Mechanisms of Attachment of Pathogens to Seeds of Selected Vegetable Species and with Different Surface Characteristics

<P>NON-TECHNICAL SUMMARY: Vegetable seeds including sprout seeds can be passive carriers and effective vectors of transmitting human and plant pathogens. They account for the movement of pathogens across a long distance and are responsible for contaminating vegetable supplies in new areas and causing vegetable-associated outbreaks of infections. This research responds to Program Area Priority A1331 with a long term goal of producing vegetable seeds particularly sprout seeds that are free of human pathogens. The project team comprised of a food microbiologist and a seed pathologist intends to use a collaborative approach to elucidate the physical and molecular mechanisms that selected human pathogens, Salmonella and enterohemorrhagic Escherichia coli (EHEC), use to attach to, colonize and establish populations on germinating seeds, sprouts and seedlings. The team will assess the differences in attachment by selected human pathogens to chemically-treated vs. untreated, and intact vs. mechanically-damaged seeds of alfalfa, fenugreek, tomato and lettuce. Pathogen migration from selected vegetable seeds with systemic infection, infestation, or accompany contamination, to sprouts and different areas of seedling tissues will be assessed. Genes and gene products that are critical for pathogen attachment and colonization to/on vegetable seeds will be identified. The effect of selected phytopathogens and biological control agents on the attachment of Salmonella and EHEC to different vegetable seeds and on pathogen colonization on emerging sprout and seedling tissues will be evaluated. The project will lead to a better understanding of pathogen attachment/colonization to/on vegetable seeds and help reduce the economic losses associated with vegetable-related, especially sprout-related outbreaks of infections.</P>
<P>APPROACH: Month 1-12: We will study the differences in attachment by selected Salmonella and EHEC strains to different types of vegetable seeds including chemically treated vs. untreated, intact vs. mechanically damaged seeds of alfalfa, fenugreek, tomato and lettuce. Seeds will be exposed to three different levels of Salmonella or EHEC strains for surface attachment. Five samples will be used for each inoculated level and each type of seed. Each experiment will be repeated twice. Month 13-24: Seeds of alfalfa, fenugreek, tomato and lettuce will be inoculated with nalidixic acid resistant strains of Salmonella or EHEC by mimicking systemic infection, infestation and accompanying contamination, respectively. The population of bacterial cells on seeds, sprouts, as well as seed coats, cotyledons, hypocotyls and radicals of the seedlings will be determined. Month 25-42: We will use a selected Salmonella and E. coli O157:H7 strain to identify the genes whose products play a crucial role in bacterial attachment to and colonization on the seeds of alfalfa, fenugreek, tomato and lettuce. Transposon mutagenesis will be performed followed by selection of Salmonella or E. coli attachment mutants. Several available differential microbiological media will be used to select the Salmonella or E. coli O157:H7 that are deficient in the expression of certain cell surface components including thin aggregative fimbriae or curli, cellulose and EPS-CA. Other genes playing an important role in Salmonella or E. coli O157:H7 attachment and colonization to/on vegetable seeds will be identified using DNA cloning and nucleotide sequencing. Month 43-60: Comparative attachment between selected wild type parental and non-attachment mutant strains of Salmonella and E. coli O157:H7 will be conducted in the presence of plant pathogens and biological control agents with a chromosome borne, spontaneous antibiotic resistance marker. </P>