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Plant Responses to Food-Borne Bacteria and Viruses and Mechanisms Used by Pathogens to Survive

Kniel-Tolbert, Kalmia
University of Delaware
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The ultimate long term goal of this project is to understand and characterize the mechanisms that allow human foodborne pathogens, specifically norovirus, pathogenic E. coli and Salmonella, to attach to, persist on/in, and colonize plants. By elucidating the mechanisms at the plant-pathogen interface using genetic and proteomic approaches, we will generate information that will be useful in inhibiting the attachment and persistence of enteric pathogens on leafy greens and other produce commodities. These results will lead to the more effective use and application of antimicrobials and good agricultural practices, ultimately reducing the number of illnesses and detrimental impact on public health.

Three specific objectives are listed below.

  1. Concurrently evaluate the persistence of three foodborne pathogens (E. coli O157:H7, Salmonella, and Norovirus) on foliar surfaces of plants (lettuce and spinach) under the same conditions to facilitate a direct comparison of the survival characteristics of these organisms in the phyllosphere.
  2. Investigate plant defense pathways during attachment and internalization of E. coli O157:H7, Salmonella and noroviruses. Determine if plants respond to these human pathogens using one or more plant-specific defense strategies.
  3. Evaluate the metabolic pathways used by pathogenic bacteria to attach to plants, specifically using a novel approach to identify the major proteins of pathogenic bacteria using proteomics.
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Non-Technical Summary: By studying concurrently three important pathogens that are also the main causes of foodborne illness associated with fresh produce we will gain a better understanding of how the plant and the microbe interact during the processes of association, attachment, internalization and survival. This is the main objective of this program. This unique research team is composed of a virologist and a bacteriologist who are experts in produce-specific food safety and a plant pathologist has significant experience in working with both phyto- and enteric pathogens. These three individuals can accomplish the objectives of the proposed research and as the main goal of the Food-borne Pathogens-Plant Interactions Foundational Program this research will generate fundamental information into the physical and molecular mechanisms that enable human pathogens to attach, internalize, grow and survive in and on fresh produce. Additionally this work will generate information on how plants interact with food-borne pathogens and if these associations affect the attachment and fate of human pathogens on fresh produce. With regards to potential long range improvement of U.S. agriculture and food systems the goals of this project will clearly be used to better enhance the safety of produce and reduce transmission of these pathogens in the field. Acquired data will be translated into applied farming practices and integrated into Good Agricultural Practices where applicable. In particular information concerning persistence and survival will impact growing and irrigation practices. Information gained by studying the plant defense response in relation to colonization by viruses and bacteria will also impact pre-harvest growing practices.

Approach: Baby spinach and Romaine lettuce plants will be grown in BSL-2 growth chambers using previously utilized methods under dynamic temperature and light conditions. Plants at 14- or 21-day stages of growth will be inoculated with the respective pathogens grown in bovine fecal suspensions. Shoot tissue will be inoculated with respective pathogenic suspensions using multiple spot inoculations (10 ?l spots) on foliar surfaces so that a total of 100 spots (1 ml) will be applied to each plant on multiple leaves. Post-homogenization samples will be analyzed by plating or a modified Most Probably Number technique or RT-PCR. Plants at the second true leaf stage will be dip-inoculated with either an eight strain cocktail of Salmonella enterica or four strain cocktail of EHEC. Plants will be kept in a controlled-environment under a day and night cycle of 12 h with a temperature of 26C and 18C, respectively. Humidity will be constant at 75%. Four leaflets will be removed from each plant seven and 14 days post-inoculation and placed into individual tubes of 500 ?l water. The leaflet will be weighed, homogenized, aliquots serially diluted, and plated on selective media for population enumeration. Weights will be used as a normalization factor to determine the cfu/g of tissue. Samples will be taken 1, 7, 14, and 30 dpi. Populations will be log transformed and means will be tested for significance using Duncan's multiple range test by SAS. Internalized virus will be quantified using plaque assay in RAW cells to determine the amount of infectious virus and by qRT-PCR to determine the amount of genomic copies of nucleic acid present. Lettuce seeds will be grown for 20 days prior to being subjected to contaminated water containing 5x10e8 RT-qPCRU/ml MNV for 24 hr, followed by removal of virus solution and replaced with virus free nutrient solution every day for up to 5 days. Total RNA will be eluted with 60 ?l RNase-free water and a two step RT-qPCR will be used to quantify the virus concentration. Humidity is a major factor controlling plant transpiration and guttation. Plants will be grown under two conditions, one with high humidity and one with lower (<70%). Romaine lettuce at the rosette stage, ~14 days old, will be dip-inoculated. Leaves will be sampled 72 h post-inoculation. Proteins will be extracted with the All prep DNA/RNA/Protein kit, precipitated, and then dissolved in a kit-supplied sample buffer. Proteins will be separated by 1-dimensional SDS/PAGE and analyzed after tryptic digestion by reversed-phase high-performance liquid-chromatography coupled to electrospray-ionization tandem mass-spectrometry. MS data will be converted to peak lists and analyzed with 2 search algorithms and validated with Scaffold. MS/MS spectra will be searched against a database consisting of protein sequences obtained from RefSeq and a second database consisting of all protein sequences from the complete genomes of Salmonella enterica and EHEC. For protein identification, at least 2 peptide matches will be required. The minimum peptide identification probability will be 95% and protein identification probability will be 99%.

Funding Source
Nat'l. Inst. of Food and Agriculture
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Escherichia coli
Chemical Contaminants
Bacterial Pathogens