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

Thomas R Williams,Maria L Marco,Anne-Laure Moyne,Linda J Harris,A Mark Ibekwe

doi:10.1371/journal.pone.0068642

Abstract

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.

Highlights

The phyllosphere, or total above-ground surfaces of plants, is a habitat for a variety of microorganisms [1]
E. coli O157:H7 persistence and total viable bacterial cell densities were measured until 28 dpi, corresponding to when the plants reached the size typically harvested for human consumption
The bacterial inhabitants of the Romaine lettuce phyllosphere were identified for plants grown in the same field during different times of year over 2 years and exposed to distinct abiotic and biotic (E. coli O157:H7) conditions

Summary

Introduction

The phyllosphere, or total above-ground surfaces of plants, is a habitat for a variety of microorganisms [1]. Leafy green produce has been associated with numerous large outbreaks of foodborne illness and is regarded to be a significant vector of human pathogens [10]. Many of these outbreaks were traced back to the Salinas Valley, CA, USA, where more than 70% of U.S.-produced lettuce is grown. Field-based studies investigating E. coli O157:H7 population dynamics on lettuce found that the pathogen does not typically colonize plants in high levels but can persist in low numbers, on a fraction of plants, long after inoculation [13,14,15,16]. Because the infective dose of E. coli O157:H7 is as few as 10 cells, even a low amount of the pathogen is considered a threat for human infection [17,18]

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