Characterization of endophytic microbial communities in store-bought kale evaluated by different plant tissue homogenization methods

Endophytic microorganisms live in intercellular and vascular spaces of plants and span the continuum from beneficial to pathogenic in both plants and animals. Increasing human consumption of fruits and vegetables has placed an emphasis on identifying and studying those microbes that colonize the internal tissues of plants, with a goal of limiting populations of enteric pathogens in store-bought foods meant for raw consumption, such as leafy greens. Culture-independent (i.e., metagenomic) methods are increasingly used to obtain an accurate snapshot of plant microbial communities, yet technical hurdles limit the accuracy and throughput of these methods. This includes the low-throughput nature of plant tissue maceration, and the prevalence of plant plastid DNA in metagenomic DNA extracts, which is typically coamplified via PCR strategies that target the bacterial 16S rRNA gene. In this study, we use kale (Brassica oleracea) as a model to explore the leafy green endophytic microbiome and to compare how two tissue maceration techniques used in traditional endophyte research compare in culture-independent microbiome studies using the Illumina Miseq platform. Three different brands of store-bought kale were surface sterilized and subjected to two tissue maceration strategies: enzyme digestion and blender processing. Analysis of 16S rRNA gene amplicon libraries revealed two highly abundant operational taxonomic units present in all libraries, one classified to the genus Pseudomonas and one to the family Enterobacteriaceae. Community structure and membership were highly similar among brands and between tissue maceration strategies, suggesting that both enzyme digestion and blender processing are suitable methods. Nonetheless, enzyme digestion may increase sample throughput and minimize steps involved in sample processing.