Interactions between leaf lesions and the phyllosphere microbiota in leafy vegetables

Leafy vegetables (baby leaves) are considered an important source and vector for transmission of foodborne pathogens to humans. Contamination can occur from farm to fork. Although ‘rare’, contamination events have a substantial impact on public health. The shigatoxigenic bacterium, Escherichia coli O157:H7, can establish on the surface (epiphyte) and interior (endophyte) of leaves. Natural openings and leaf lesions serve as entry points, and internalised bacterial cells are shielded from rinse water and sanitisers. In this thesis, a new method for scrutinising leaf lesions in leafy vegetables was developed and used to link leaf damage to the dynamics of E. coli O157:H7gfp+ and the indigenous microbiota in the phyllosphere, and to evaluate use of calcium fortification of leafy vegetables for damage reduction. The new approach combines trypan blue dye staining of whole leaves with digital image analysis for detection and automated quantification of damage, enabling assessment of lesion size, shape and position. Number of lesions and relative lesion area were found to be crop-specific and increased along the production chain, while diversity of the leaf-associated microbial community decreased upon entry of baby leaves to the cold chain. The size of individual lesions and damaged leaf area affected the depth of invasion into plant tissue, dispersal to adjacent areas and number of culturable E. coli O157:H7gfp+ directly after inoculation. However, differences in culturable E. coli O157:H7gfp+ retrieved from leaf macerate evened out after 2 days post-inoculation (dpi). Leaf spraying with calcium decreased the number of lesions and damaged area on spinach leaves, lowering log CFU E. coli cm-2 detached at 0 and 1 dpi. Overall, the results in this thesis question the assumption that macroscopically intact leaves are free of lesions and safe. The method developed can assist in establishment of hurdles for preventing transmission of foodborne pathogens via baby leaves.