Baseline survey of the anatomical microbial ecology of an important food plant: Solanum lycopersicum (tomato)

Abstract

Background: Research to understand and control microbiological risks associated with the consumption of fresh fruits and vegetables has examined many environments in the farm to fork continuum. An important data gap however, that remains poorly studied is the baseline description of microflora that may be associated with plant anatomy either endemically or in response to environmental pressures. Specific anatomical niches of plants may contribute to persistence of human pathogens in agricultural environments in ways we have yet to describe. Tomatoes have been implicated in outbreaks of Salmonella at least 17 times during the years spanning 1990 to 2010. Our research seeks to provide a baseline description of the tomato microbiome and possibly identify whether or not there is something distinctive about tomatoes or their growing ecology that contributes to persistence of Salmonella in this important food crop.

Results: DNA was recovered from washes of epiphytic surfaces of tomato anatomical organs; leaves, stems, roots, flowers and fruits of Solanum lycopersicum (BHN602), grown at a site in close proximity to commercial farms previously implicated in tomato-Salmonella outbreaks. DNA was amplified for targeted 16S and 18S rRNA genes and sheared for shotgun metagenomic sequencing. Amplicons and metagenomes were used to describe "native" bacterial microflora for diverse anatomical parts of Virginia-grown tomatoes.

Conclusions: Distinct groupings of microbial communities were associated with different tomato plant organs and a gradient of compositional similarity could be correlated to the distance of a given plant part from the soil. Unique bacterial phylotypes (at 95% identity) were associated with fruits and flowers of tomato plants. These include Microvirga, Pseudomonas, Sphingomonas, Brachybacterium, Rhizobiales, Paracocccus, Chryseomonas and Microbacterium. The most frequently observed bacterial taxa across aerial plant regions were Pseudomonas and Xanthomonas. Dominant fungal taxa that could be identified to genus with 18S amplicons included Hypocrea, Aureobasidium and Cryptococcus. No definitive presence of Salmonella could be confirmed in any of the plant samples, although 16S sequences suggested that closely related genera were present on leaves, fruits and roots.

Figure 1

Phyla associated with tomato anatomy. Phyla associated with shotgun metagenomic data using M5NR for annotation (Mg Rast version 3.2) with a maximum e-value of 1e-5 and minimum identity of 80%, over 100 bases.

Figure 2

Number of OTUs per sequences sampled and principal component gradient of unique phylogentic diversity. A. Rarefaction curves showing diversity of OTUs at 95% associated with tomato organs; roots, leaves (top and bottom), fruits and flowers. B. Gradient of unique phylogenetic diversity between bacterial communities associated with each tomato organ.

Figure 3

Bacterial diversity in roots, bottom leaves, stems, tomatoes, flowers and top leaves of tomato plants using 16SrRNA. Bacterial diversity associated with diverse tomato organs (16S).

Figure 4

Fungal diversity in roots, bottom leaves, stems, tomatoes, flowers and top leaves of tomato plants using 18SrRNA. Fungal diversity associated with diverse tomato organs (18S).

Figure 5

Tree based examination of Salmonella 16S sequences. Phylogenetic placement of putative Salmonella 16S rRNA gene sequences from different anatomical regions of tomato plants. Blue sequences are Salmonella reference samples (Additional file 2: Table S2) and red sequences are from the tomato anatomy data. A single tip label is used in instances where a clade consists of predominantly one taxa. Phylogenetic placement of putative Salmonella 16S rRNA gene sequences from different anatomical regions of tomato plants. Blue sequences are Salmonella reference samples (Additional file 2: Table S2) and red sequences are from the tomato anatomy dataset.

Figure 6

The clustering of individuals using the program STRUCTURE corroborate the phylogenetic results in that Salmonella reference samples are primarily distinct from the isolates identified as being putative Salmonella based on BLAST results (Figure5). At K = 2, the reference sequences belong to one cluster and the anatomy samples comprise the second cluster.

Figure 7

Taxonomic distribution of representative genera on the tomato plant using 16S with SitePainter. Images display the geographical location of observed genera (A) Buchnera, (B) Erwinia, (C) Pantoea, (D) Other and (E) Unassigned, on tomato plants. The sites are colored by abundance, where red represents high abundance, blue represents low abundance and purple represents medium range. The graphic was generated using 16S sequences with SitePainter [34].