The plant cuticle contains waxes that make it difficult for microorganisms to attach to plant tissues. This layer repels water that might contain immigrating microbes. The cuticle also protects the plant from environmental stresses. For plant and human pathogen attachment, the cuticle usually must be penetrated (Beattie 2002). Damaged fruits and vegetables might harbor human pathogens more than intact fruits and vegetables. Wounded apples had a 1 log10 to 3 log10 increase in E. coli concentration 2 d after infection compared to noninjured apples (Janisiewski and others 1999a). The increase in population was greater with a small inoculum than a large inoculum. It is possible that E. coli adjusted to the microenvironment in the wounds, allowing it to reproduce more readily, especially after the first 48 h postinoculation (Janisiewski and others 1999a). Dingman (2000) also showed about a 2 log10 increase in E. coli O157 within 48 h postinoculation in damaged tissue of apples but used higher starting concentrations. There was a significant increase in pH of damaged apples as compared to similar varieties that were not damaged (Dingman 2000). On damaged honeydew melons and apples, Salmonella Enteriditis replicated at 10 °C and 20 °C. The pathogen had a greater than 5 log10 increase over the 168 h study when incubated at 20 °C on the honeydew slices (Leverentz and others 2001). We hypothesize that it is possible that damaged tissue can enhance microbial proliferation in 2 ways: first, the tissue might be more readily colonized by human pathogenic enterobacteria because of changes in the microenvironment or, alternatively, the microenvironment conditions change independent of bacterial colonization, such as through enzyme or nutrient leakage.
Wounds can also act as sites of coinfection with other microorganisms that can alter the microenvironment and stimulate proliferation of E. coli O157. E. coli O157:H7 had increased in number 3 log10 from the initial dose in wounded apple tissue in the presence of Glomerella cingulata (Riordan and others 2000). This fungal plant pathogen erodes the cuticle of apples, providing a way for E. coli O157 to enter wounds and further alter the environment to allow for proliferation (Riordan and others 2000). Brandl and Mandrell (2002), using confocal laser scanning microscopy, showed that Salmonella Thompson was in lesions on cilantro plants. There is additional evidence that human enterobacterial pathogens are more likely to be found in the presence of fungal or bacterial plant pathogens than in noninfected retail produce (Wells and Butterfield 1997, 1999).
Since E. coli O157 and Salmonella, transient species in the plant phyllosphere, proliferate to greater numbers in damaged plants than whole plants, it stands to reason that there must be an ecological disturbance on the damaged plant surface. Epiphytic microorganisms normally present in the harsh conditions of the plant phyllosphere use nutrients leached by the plant for survival and proliferation. Measuring carbon utilization can determine the structure of a microbial community in the plant phyllosphere (Wilson and Lindow 1994). Understanding the interactions in the epiphytic flora might assist in understanding how human pathogens act in the phyllosphere.
The average epiphytic aerobic heterophilic bacterial population is between 105 and 107 cfu/g of leaf (Mercier and Lindow 2000). Early studies showed approximately 2 × 106 cfu/g of leaf tissue were present on unwashed cabbage leaves (Lund 1992). Lettuce had approximately 105 cfu of aerobic bacteria/g of leaf tissue (Lund 1992). Not all microorganisms have adapted to survive in this harsh habitat. Thompson and others (1993) cultured an average of 13 bacterial species on different leaf types as compared to 40 different species in the soil where plants were grown. The most common group of epiphytic microorganisms present in the phyllosphere of edible plants is gram-negative bacteria (Lund 1992). Colonization is not spatially distributed uniformly across the entire leaf surface. Instead, bacteria cluster in specific regions. The most common areas of bacterial aggregations are located at the base of trichomes, around the stomata, and along veins in the leaves. These areas allow the best proliferation conditions for epiphytic bacteria (Beattie and Lindow 1999; Leveau and Lindow 2001). Cooley and others (2003) found E. coli O157:H7 clustered near the veins on the surface of Arabidopsis thaliana. Salmonella Thompson clustered in regions along the veins of cilantro plants. It was concluded that this region has greater wettability than other areas of the plant, thus increasing the likelihood of nutrient leaching and water availability (Brandl and Mandrell 2002). Knowing what nutrients leach and how they are made available to human pathogens can help in the development of biocontrol agents for human pathogens in fresh produce. Janisiewski and others (1999a) showed that a formula of antagonist microbes used to slow decay of stored apples also decreased E. coli O157:H7 present in wounds of apples.
The predominant sugars leached from the surface of plants are glucose, sucrose, and fructose (Mercier and Lindow 2000). Jaegar and others (1999) found rhizosphere microorganisms were in largest numbers near the root tips where the highest concentration of sucrose diffused into the soil. Plants also leach minerals, amino acids, sugar alcohols, pectic substances, proliferation hormones, and vitamins (Tukey 1970). These leachates supply the carbon and nitrogen sources for the microbial flora present on or in the plant tissue. It is possible that after the bacteria attach, they multiply in areas around these nutrient sources (Wachtel and others 2002).
The amount and type of substances leached by different plants vary considerably. Bean leaves release as much as eight times more potassium than sugar beet leaves. This, in turn, can influence how much bacterial colonization occurs on a plant. Dingman (2000) found damaged Macintosh apples inhibited the proliferation of E. coli O157 2 d to 3 d after infection, but damaged Red Delicious apples showed increases in the E. coli O157 population. It is likely that Macintosh apples released a compound that inhibited the proliferation of the human pathogen (Dingman 2000). Reinders and others (2001) showed that caffeic acid, a common phenolic compound in apples, inhibited proliferation of E. coli O157.
Very young, growing leaves release limited amounts of leachate compared to those that are near senescence (Tukey 1970). Thompson and others (1993) showed microbial communities on sugar beets were greater on old and senescent leaves than on young leaves. Jacques and Morris (1995) observed more microbial contamination on older leaves of endive than on the younger inner leaves. Plant pathogens caused more decay on these older leaves than on the young leaves, further contributing to leaching and other microenvironmental changes on the plant surface that would favor the survival and proliferation of human pathogens (Jacques and Morris 1995).
Injury to leaves also increases the amount of leaching from leaves (Tukey and Morgan 1963). There is evidence that suggests damaged tissue is more likely to positively affect the proliferation of E. coli O157 than healthy tissue (Janisiewski and others 1999a; Dingman 2000; Riordan and others 2000). The type of damage can be an important factor. Kenney and others (2001) showed no significant difference in E. coli O157 concentration on the surface of bruised or nonbruised apples, but Dingman (2000) extracted the juice from the bruised tissue and showed E. coli O157 proliferated in the nutrient solution. Janisiewski and others (1999a) found proliferation in the cut surface of an apple, as did Riordan and others (2000). Confocal laser scanning microscopy showed that E. coli O157 attached to lettuce tissue more readily at cut edges than on intact lettuce tissue (Seo and Frank 1999). It is possible that bruising alone might not cause increased proliferation, but the cracking of the surface that allows nutrient release might be needed. This could be associated with the pathogen's inability to penetrate the cuticle that is necessary for attachment (Beattie 2002).
Survival of microorganisms in the phyllosphere is often limited by availability of carbon and nitrogen sources leached from the plant. Across several vegetable samples tested, carbohydrates range from 3.4 to 74 μg/mL and amino acids range from 3.0 to 52 μg/mL in leaf exudate (Morris and Rouse 1985). Different plants do not replenish these pools at the same rate required by the microbes, so competition for nutrients is very important in epiphyte survival (Leveau and Lindow 2001). The availability of sugars is not uniform around the leaf, so when these pools are depleted, rapid proliferation of the epiphytic microorganisms usually stops. Jablasone and others (2005) determined that large counts of E. coli O157:H7 associated with alfalfa sprouts were due to nutrients in the exudate released by emerging seedlings. A decrease in bioluminescence over time was likely associated with changes in the nutrient patterns of the plant exudate (Jablasone and others 2005).
The type and amount of exudates released by plants are limiting factors in the survival and proliferation of epiphytic organisms. The ability of leaf surface organisms to find and use these nutrient sources is a very important determinant of epiphytic fitness. For resident epiphytes, the limiting factor for proliferation is nitrogen (Lindow 1991). It is not known if nitrogen is a limiting factor for enteric pathogens because as a casual species to the phyllosphere, human pathogenic bacteria might use carbon and nitrogen sources not commonly used by epiphytes. It is necessary to know what extracts are being released from the plant that might give these enteric pathogens a competitive advantage when they are present on produce associated with outbreaks.
Spatial experiments have shown that epiphytic bacteria grow better in areas where there is high wettability of the tissue. The population size and distribution of phyllosphere microorganisms might be altered by the amount of usable water present on the leaf surface (Bunster and others 1989). There is also evidence that high relative humidity increases bacterial communities on plant surfaces (Leben 1988), so besides nutrients, water is a factor in bacterial proliferation in the phyllosphere. Cooley and others (2003) found at 100% humidity and in the absence of competition, E. coli O157 and Salmonella enterica grew to as much as 107 cfu/g of leaf tissue in the phyllosphere of Arabidopsis thaliana.
Chet and others (1973) observed Pseudomonas lachrymans was attracted by chemotaxis to water droplets from different plant sources. Some plant epiphytes can release biosurfactants that changes leaf wettability in a way that makes it easier for these organisms to use water. It has also been demonstrated that even after these biosurfactant-producing organisms have become less dense on the leaf surface, the biosurfactant activity continues; thus water availability is still increased for remaining bacteria (Bunster and others 1989). This is an important food safety issue because plants that have been previously diseased by pathogens capable of producing biosurfactants have increased leaf wettability. This increased wettability favors proliferation of enteric pathogens that contact these plants.
A competitive advantage can be gained by an organism that grows quickly, thus establishing dominance when nutrient levels are high or by being able to grow when there are few nutrients remaining. Competitors who have highly efficient modes of nutrient uptake or ability to produce antimicrobial compounds have a competitive advantage (Beattie and Lindow 1994). Schuenzel and Harrison (2002) found 3% of epiphytes isolated from produce contained inhibitory compounds that acted against 1 or more of the following pathogens: Staphylococcus aureus, E. coli O157, Salmonella Montevideo, and Listeria monocytogenes. Isolates from shredded lettuce were most likely to produce inhibitory compounds that were effective against all 4 pathogens. Most of the inhibitory epiphytes were gram-negative, with the highest percentage being pseudomonads (Schuenzel and Harrison 2002).
If organisms use different carbon and energy sources, microbial populations might actually coexist in the phyllosphere and might enhance the epiphytic fitness of other populations. In transgenic plants that released excess opines, opine-catabolizing pseudomonads coexisted with other pseudomonads that used different carbon sources. In the wild-type variety, these 2 microorganisms would normally compete for nutrients (Wilson and others 1995). Wilson and Lindow (1995) used salicylate as the sole carbon source for one of the Pseudomonas strains so that it did not compete with a known competitor and found similar nutrition partitioning between the species. Wilson and Lindow (1994) hypothesized that coexistence could occur between epiphytes that have different nutrient resource patterns. If 2 or more organisms use different carbon sources, they all might be able to survive in the same region of the leaf surface. Most cohabitation of microbes is not complete coexistence (reaching same population sizes with or without the other species) or complete competition (sharing the same nutrients), but a coexistence of microbes between the 2 extremes (Wilson and Lindow 1994). It is evident that finding what nutrients are available and how they are utilized by human pathogens is essential in devising a strategy to control human pathogens in the phyllosphere by use of biocontrol agents.
Plant lesions in which E. coli O157 and other pathogens proliferate might be a source of rich nutrients (Dingman 2000; Riordan and others 2000). High concentrations of Pseudomonas syringae acted as an antagonist to E. coli O157 in these wounds, possibly competing for the same carbon and energy sources. When P. syringae was not present, E. coli O157 had at least a 2 log10 increase in concentration compared to a coinoculation (Janisiewski and others 1999b). Liao and Fett (2001) found 6 out of 120 resident epiphytes inhibited the proliferation of at least 1 of the following: E. coli, L. monocytogenes, Salmonella Chester, or Erwinia carotovora. Two Pseudomonas species reduced the proliferation of both E. coli and L. monocytogenes (Liao and Fett 2001). These organisms were found to be present in the phyllosphere of different types of vegetables. Enterobacter asburiae competed with E. coli O157 and Salmonella enterica (Cooley and others 2003). Janisiewski and others (1999b) found no evidence of compounds produced by Pseudomonas that inhibited E. coli O157. Pectin-degrading epiphytes of bell peppers, romaine lettuce, and baby carrots did not inhibit proliferation of human pathogens (Liao and Fett 2001). Some strains of E. coli might also participate in pectin degradation or enhance the ability of other organisms to degrade pectin. Dongowski and others (2000) showed increased pectin degradation when Bacteroides thetaiotaomicron (a known pectin-degrading microbe) was mixed with E. coli than when B. thetaiotaomicron was alone in culture. While some residents of the phyllosphere act as antagonists of enteric pathogens, some might actually have neutral or symbiotic association with these types of casual residents.