This study provides the first next-generation sequencing survey of the bacterial community in the tomato fruit surface. As such it has confirmed the presence of taxa previously found to inhabit the phyllosphere of this crop species, as well as identified many others not yet encountered in this environment. The three most abundant bacterial classes in the tomato fruit surface environments compared in this study were Gamma, Alpha and Betaproteobacteria. These were also found in higher abundance in the phyllosphere of other plant species, although the relative abundances for these classes vary [16–18, 27]. Genera here found in high abundance in the tomato fruit surface, such as Pantoea and Enterobacter, are also abundant in the phyllosphere of certain Atlantic rainforest tree species and cottonwood, indicating a wide distribution across different plant species [16, 18]. Bacterial genera found in our 2009 fruit surface samples were also identified among the culturable bacteria on leaves of field-grown tomatoes, including Pseudomonas, Pantoea, Sphingomonas, Massilia, Xhantomonas and Curtobacterium . Two additional genera, Burkholderia and Leuconostoc, showed high abundance in our study. Burkholderia was the most abundant genus in our groundwater samples, representing 75% of the sequences, and might have been introduced in the environment through groundwater applications. Leuconostoc has been previously described as the predominant lactic acid bacteria on tomato fruit surfaces .
Similar bacterial classes and genera were found in high abundance in samples collected in 2008 and 2009, with the largest differences corresponding to the unclassified sequences. Several different reasons could account for this variation, including differences in DNA extraction, sequencing sample preparation and primers used in both years, as well as potential growing season effects.
Of special interest is the high proportion of sequences identified as Enterobacteriaceae, given that this family includes important human pathogenic bacteria like Salmonella and E. coli. Similar representation of this family was obtained in the phyllosphere of Trichilia spp. and Pinus ponderosa, but not in that of Campomanesia xanthocarpa [16, 27]. The high adaptability of this family to the tomato fruit surface environment might be associated to the higher risk of disease outbreaks associated with this crop.
Differences between fruit surface environments do not appear to be linked to the water applications, indicating that plant conditions allow for only some of the bacterial groups present in water to establish themselves. Similar results were obtained when the fruit surface communities living on apple trees under conventional and organic management were compared, where only low abundance groups differed between the two environments . Similarly, no effect on the levels of fecal and total coliforms was observed when reclaimed water with higher coliform counts, and well water were sprayed on six horticultural crops .
Several factors determine whether the microorganisms arriving on the leaf surface can become established, including leaf characteristics, environmental factors and properties of the microorganisms themselves . Pesticides are known to differentially impact bacterial survival and growth. In a study conducted to determine the effect of pesticides on bacterial survival, Salmonella spp. were best able to survive and Listeria spp. were least able to survive in pesticide solutions, among all the bacteria tested. Bravo, the fungicide applied closest to the sampling date in this study, has been found to reduce bacterial growth, although it was less inhibitory than other products tested . The addition of pesticides to the different water sources used in this study might have reduced bacterial community differentiation in the two resulting fruit environments. The smooth texture of tomato skin may also prevent attachment and result in bacteria being washed away by rain or spray water.
Although our results point to the lack of major effects of the two water sources used for pesticide applications, confirming this at the species level for human enteric pathogens such as Salmonella, would be crucial for establishing the potential safety of surface water use for contact applications. In addition, our sampling depth analysis suggests that deeper sampling is needed for all the environments, but especially for the more diverse ws, to capture at least 90% of the community members
Recent studies of analysis methodologies in bacterial diversity and metagenomics projects have revealed that small modifications or substitution of similar tools may potentially result in significant changes in the overall biological conclusions [35–37]. In the rapidly evolving field of genomics, there are few concrete standards, and the sophisticated computational protocols being developed certainly will always be sensitive to some uncertainty in the analysis parameters. To examine the sensitivity of our results to the methodology employed, we re-ran our analysis using two parallel 16S rRNA protocols from the CloVR package and found large agreement with our major results. Additionally, the 454 platform itself has ongoing issues regarding artificial replicate generation  and homopolymer identification errors , both of which contribute to overestimation of species-level diversity in 16S rRNA-based studies. Though it is likely that our estimates of absolute species-level diversity are indeed inflated, the consistency in relative diversity differences between samples across multiple analyses is encouraging and lends support to the validity of our initial computational results and final biological and ecological conclusions.