterohemorrhagic Escherichia coli was the causal agent for major outbreaks in Europe with leafy green vegetables and sprouts. To improve food safety, microbial antagonism has received attention during recent years and could be one of the solution to prevent contamination of food borne pathogens on fresh produce. Here we investigate the antagonistic effect of three bacterial strains (Pseudomonas orientalis, P. flavescens and Rhodococcus sp.) isolated from spinach leaves against E. coli O157:H7gfp + under laboratory and greenhouse conditions. RESULTS: Our results shows that significantly less culturable E.coli O157:H7gfp + were retrieved from the spinach canopy subjected to antagonist seed treatment than canopy inoculation. Seeds inoculated with Rhodococcus sp. significantly reduced growth of E. coli O157:H7gfp + compared with the other antagonists. The result from the in vitro study shows a significant reduction of growth of E. coli O157:H7gfp+, but only after 44 h when E. coli O157:H7gfp + was propagated in a mixture of spent media from all three antagonists. CONCLUSIONS: The antagonistic effect on phyllospheric E.coli O157:H7gfp + observed after seed inoculation with Rhodococcus sp. might be an indication of induced resistance mechanism in the crop. In addition, there was a small reduction of culturable E.coli O157:H7gfp + when propagated in spent media from all three antagonists. Nutritional conditions rather than metabolites formed by the three chosen organisms appear to be critical for controlling E. coli O157:H7gfp+.

Growth and survival of aerobic and Gram-negative bacteria on fresh spinach in a Chinese supply chain from harvest through distribution and refrigerated storage.

Spinach is a highly perishable product that degrades over time, including due to bacteria contaminating the product prior to packaging, yet the dynamics of bacterial spoilage and factors that affect it are not well understood. Notably, while China is the top producer of spinach globally, there is limited available microbiological data in the literature for spinach supply chains in China. The overall goal of this foundational study was to establish a baseline understanding of bacterial population dynamics on spinach from harvest to 10 days postprocessing for a Chinese supply chain that includes distribution via traditional grocery (a local physical store) and eCommerce (an online store). To this end, organic spinach samples were collected at different stages in a Chinese supply chain by following the same 3 lots, starting at point-of-harvest through processing and distribution via a local grocery store and eCommerce. After distribution, the same 3 lots were stored at 4 °C with microbiological testing performed on multiple days up to day 10 postprocessing, simulating storage at the point-of-consumer. Results showed aerobic plate counts and total Gram-negative counts did not significantly differ across stages in the supply chain from harvest through processing. However, packaged spinach from the same processing facility and lots, exhibited different patterns in bacterial levels across 0 to 10 days postprocessing, depending on whether it was distributed via the local grocery store or eCommerce. Evaluation of bacterial populations performed on a subset of the packaged spinach samples indicated Gram-negative bacteria, in particular Pseudomonas, were predominant across all days of testing (days 0, 3, and 10 postprocessing), with populations differing at the genus level by day. Overall, this study improves our understanding of the dynamics of bacterial populations on spinach and provides baseline data needed for future studies.

Microbiological safety of spinach throughout commercial supply chains in Gauteng Province, South Africa and characterization of isolated multidrug-resistant Escherichia coli.

AIM: To investigate the microbiological quality, potential foodborne pathogen presence, and to phenotypically (antimicrobial resistance [AMR] profiles) and genotypically (DNA fingerprints and diarrhoeagenic genes) characterize Escherichia coli isolated throughout spinach production systems from farm-to-sale. METHODS AND RESULTS: Samples (n = 288) were collected from two commercial supply chains using either river or borehole irrigation water. E. coli was enumerated throughout the chain where river water was directly used for overhead irrigation at levels between 0.00 and 3.22 log colony forming unit (CFU) g-1 . Following enrichment, isolation and matrix-assisted laser desorption ionization time-of-flight mass spectrometry identification, E. coli was isolated from 22.57% (n = 65/288) of all samples. Salmonella spp. were isolated from 3% (n = 9/288) of river and irrigation water samples on one farm, and no Listeria monocytogenes was detected throughout the study. Of the 80 characterized E. coli isolates, one harboured the stx2 virulence gene, while 43.75% (n = 35) were multidrug resistant. Overall, 26.30% of the multidrug-resistant E. coli isolates were from production scenario one that used river irrigation water, and 17.50% from the second production scenario that used borehole irrigation water. A greater percentage of resistance phenotypes were from water E. coli isolates (52.50%), than isolates from spinach (37.50%). E. coli isolates from spinach and irrigation water clustered together at high similarity values (>90%) using enterobacterial repetitive intergenic consensus-polymerase chan reaction analysis. CONCLUSIONS: This study reported the presence of multidrug-resistant environmental E. coli throughout spinach production from farm, during processing and up to retail. Furthermore, the similarity of multi-drug resistant E. coli isolates suggests transfer from irrigation water to spinach in both scenarios, reiterating that irrigation water for vegetables consumed raw, should comply with standardized microbiological safety guidelines. SIGNIFICANCE AND IMPACT OF STUDY: Multidrug-resistant E. coli presence throughout spinach production emphasizes the necessity of increased surveillance of AMR in fresh produce and the production environment within a One Health paradigm to develop AMR mitigation strategies.

Validation of the 3M™ Molecular Detection Assay 2 - STEC Gene Screen (stx) for the Detection of Shiga Toxin Gene (stx1 and/or stx2) in Fresh Raw Ground Beef and Fresh Spinach: AOAC Performance Tested MethodSM 071903.

BACKGROUND: The 3M™ Molecular Detection Assay 2 - STEC Gene Screen (stx) method is based on gene amplification by the use of real time loop-mediated isothermal amplification when used with the 3M Molecular Detection System for the rapid and specific detection of Shiga toxin gene (stx1 and/or stx2) from Shiga toxin-producing Escherichia coli (STEC) in enriched foods. The stx assay does not differentiate between stx1 and stx2 but detects the presence of stx1 and/or stx2. OBJECTIVE: The 3M Molecular Detection Assay 2 - STEC Gene Screen (stx) method was evaluated for AOAC®  Performance Tested MethodsSM certification. METHODS: Matrix studies, inclusivity/exclusivity, robustness testing, product stability, and lot-to-lot variability testing were conducted to assess the method's performance. RESULTS: The 3M Molecular Detection Assay 2 - STEC Gene Screen (stx) demonstrated equivalent results to the United States Department of Agriculture/Food Safety and Inspection Service Microbiology Laboratory Guidebook Chapter 5C.00 reference method for fresh raw ground beef, and the U.S. Food and Drug Administration Bacteriological Analytical Manual Chapter 4A reference method for fresh spinach. The 3M Molecular Detection Assay 2 - STEC Gene Screen (stx) detected all STEC E. coli strains (E. coli strains with stx1 and/or stx2 genes) and did not detect any of the 45 strains from the exclusivity panel. Robustness testing indicated that small variations in critical test parameters did not adversely affect the assay's performance. Product consistency and stability testing demonstrated no differences between the lots evaluated. CONCLUSION: The data collected in these studies demonstrate that the 3M Molecular Detection Assay 2 - STEC Gene Screen (stx) is a reliable method for the rapid and specific detection of Shiga toxin-producing E. coli in raw ground beef and spinach. HIGHLIGHTS: The 3M Molecular Detection Assay 2 - STEC Gene Screen (stx) method is suitable for the rapid and specific detection of Shiga toxin-producing E. coli in fresh raw ground beef, and spinach.

Lysinibacillus xylanilyticus Strain GIC41 as a Potential Plant Biostimulant.

To identify Lysinibacillus strains with the potential to function as plant biostimulants, we screened 10 previously isolated Lysinibacillus strains from the rhizosphere and soil for their plant growth-promoting (PGP) effects. In vitro tests showed that all strains produced indole-3-acetic acid. In primary screening, the PGP effects of these strains were assessed on spinach seedlings grown on Jiffy-7 pellets; strains GIC31, GIC41, and GIC51 markedly promoted shoot growth. In secondary screening, the PGP efficacies of these three strains were examined using spinach seedlings grown in pots under controlled conditions. Only GIC41 exerted consistent and significant PGP effects; therefore, it was selected for subsequent experiments. The results of 6-week glasshouse experiments revealed that GIC41 markedly increased shoot dry weight by ca. 12-49% over that of the control. The impact of fertilization levels on the PGP efficacy of GIC41 was investigated using pot experiments. The application of a specific level of fertilizer was required for the induction of sufficient PGP effects by this strain. The phylogenetic ana-lysis based on the 16S rDNA sequence identified GIC41 as L. xylanilyticus. Collectively, these results show the potential of strain GIC41 to function as a plant biostimulant.

Extracellular DNA as an elicitor of broad-spectrum resistance in Arabidopsis thaliana.

Like in mammals, the plant immune system has evolved to perceive damage. Damaged-associated molecular patterns (DAMPs) are endogenous signals generated in wounded or infected tissue after pathogen or insect attack. Although extracellular DNA (eDNA) is a DAMP signal that induces immune responses, plant responses after eDNA perception remain largely unknown. Here, we report that signaling defenses but not direct defense responses are induced after eDNA applications enhancing broad-range plant protection. A screening of defense signaling and hormone biosynthesis marker genes revealed that OXI1, CML37 and MPK3 are relevant eDNA-Induced Resistance markers (eDNA-IR). Additionally, we observed that eDNA from several Arabidopsis ecotypes and other phylogenetically distant plants such as citrus, bean and, more surprisingly, a monocotyledonous plant such as maize upregulates eDNA-IR marker genes. Using 3,3'-Diaminobenzidine (DAB) and aniline blue staining methods, we observed that H2O2 but not callose was strongly accumulated following self-eDNA treatments. Finally, eDNA resulted in effective induced resistance in Arabidopsis against the pathogens Hyaloperonospora arabidopsidis, Pseudomonas syringae, and Botrytis cinerea and against aphid infestation, reducing the number of nymphs and moving forms. Hence, the unspecificity of DNA origin and the wide range of insects to which eDNA can protect opens many questions about the mechanisms behind eDNA-IR.

The role of l-arabinose metabolism for Escherichia coli O157:H7 in edible plants.

Arabinose is a major plant aldopentose in the form of arabinans complexed in cell wall polysaccharides or glycoproteins (AGP), but comparatively rare as a monosaccharide. l-arabinose is an important bacterial metabolite, accessed by pectolytic micro-organisms such as Pectobacterium atrosepticum via pectin and hemicellulose degrading enzymes. However, not all plant-associated microbes encode cell-wall-degrading enzymes, yet can metabolize l-arabinose, raising questions about their use of and access to the glycan in plants. Therefore, we examined l-arabinose metabolism in the food-borne pathogen Escherichia coli O157:H7 (isolate Sakai) during its colonization of plants. l-arabinose metabolism (araBA) and transport (araF) genes were activated at 18 °C in vitro by l-arabinose and expressed over prolonged periods in planta. Although deletion of araBAD did not impact the colonization ability of E. coli O157:H7 (Sakai) on spinach and lettuce plants (both associated with STEC outbreaks), araA was induced on exposure to spinach cell-wall polysaccharides. Furthermore, debranched and arabinan oligosaccharides induced ara metabolism gene expression in vitro, and stimulated modest proliferation, while immobilized pectin did not. Thus, E. coli O157:H7 (Sakai) can utilize pectin/AGP-derived l-arabinose as a metabolite. Furthermore, it differs fundamentally in ara gene organization, transport and regulation from the related pectinolytic species P. atrosepticum, reflective of distinct plant-associated lifestyles.

Incorporation of Antimicrobial Bio-Based Carriers onto Poly(vinyl alcohol-co-ethylene) Surface for Enhanced Antimicrobial Activity.

A biobased rechargeable antimicrobial modification approach was developed using a covalent immobilization of food grade yeast cell wall particles on a model plastic film. We demonstrate the applications of this modification approach on poly(vinyl alcohol-co-ethylene) surface to inactivate inoculated bacteria with or without the presence of organic content, reducing the cross-contamination between food contact surface and model fresh produce, and inhibiting the growth of biofilms on the film surface. These biobased cell wall particle modified plastic films can enhance the binding of chlorine to the plastic surface in the form of N-halamine, extend the stability of chlorine against high organic content and ambient storage, and improve the rechargeability of the plastic films. Upon charging with chlorine, these modified plastic films inactivated 5 log of model Gram-negative bacteria (Escherichia coli O157:H7) and Gram-positive bacteria (Listeria innocua used as a surrogate of pathogenic Listeria monocytogenes) within 2 min of surface inoculation in water and within 20 min in an organic-rich aqueous environment. The modified plastic films prevented the transfer of bacteria and eliminated cross-contamination from the contaminated films to a spinach leaf surface, while 3 log CFU/leaf of bacteria were transferred from a contaminated native film to a noninoculated spinach surface. In addition, these modified plastic films reduced the adhesion of L. innocua cells by 2.7-3.6 log CFU/cm2 compared with control films during extended incubation for biofilm formation. Overall, this study demonstrates the feasibility of this biobased food grade modification approach to reduce microbial contamination and improve produce safety in the food processing industry.

Influence of seasonal changes and salinity on spinach phyllosphere bacterial functional assemblage.

The phyllosphere is the aerial part of plants that is exposed to different environmental conditions and is also known to harbor a wide variety of bacteria including both plant and human pathogens. However, studies on phyllosphere bacterial communities have focused on bacterial composition at different stages of plant growth without correlating their functional capabilities to bacterial communities. In this study, we examined the seasonal effects and temporal variabilities driving bacterial community composition and function in spinach phyllosphere due to increasing salinity and season and estimated the functional capacity of bacterial community16S V4 rRNA gene profiles by indirectly inferring the abundance of functional genes based on metagenomics inference tool Piphillin. The experimental design involved three sets of spinach (Spinacia oleracea L., cv. Racoon) grown with saline water during different seasons. Total bacteria DNA from leaf surfaces were sequenced using MiSeq® Illumina platform. About 66.35% of bacteria detected in the phyllosphere were dominated by four phyla- Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria. Permutational analysis of variance (PERMANOVA) showed that phyllosphere microbiomes were significantly (P < 0.003) affected by season, but not salinity (P = 0.501). The most abundant inferred functional pathways in leaf samples were the amino acids biosynthesis, ABC transporters, ribosome, aminoacyl-tRNA biosynthesis, two-component system, carbon metabolism, purine metabolism, and pyrimidine metabolism. The photosynthesis antenna proteins pathway was significantly enriched in June leaf samples, when compared to March and May. Several genes related to toxin co-regulated pilus biosynthesis proteins were also significantly enriched in June leaf samples, when compared to March and May leaf samples. Therefore, planting and harvesting times must be considered during leafy green production due to the influence of seasons in growth and proliferation of phyllosphere microbial communities.

Disrupting Irreversible Bacterial Adhesion and Biofilm Formation with an Engineered Enzyme.

Biofilm formation is often attributed to postharvest bacterial persistence on fresh produce and food handling surfaces. In this study, a predicted glycosyl hydrolase enzyme was expressed, purified, and validated for the removal of microbial biofilms from biotic and abiotic surfaces under conditions used for chemical cleaning agents. Crystal violet biofilm staining assays revealed that 0.1 mg/ml of enzyme inhibited up to 41% of biofilm formation by Escherichia coli O157:H7, E. coli 25922, Salmonella enterica serovar Typhimurium, and Listeria monocytogenes. Furthermore, the enzyme was effective at removing mature biofilms, providing a 35% improvement over rinsing with a saline solution alone. Additionally, a parallel-plate flow cell was used to directly observe and quantify the impact of enzyme rinses on E. coli O157:H7 cells adhering to spinach leaf surfaces. The presence of 1 mg/liter enzyme resulted in nearly 6-times-higher detachment rate coefficients than a deionized (DI) water rinse, while the total cells removed from the surface increased from 10% to 25% over the 30-min rinse time, reversing the initial phases of biofilm formation. Enzyme treatment of all 4 cell types resulted in significantly reduced cell surface hydrophobicity and collapse of negatively stained E. coli 25922 cells imaged by electron microscopy, suggesting potential polysaccharide surface modification of enzyme-treated bacteria. Collectively, these results point to the broad substrate specificity and robustness of the enzyme for different types of biofilm stages, solution conditions, and pathogen biofilm types and may be useful as a method for the removal or inhibition of bacterial biofilm formation. IMPORTANCE In this study, the ability of an engineered enzyme to reduce bacterial adhesion and biofilm formation of several foodborne pathogens was demonstrated, representing a promising option for enhancing or replacing chlorine and other chemical sanitizers in food processing applications. Specifically, significant reductions of biofilms of the pathogens Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes are observed, as are reductions in initial adhesion. Enzymes have the added benefits of being green, sustainable alternatives to chemical sanitizers, as well as having a minimal impact on food properties, in contrast to many alternative antimicrobial options such as bleach that aim to minimize food safety risks.