The presence, size and importance of bacterial communities on plant leaf surfaces are widely appreciated. However, information is scarce regarding their composition and how it changes along geographical and seasonal scales. We collected 106 samples of field-grown Romaine lettuce from commercial production regions in California and Arizona during the 2009-2010 crop cycle. Total bacterial populations averaged between 10(5) and 10(6) per gram of tissue, whereas counts of culturable bacteria were on average one (summer season) or two (winter season) orders of magnitude lower. Pyrosequencing of 16S rRNA gene amplicons from 88 samples revealed that Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria were the most abundantly represented phyla. At the genus level, Pseudomonas, Bacillus, Massilia, Arthrobacter and Pantoea were the most consistently found across samples, suggesting that they form the bacterial 'core' phyllosphere microbiota on lettuce. The foliar presence of Xanthomonas campestris pv. vitians, which is the causal agent of bacterial leaf spot of lettuce, correlated positively with the relative representation of bacteria from the genus Alkanindiges, but negatively with Bacillus, Erwinia and Pantoea. Summer samples showed an overrepresentation of Enterobacteriaceae sequences and culturable coliforms compared with winter samples. The distance between fields or the timing of a dust storm, but not Romaine cultivar, explained differences in bacterial community composition between several of the fields sampled. As one of the largest surveys of leaf surface microbiology, this study offers new insights into the extent and underlying causes of variability in bacterial community composition on plant leaves as a function of time, space and environment.

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The Food Safety Modernization Act (FSMA) includes a time-to-harvest interval following the application of non-compliant water to pre-harvest produce to allow for microbial die-off. However, additional scientific evidence is needed to support this rule. This study aimed to determine the impact of weather on the die-off rate of E. coli and Salmonella on spinach and lettuce under field conditions. Standardized, replicated field trials were conducted in California, New York, and Spain over two years. Baby spinach and lettuce were grown and inoculated with a ∼104 CFU/mL cocktail of E. coli and attenuated Salmonella. Leaf samples were collected at 7 timepoints (0-96h) following inoculation; E. coli and Salmonella were enumerated. The associations of die-off with study design factors (location, produce type, and bacteria) and weather were assessed using log-linear and biphasic segmented log-linear regression. A segmented log-linear model best fit die-off on inoculated leaves in most cases, with a greater variation in the segment 1 die-off rate across trials [-0.46 (95% confidence interval (95% CI): -0.52, -0.41) to -6.99 (95% CI: -7.38, -6.59) log10 die-off/day] compared to the segment 2 die-off rate [0.28 (95% CI: -0.20, 0.77) to -1.00 (95% CI: -1.16, -0.85) log10 die-off/day]. A lower relative humidity was associated with a faster segment 1 die-off and an earlier breakpoint (the time when segment 1 die-off rate switches to the segment 2 rate). Relative humidity was also found to be associated with whether die-off would comply with FSMA's specified die-off rate of -0.5 log10 die-off/day. Importance The log-linear die-off rate proposed by FSMA is not always appropriate, as the die-off of foodborne bacterial pathogens and specified agricultural water quality indicator organisms appear to commonly follow a biphasic die-off pattern with an initial rapid decline followed by a period of tailing. While we observed substantial variation in the net culturable population levels of Salmonella and E. coli at each time point, die-off rate and FSMA compliance (i.e., at least a 2 log10 die-off over 4 days) appear to be impacted by produce type, bacteria, and weather; die-off on lettuce tended to be faster than that on spinach, die-off of E. coli tended to be faster than that of attenuated Salmonella, and die-off tended to become faster as relative humidity decreased. As such, the use of a single die-off rate for estimating time-to-harvest intervals across different weather conditions, produce types, and bacteria should be revised.