Molecular Analysis of Endophytic Bacteria Associated with Lettuce.

Bacteria associated with lettuce and spinach have been linked to outbreaks of infectious diseases in humans. While studies have examined the culturable bacteria residing on lettuce surfaces, few have evaluated the non-culturable bacteria within the plant. This study used molecular techniques to examine the bacterial community present in five different salad greens (green leaf lettuce, red leaf lettuce, romaine lettuce, iceberg lettuce, and baby spinach). Both conventionally and organically grown samples of each salad type were examined, and samples were processed as purchased and following surface sterilization. DNA was extracted from homogenized samples and polymerase chain reaction (PCR) was used to amplify bacterial 16S rRNA gene fi-agments. Co-amplified plant mitochondrial DNA was excluded and PCR products reamplified to obtain only bacterial DNA. These amplification products were subsequently amplified with bacterial specific primers suitable for denaturing gradient gel electrophoresis (DGGE), which was used to initially examine differences in bacterial community structure across samples. Purified 16S rRNA gene amplifications were also cloned into plasmid vectors to generate clone libraries for the surface sterilized samples. Clones from each library were sequenced to identify dominant endophytic bacteria within each leaf vegetable type. Based on bacterial band intensities, the largest amount of bacterial DNA was obtained fi-om baby spinach, which also gave the largest number of bacterial species identified. This suggests that baby spinach may be a more nutrient rich environment for bacterial growth than the other lettuce varieties. DGGE showed various differences between samples, although sterilized and unsterilized samples of regularly grown green leaf lettuce had identical bacterial community structure, suggesting that the bacteria associated with this plant are primarily endophytes. Across all samples, the most dominant bacteria were unclassified species of Pseudomonas, Stenotrophomonas and Ralstonia, as well as Ralstonia solanacearum. These bacteria are primarily related to plant diseases, either as a causal agent or as a potential biological control mechanism for improved plant survival and growth. This study concluded that the endophytic bacteria associated with salad greens are essentially plant-related and may function in both the survival as well as the infection of these lettuces.