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The development of a novel assay to sensitively detect biosurfactant production by bacteria recovered from plants has provided evidence that they may greatly alter the nature of the plant surface, thereby altering the behavior of human pathogens, enabling them to more easily invade leaves. <P>The objectives of this study are thus to isolate and characterize the diversity of epiphytic bacteria and their biosurfactants on produce plant species. We further will investigate the production of biosurfactants on the leaf surface, and the effect of a rich indigenous epiphytic population on the leaf's physical properties. To address the effects of indigenous biosurfactant producers on plants we will examine the effect of biosurfactant production on the attachment, movement, and internalization of human pathogens such as E. coli or Salmonella enterica.
Non-Technical Summary: Colonization of fresh produce by human pathogens such as E. coli and Salmonella enterica is a growing food safety issue. The behavior of such human pathogens on produce is, however, strongly influenced by the other resident bacterial colonists of the plant surface. Specifically, epiphytic bacteria that produce biosurfactants that alter the interaction of water with the plant can facilitate the invasion of pathogens into the plant where they can not be decontaminated during postharvest treatments. We have developed a novel method to identify biosurfactant producers on plants that reveals that they are very common in this habitat and that they produce surfactants with unique properties that will facilitate their interactions with plants. We will use this assay to determine the seasonal and regional variation in biosurfactant producers on spinach, romaine lettuce, and tatsoi in the region of commercial leafy vegetable production in Northern California and determine the variety of biosurfactants produced. Bacteria producing different classes of biosurfactants will be assessed for their ability to enhance the invasion of E. coli and Salmonella enterica into the edible plant varieties both in greenhouse and field studies. Likewise the effect of the biosurfactants on the viability and retention of the pathogens on plants will be assessed. Understanding the contribution of such extrinsic factors as the indigenous biosurfactant-producing bacterial microflora will enable better estimates of the risks associated with contamination of human pathogens to be developed. <P> Approach: Using the novel oil-spray assay for biosurfactants that we have developed, initially, we will use this assay to screen isolates obtained from field-grown baby-leaf romaine lettuce, baby flat-leaf spinach, and tatsoi from field sites in northern California. We will further characterize the variety of surfactants that these strains produce such as by determining their hydrophilic-lipophilic balance (HLB) so that in the future, the effects of novel biosurfactants on human pathogens might be easily predicted based on their properties. To enable subsequent studies of the effect of surfactants themselves on neighboring human pathogenic bacteria, we will create surfactant-deficient mutants of at least one representative strain for each observed class of biosurfactant. Biosurfactant-deficient mutants and WT strains will be tested in the greenhouse, and in field experiments to determine the role of that given surfactant in plant colonization by the bacteria. We will develop reporter gene fusions to biosurfactant-producing genes to determine conditions affecting surfactant production in culture and in planta. Direct assessment of the effects of surfactants on the properties of the leaf surface will be assessed by applying droplets of water of different sizes to leaf surfaces. Tests in the greenhouse and the field will show how biosurfactant alter the behavior of human pathogens. Initial field studies will be to characterize attachment and invasion on the three distinctive edible plant species without modification of the microbial flora to provide a baseline for subsequent studies to test the impact of surfactant producers. Invasion of these inoculated plant species by human pathogens under several controlled conditions and in the field. Plants without an altered epiphytic microbial community will be inoculated with human pathogens and their subsequent ease of removal and invasion into plants will be assessed. The effects of application of extracted biosurfactants from bacteria on the behavior of the human pathogens and of commonly used agricultural surfactants will be measured. Plants will be challenge inoculated with human pathogens after the plants are treated with the various surfactants. The effect of indigenous surfactant producers on human pathogen behavior will be measured by establishing populations of a given strain on plants under field conditions and then challenging the plants by inoculating human pathogens. Invasion and ease of removal from leaves will be assessed. Plants treated with the biosurfactants and untreated controls will be harvested and exposed to human pathogens and then subjected to postharvest storage conditions and the numbers and location of the human pathogens on these plants will be compared. The overall experimental design will be to compare human pathogen mortality, invasion, and adhesion on leaves that are A) uncolonized (colonized only with small numbers of epiphytic bacteria, and few biosurfactant producers), B) colonized with large numbers of a given surfactant producer, or C) colonized by a mutant unable to produce surfactant.