Season, irrigation, leaf age, and Escherichia coli inoculation influence the bacterial diversity in the lettuce phyllosphere

PLoS One. 2013 Jul 2;8(7):e68642. doi: 10.1371/journal.pone.0068642. Print 2013.


The developmental and temporal succession patterns and disturbance responses of phyllosphere bacterial communities are largely unknown. These factors might influence the capacity of human pathogens to persist in association with those communities on agriculturally-relevant plants. In this study, the phyllosphere microbiota was identified for Romaine lettuce plants grown in the Salinas Valley, CA, USA from four plantings performed over 2 years and including two irrigation methods and inoculations with an attenuated strain of Escherichia coli O157:H7. High-throughput DNA pyrosequencing of the V5 to V9 variable regions of bacterial 16S rRNA genes recovered in lettuce leaf washes revealed that the bacterial diversity in the phyllosphere was distinct for each field trial but was also strongly correlated with the season of planting. Firmicutes were generally most abundant in early season (June) plantings and Proteobacteria comprised the majority of bacteria recovered later in the year (August and October). Comparisons within individual field trials showed that bacterial diversity differed between sprinkler (overhead) and drip (surface) irrigated lettuce and increased over time as the plants grew. The microbiota were also distinct between control and E. coli O157:H7-inoculated plants and between E. coli O157:H7-inoculated plants with and without surviving pathogen cells. The bacterial inhabitants of the phyllosphere therefore appear to be affected by seasonal, irrigation, and biological factors in ways that are relevant for assessments of fresh produce food safety.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Agricultural Irrigation / methods*
  • DNA, Bacterial / chemistry
  • DNA, Bacterial / genetics
  • Enterobacteriaceae / classification
  • Enterobacteriaceae / genetics
  • Enterobacteriaceae / physiology
  • Escherichia coli O157 / classification
  • Escherichia coli O157 / genetics*
  • Escherichia coli O157 / physiology
  • Genetic Variation
  • Host-Pathogen Interactions
  • Humans
  • Humidity
  • Lettuce / growth & development*
  • Lettuce / microbiology
  • Microbiota / genetics
  • Microbiota / physiology
  • Phylogeny
  • Plant Leaves / growth & development*
  • Plant Leaves / microbiology
  • RNA, Ribosomal, 16S / genetics
  • Seasons*
  • Sequence Analysis, DNA
  • Temperature


  • DNA, Bacterial
  • RNA, Ribosomal, 16S

Grant support

This project was partially supported by the Agriculture and Food Research Initiative Grant 2010-65201-20572 from the USDA National Institute of Food and Agriculture ( This project was also supported by The Center for Produce Safety (, the California Lettuce and Leafy Greens Research Board (, and the U.S. Food and Drug Administration, Project No. U01-002-527 ( The funders had no role in study design, data collections and analysis, decision to publish, or preparation of the manuscript.