**2. Leafy greens are an easy target for contamination with pathogens: mechanisms of microbial contamination**

#### **2.1 General considerations**

Leafy greens are known as an important vector for microbial hazards responsible for foodborne outbreak illnesses and almost 20% of leafy greens contamination with pathogens takes place on-farm [19, 20]. In leafy greens, *E. coli* O157:H7 is found to be more frequent than other pathogens due to its ability to contaminate mostly via biofilm formation on the produce surface which could explain the large number *E. coli* O157:H7 related outbreaks [21]. The on-farm fate of enteric pathogens on leafy greens depends on multiple conditions that the pathogenic bacteria are facing in the soil-produce environment, and on the pathogen's ability and strategies to survive and contaminate the fresh produce, such as biofilm formation or internalization. In the preharvest stage, due to the pathogen-produce interaction pathways and mechanisms, some of the pathogens could become endopathogenic in leafy greens—a stage which raises serious food safety concerns since the post-harvest decontamination treatments have almost a null chance to reduce the numbers of viable cells to a harmless level [22]. The "*points-of-entry*" used by pathogens to contaminate the leafy greens are the plant's rhizosphere and/or phyllosphere. Due to its richness in nutrients (root exudates including compounds released as a consequence of root cell metabolism or after lysis of plant cells), the root zone (rhizosphere) is an excellent environment for pathogens and it could support the presence of 106 to 109 bacteria per gram of roots [23]. On the phylloshere, *E. coli* O157:H7 is capable of attaching on these plant's parts, and can remain viable for weeks to months, and even multiply if the environmental conditions are favorable (i.e., warm temperatures, high humidity, adequate nutrients, plant's leaves' characteristics and integrity) [21]. Compared to the rhizosphere, the leaves surface nutrients are scarce. The nutrients found on leaves, probably originating from mesophyll and epidermal cell exudates that leak onto the leaves surface from wounds and broken trichomes, are not distributed homogenously. Since the phyllosphere is subjected

to many stress factors which can have rapid fluctuations will affect the bacterial survival: temperature, solar radiation and humidity, phyllospheres typically could support fewer than 103 to 107 pathogen per gram of leaf [19]. Therefore, understanding the pathogen contamination pathways and mechanisms will provide important information to fresh produce growers for either adopting preventive actions or protecting their produce during the pre-harvest stage.
