The leaf microbiota has major influences on the quality of ready-to-eat lettuce. While studies investigating the epi- and endophytic microbiota of lettuce have been published, no protocols focusing only on the epiphytic microbiota exist. As the epiphytic microbiota may be especially influenced by technological steps in the production of ready-to-eat lettuce, an in-depth knowledge of these microorganisms is essential with regard to consumer safety and spoilage. Currently it is not clear to what extent results gained from single samples are representative of the community composition. A technique for the separation of bacterial cells from the leaf surface was applied to green oak leaf lettuce. The bacterial diversity was analysed in triplicate with high throughput Roche 454 sequencing of prokaryotic 16S rRNA genes to analyse the intra-sample variation. Sequence analysis revealed members of the phyla Acidobacteria, Actinobacteria, Bacteroidetes, Firmicutes, Gemmatimonadetes, Proteobacteria and Verrucomicrobia, and of the candidate division WYO. The ten most abundant proteobacterial genera in all three samples were Alkanindiges (24.6%), Pseudomonas (11.3%), Sphingomonas (8.6%), Janthinobacterium (8.3%), Acinetobacter (4.3%), Polaromonas (1.3%), Erwinia (1.1%), and Methylobacterium (1.1%). The genera Pedobacter (2.5%) and Hymenobacter (1.4%) dominated the phylum Bacteroidetes. The intra-sample variation was less than 0.7% for seven of these most abundant genera with the exception of Pseudomonas, Janthinobacterium and Alkanindiges, where larger standard deviations were obtained. This low intra-sample variation demonstrates that the established technique based on oak leaf lettuce is suitable for the culture-independent analysis of the epiphytic bacterial microbiota of produce.
Oak leaf lettuce; microbiota; biodiversity; next generation sequencing; Alkanindiges