Progress 02/01/24 to 01/31/25
Outputs
Target Audience:The target audience includes scientists, extension agents, graduate students, government officials, industry professionals such as CEA growers interested and working in environmental horticulture, food safety, nutrition, food science, post-harvest physiology and plant science. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Three undergraduates have been technically trained on the microbial and phytochemical analysis aspects of the project. A graduate student has been trained on all aspects (field, growth chaamber, laboratory)of this project.The graduate student has had the opportunity to mentor undergradaute students, collect and manage data, analyse and interpret data, write abstracts, prepare and present posters and prepare scientific manuscripts. How have the results been disseminated to communities of interest?Results have been shared at the International Association for Food Protection Annual Meetings at the US and European meetings. Results ahve also been published in scientific journals. What do you plan to do during the next reporting period to accomplish the goals?More high tunnel trials are planned for the next reporting period. These will provide material to evaluate plant responses to drought and their effects on food safety, quality andshelf-life outcomes. Several publications are also planned and will be submitted in the coming year.
Impacts
What was accomplished under these goals?
Objective 1: Evaluate abiotic stress elicitation of secondary metabolites via regulated drought and other abiotic stresses onfood safety outcomes and shelf-lifeof a variety of lettuce and kale cultivars grown to the juvenile (baby leaf) stage. Several experiments were conducted in the field to progress in accomplishing this goal. Plants were grown in high tunnels at the Central Maryland Research and Education Centre, Upper Marlboro, MD, under restricted watering regimes. Three watering schedules were implemented, consistent watering, intermittent water restriction and one-time water restriction. For the soil type in the high tunnel, this allowed us to achieve soil moisture levels between 20% to 8%. The two watering restricted regimes gave mixed and inconsistent results such that we determined that two watering schedules, high - 20% and low 10%, would be used in subsequent experiments. Foodborne pathogen association experiments were conducted on the kale and lettuce grown in high tunnels. For leaf surface association with foodborne pathogens: Romaine lettuce 'Rouge d'Hiver' was direct seeded along 60 cm wide beds. Two irrigation treatments were set up in a randomized design based on regular watering (High) and water restriction (Low) and volumetric soil water content, (VWC) recorded continuously prior to harvesting at 26 days post-germination. Samples were transported to the lab for bacterial spot (surface) inoculations with rifampicin-adapted Escherichia coli O157:H7, Salmonella Enteritidis and Listeria monocytogenes. We evaluated an 18 h incubation at room temperature (RT), and a 3 or 6 day incubation at 4°C. Leaves were stomached for 1 min and serial dilutions plated on tryptic soy agar with rifampicin for colony counting. When incubated at RT, fewer E. coli O157 counts were detected on leaves grown under low irrigation, while Listeria and Salmonella were unaffected. Under refrigerated storage, soil moisture was a factor only for Salmonella. Fewer Salmonella were recovered from low-irrigation leaves stored at 3 and 6 d compared to high-irrigation lettuce. Listeria and E. coli O157:H7 counts were unaffected by soil moisture but bacterial levels decreased from 3 to 6 days in storage. For foodborne pathogens internalized into leaves: Kale 'Red Russian' was used for experiments with pathogen infiltration into leaves. Two irrigation regimes were set up in a randomized design based on regular (High) and restricted (Low) watering. Volumetric soil water content was recorded continuously up to harvesting at 26 days post-germination. On harvest day, three sets of two leaves per treatment were placed into bags and transported to the lab on ice. Inocula were prepared for rifampicin-adapted Escherichia coli O157:H7, Salmonella Enteritidis and Listeria monocytogenes and leaves infiltrated with inoculum using a needleless syringe. Leaves were incubated for 1, 2 and 4 days at 4°C, then homogenized in peptone water and analysed by plate counting. Overall, counts varied by pathogen in the order from highest to lowest of Listeria, E. coli O157:H7 and Salmonella. Statistical analyses of these data identified soil moisture and storage time as factors in Salmonella persistence. Salmonella in low-irrigation kale after 4 days of storage was most affected by soil moisture as compared to high-irrigation kale. Only storage time was a factor for Listeria. An interaction between storage time and soil moisture was detected, however, whereby Listeria counts decreased after 4 days of storage compared to 1 and 2 days within high-irrigation kale only. E. coli O157:H7 was unaffected by soil moisture or storage time. Objective 2:Analyze the metabolomethrough identification and quantification of flavonoids, phenolics and glucosinolates (kale only) in leaf tissue and leaf surfacemetabolomesin select juvenile lettuce and kale plants induced in response to regulated abiotic stress. A method was developed to quantify phenolic compounds using a profiling spectrophotometric method. Standard curves of phenolic compounds apigenin, catechin, gallic acid, quercetin and Chlorogenic acid were made and used to calculate equivalent concentrations at specific wavelength absorbances. An HPLC protocol was also developed. The quantification methods were validated against a colorimetric technique. From chamber experiments, we explored how E. coli O157 and Salmonella affect plant specialized metabolites in pre-harvest and post-harvest baby romaine lettuce. This addresses a research gap regarding whether E. coli O157:H7 and Salmonella enterica induce lettuce metabolite responses at different stages of harvest. In several repeated experiments, Romaine lettuce 'Carlsbad' was grown for 4 weeks under controlled conditions and used as pre-harvest (live) plants, or for simulated post-harvest processed leaves. Leaves were cut, washed with SaniDate15®, dried, bagged and stored for 7 days at 4°C. Using a needleless syringe, pre- and post-harvest leaves were infiltrated with water (control), and rifampicin-adapted E.coli O157 and Salmonella Enteritidis. Following incubation, samples were enumerated for foodborne pathogens by plate counting. Lyophilized leaf tissue was extracted in aqueous methanol for a newly developed spectrophotometric analysis as described previously. Overall, pathogen reduction was higher on pre-harvest 'Carlsbad' for both E. coli O157 and Salmonella, compared to post-harvest leaves. For all metabolites tested, phenolic compound equivalent (PCE) values were higher 4 hours post inoculation with live Salmonella in pre-harvest plants. The response to E. coli was more muted or inconsistent. Ater 24 h post-inoculation, many metabolite levels went back to baseline or decreased lower than baseline. PCEs did not change in post-harvest leaves. From the high tunnel experiments, leaf tissue has been collected to evaluate how plant secondary metabolites changed in response to different watering regimes. Plant tissue has been lyophilized and will be extracted for spectrophotometric and chromatographic analysis. Objective 3:Assesssensory propertiesandconsumer preferenceof select lettuce and kale varieties grown under regulated abiotic stress. A high tunnel study was conducted with kale and lettuce grown under variable watering regimes. The lettuce was harvested, stored at 4°C, washed in hypochlorite solution and prepared for evaluation by an eight-member trained panel. The descriptive analysis included 23 attributes for kale and 18 attributes for lettuce. Data are being analyzed.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Hudson, C. and Micallef, S. (2024), Epiphytic, Attached, and Internal Escherichia coli O157:H7 Subpopulations Associating With Romaine Lettuce Are Strain-Dependent and Affected by Relative Humidity and Pre- and Postharvest Plant State. J Food Saf, 44: e13169. https://doi.org/10.1111/jfs.13169
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Micallef SA. Differential enteric pathogen modulation on various lettuce and tomato cultivars mediated by plant metabolites. In Symposium: Plant Genotypes and Phenotypes as an Intrinsic Approach to Enhance the Microbial Safety of Produce. IAFP Europe, 30 April 2 May 2024, Geneva, Switzerland.
- Type:
Other Journal Articles
Status:
Other
Year Published:
2025
Citation:
Hudson C, Micallef SA. Spectrophotometric profiling and quantification of plant phenolics reveals lettuce metabolite response to enteric pathogens. Journal of Agricultural and Food Research. - preparing for submission.
- Type:
Conference Papers and Presentations
Status:
Under Review
Year Published:
2025
Citation:
Hudson C, Diksha Klair, Donald Murphy, Guy H. Kilpatric, Micallef SA. Impact of Pre-Harvest Water Stress on Enteric Pathogen Persistence on Baby Romaine Lettuce in Storage. International Association for Food Protection (IAFP2025) Annual Meeting, 27-30 July 2025, Cleveland, OH.
- Type:
Conference Papers and Presentations
Status:
Under Review
Year Published:
2025
Citation:
Hudson C, Diksha Klair, Donald Murphy, Guy H. Kilpatric, Micallef SA. Persistence of Internalized Enteropathogens in Baby Leaf Kale as Influenced by Pre-Harvest Water Stress and Post-Harvest Storage Conditions. International Association for Food Protection (IAFP2025) Annual Meeting, 27-30 July 2025, Cleveland, OH.
- Type:
Conference Papers and Presentations
Status:
Under Review
Year Published:
2025
Citation:
Hudson C, Micallef SA. Specialized Metabolite Responses to Internalized Enteropathogens in Baby Romaine Lettuce Carlsbad Pre- and Post-harvest. International Association for Food Protection (IAFP2025) Annual Meeting, 27-30 July 2025, Cleveland, OH.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Hudson C, Micallef SA. Responses in Plant Secondary Metabolite Accumulations in Pre- and Post-Harvest Baby Romaine Lettuce Leaves Induced by Escherichia coli O157:H7. International Association for Food Protection (IAFP2024) Annual Meeting, 14-17 July 2024, Long Beach, California.
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Progress 02/01/23 to 01/31/24
Outputs
Target Audience:The target audience includes scientists, extension agents, graduate students, government officials, industryprofessionals such as CEA growers interested and working in environmental horticulture, food safety, nutrition, food science, post-harvest physiologyand plant science. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One PhD student is being trained under this grant, learning laboratory skills in plant physiology, plant phytochemistry and food safety microbiology. Student is learning experimental design, data management and statistical analyses and expanding scientific knowledge in all the areas mentioned. The student had the opportunities to present data at an international conference and preparemanuscripts for publication. One undergraduate studentlearnedbacterial culturingtechniques and how to measure plant growth parameters, as well as preparing plant tissue for phytochemical extractions and spectrophotometric measurements to determine levels of phytocompounds in samples.Student was introduced to food safety microbiology concepts and gained laboratory skills in this area. How have the results been disseminated to communities of interest?The results were disseminated via scientific meetings at the International Association for Food Protection (IAFP) 2023 and theFlorida Association for Food Protection, Annual Educational Conference, both attended by academicians, researchers, industry stakeholders and government officials. A presentation was also made at a Controlled Environment Agriculture focused Extension Meeting attended by Extension Specialists, Industry partners, growers and researchers and academicians. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: In the next period, we will evalaute effects of abiotic elicitation of plant secondary metabolites on internal Salmonella andListeria monocytogenes. We plan to collect more phytonutrient data (e.g., vitamin C, glucosinolates, sugars). We plan to commence work on specific aim 1.4. Quality/shelf-life assessments which will include microbial assays to assess microbiological quality of lettuce,and evaluate product quality and shelf-lifeby measuring electrolyte leakage and enzymatic activity to assess . Objective 2: We plan to conduct more targeted profiling (flavonoids in lettuce cultivars) of leaf tissue and surface compounds, identify specific phytocompounds that are induced by abiotic elicitation and evaluate microbiological effects of these compounds on bacterial viability and cell membrane integrity. Objective 3: We plant to conduct a water restriction trial on lettuce and kale in high tunnels in the field in late spring. Three watering regimes will be evaluated, optimal watering (controls) and two regulated water restriction regimes. Plants will be evalauted for plant secondary metabolite induction and the effect on foodborne pathogens will be assessed. A second high tunnel trial will be conducted in early fall. These trials will inform the products to be produced for the sensory evaluation to happen later in the year. A sensory evaluation with a trained panel will be conducted in October/November and a full suite of physicochemical measurements will be conducted on the product in the winter.
Impacts
What was accomplished under these goals?
Objective 1: Abiotic stress application, consisting of irrigation water restriction to leafy vegetableswas optimized based on proline induction and in relation to elicitation of total phenolic and total flavonoid accumulation, while maintaining fresh weight of plants. The optimization was done on various Romaine lettuce varieties, loose-leaf lettuce and kale, all grown to the baby leaf stage. Phytochemical measurements were conducted using a colorimetric assay to obtain total levels accumulated in leaf tissue. UV-spectral analysis corroborated the colorimetric measurements and further informed the effects of abiotic stress elicitation by revealing changes in the amounts and spectra detected at different wavelengths. These data will also help in targeted phytochemical profiling in objective 2. For the food safety sub-objective, protocols were generated to accurately measure various cell fractions of enteric pathogens applied to leaves. These included the entire bacterial population, the epiphytic population, the strongly attached and internalized population and the internalized population only. Food safety assessments were then carried out with a lettuce outbreak strain of E. coli O157:H7 and with Salmonella Newport, Typhimurium and Enteritidis. Data included epiphytic Salmonella persistence data on lettuce and kale, and E. coli O157:H7 data that includes both epiphytic and internal cell fractions. Both cultivar and water restriction were factors included in the analyses on various lettuce varieties and kale. Persistence of E. coli O157:H7 was also compared between pre- and post-harvest plants, assessed by means of the four cell fractions described above. The effect of relative humidity inE. coliO157:H7 persistence was also evaluated on pre-harvest plants. For this aim, a protocol for small scale post-harvest wash with Sanidate® followed by storage at 4C was established. This will also be used in sub-objective 1.4 (shelf life assessment). Objective 2: Metabolite profiling was conducted on kale and lettuce leaf tissue and leaf surface wash via electrospray ionization-mass spectrometry (ESI-MS), revealing differences between leaf tissue and leaf surface compounds, differences by plant developmental stage and at the baby kale stage, differences by watering regime. Specific peaks indicated the up- and down-accumulation of metabolites both inside the leaf and on the leaf surface. These data are being analyzed for identification of specific metabolites. Methanolic extracts are also being used to evaluate the effect of plant secondary metabolites on pathogenic bacterial viability and cell membrane integrity. Objective 3: In this objective, flavonoid accumulations on Romaine lettuce are being optimized for field growing conditions in high tunnels. Growth chamber experiments were conducted to compare two water restriction protocols, involving an intermittent water restriction regime and a one-time exposure to water restriction. Controls were optimally watered throughout their growth cycle. Moreover, plants underwent a period of stress recovery, which involved a full irrigation after the water restriction interval. The induced levels of total flavonoids following water restriction remained elevated one day after recovery from stress but declined 3 days after recovery from stress to control levels. Therefore, extended watering restriction is being tested. These data are being used to develop a plan for sensory evaluation using a trained panel.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2023
Citation:
Micallef SA, 2023. Advances in understanding contamination of fresh produce by Salmonella. Chapter 1, pp. 3-32. In: Advances in ensuring the microbiological safety of fresh produce. Edited by Professor Karl R. Matthews. Series: Burleigh Dodds Series in Agricultural Science No. 136. Burleigh Dodds Science Publishing Limited. ISBN 978-1-80146-270-9 (PDF) . DOI: 10.19103/AS.2023.0121.01
- Type:
Other
Status:
Published
Year Published:
2023
Citation:
Micallef SA. Pre-harvest Internalization: Water-mediated Biological Internalization of Pathogens into Produce. Organized by: IAFP's Fruit and Vegetable Safety and Quality PDG. IAFP Webinar Series: 23 October 2023.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
Micallef SA. Controlled Environment: Food safety issues when growing greenhouse/CEA leafy greens. Northeast Greenhouse Conference & Expo. Manchester, NH. 8-9 November 2023.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
Micallef SA. What doesnt kill you makes you stronger: Plant stress impacts on nutrition and safety of leafy greens. Florida Association for Food Protection, Annual Educational Conference. 16-18 May 2023, Orlando, Florida.
- Type:
Other
Status:
Published
Year Published:
2023
Citation:
Poster: Epiphytic and Internalized Fractions of Escherichia coli on Inoculated Live Lettuce Plants and Harvested Leaves. Hudson CL, Micallef SA. International Association for Food Protection (IAFP2023) Annual Meeting, 16-19 July 2023, Toronto, Canada.
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Progress 02/01/22 to 01/31/23
Outputs
Target Audience:Food microbiologists, food safety scientists, food scientists, controlled agricutlure growers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One PhD student is being trained under this grant, learning laboratory skills in plant physiology, plant phytochemistry and food safety microbiology, experimental design andscientific knowledge in all the areas mentioned. AnotherPhD student published her first two papers as first author that enhanced her professional development and writing skills. One MS student learnt greenhouse and hydroponic management/cultivation of plants and how to apply regulated plant stresses as well as numerous laboratory skills in the areas mentioned above. Two undergradaute students learnt food safety microbiology laboratory skills. How have the results been disseminated to communities of interest?Results were disseminated via scientific meetings at the International Association for Food Protection 2022, FoodMicro meeting 2022 and ASM Microbe 2022. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: We will apply stresses to lettuce and evaluate the impact onE. coliO157:H7 associating epiphytically and internalized bacteria. We will also be measuring plant growth parameters, micronutrientand phytochemical accumulation in lettuce plants and conducting statistical analysis to determine possible associations. We will continue to decipher how plant stress impacts plant phytochemical profiles and other plant characteristics. We will also start to evaluate how plant changes in resposeto stress affect shelf-life of harvested leafy greens. Objective 2: We planto conduct TOF- MS/MSto idenitfy and quantify phytocompounds that accumulate differentially under the regulated stress conditions. Objective 3: We plan to conduct our first sensory evaluation on select lettuce cultivars that are subjected to a variety of regulated stresses to evalaute changes in texture, quality, flavorand consumer preferences.
Impacts
What was accomplished under these goals?
Objective 1: We are evaluatingabiotc stress elicitation of specialized metabolites via a variety of stresses applied to leafy green crops grown in controlled environments. These have included regulated drought, salinity, heat and cold stresses. We have assessed plant growth, phytochemical accumulation and food safety outcomes usingSalmonella following drought and salinity stress in kale and lettuce. We have also started measuring plant growth parameters and phytochemical accumulation in response to cold and heat stress in various lettuces. We have optimized our protocols for measuiringE. coliO157:H7 in live lettuce plants and harvested lettuce leaves, measuring both epiphytic and internalized bacterial fractions. Objective 2:We have started using ESI-MS to evaluate the metabolome of plants under stress. We see shifts in both leaf tissue and leaf surface metabolomes in baby lettuce and kale. Objective 3: Work on this objective has not yet started.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Liu X, Li Y, Micallef SA, 2022. Developmentally related and drought-induced shifts in the kale metabolome limited Salmonella enterica association, providing novel insights to enhance food safety. Food Microbiology 108: 104113. https://doi.org/10.1016/j.fm.2022.104113
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Liu X, Li Y, Micallef SA. Natural variation and drought-induced differences in metabolite profiles of red oak-leaf and Romaine lettuce modulated the interaction with Salmonella enterica. International Journal of Food Microbiology accepted 25 October 2022. https://doi.org/10.1016/j.ijfoodmicro.2022.109998
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2022
Citation:
Talk: Micallef SA, Liu X, Hsu CK. Plant growth-promoting rhizobacterium Pseudomonas spp. shifts plant phytochemical profiles, affecting Salmonella enterica association with baby kale leaves. International Association for Food Protection, IAFP 2022 Annual Meeting, 30 July-3 August 2022, Pittsburgh, PA
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2022
Citation:
Poster: Micallef SA, Liu X, Hsu C-K. Rhizosphere Pseudomonas spp. modulated metabolite shifts caused by abiotic stress and limited Salmonella association with juvenile kale. ASM Microbe 2022, 9-13 June 2022, Washington D.C.
- Type:
Other
Status:
Published
Year Published:
2022
Citation:
Poster: Micallef SA, Liu X, Hsu C. Beneficial Pseudomonas spp. protected kale from salt stress and influenced association with Salmonella enterica. International Association for Food Protection, IAFP 2022 Annual Meeting, 30 July-3 August 2022, Pittsburgh, PA
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2022
Citation:
Talk: Abiotic elicitation of plant secondary metabolites limits Salmonella enterica in lettuce and kale. FoodMicro 2022, 28-31 August 2022, Athens, Greece.
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