Article Figures & Data
- FIG 1
Characterization of the phyllosphere microbiota on laboratory-grown, field-grown, and laboratory-acclimated lettuce. (A, B, and C) Romaine lettuce cultivar Braveheart germinated in Salinas Valley soil in a controlled environment with field-like humidity and temperature conditions was used to compare the phyllosphere communities on 6-week-old plants to those on field-grown lettuce at the same developmental stage. (D) Whole lettuce plants collected from the field 2 weeks postgermination were acclimated to laboratory conditions for 2 weeks, and the bacteria on those plants were compared to the bacteria on laboratory-grown plants and untreated field plants. (A) Bacteria on laboratory- and field-grown lettuce were enumerated on TSA and by real-time PCR quantification of 16S rRNA gene numbers. (B) Averages ± standard errors of weighted and unweighted UniFrac community distances of the bacterial communities on field and laboratory lettuce as identified by 454 pyrosequencing. (C) Averages ± standard errors of the relative proportions of the top 10 families identified from 454 pyrosequencing of the bacterial 16S rRNA genes. (D) Principal coordinate analysis using weighted UniFrac community distances. An asterisk indicates significance by Student’s t test (**, P ≤ 0.01; *, P ≤ 0.05).
- FIG 2
Phyllosphere microbiota transplantation with fresh and cryopreserved microbial communities. The microbiota associated with field-grown plants was inoculated directly or after cryopreservation into the phyllosphere of laboratory-grown plants, and the resulting communities were quantified on TSA and identified by pyrosequencing. (A, C and E) Culturable cell amounts were enumerated on TSA agar on either control plants or plants inoculated with freshly collected field microbiota from 2011 (A) or 2012 (E) or cryopreserved cells from the same field plants used in 2011 (C). Each time point shows the average value ± standard error for 3 plants. (B, D, and F) The bacterial communities on field plants in 2011 (B and D) and 2012 (F) and on laboratory-grown transplanted plants were compared to those on the laboratory-grown control plants by principal coordinate analysis using the weighted UniFrac metric. Freshly collected microbiota from 2011 (B) or 2012 (F) or cryopreserved cells from 2011 (D) were applied. (B and D) Open symbols represent plants sampled before any treatment was applied. (F) Other field plants collected at the same time are included. An asterisk indicates significance by Student’s t test (**, P ≤ 0.01; *, P ≤ 0.05).
- FIG 3
UPGMA comparison of transplantation techniques. A phylogenetic tree using the weighted UniFrac distances was created in QIIME using sequences belonging to untreated laboratory-grown lettuce (C), laboratory-grown plants dipped in buffer (C_Fr) or buffer plus glycerol (C_Gly), untreated field-grown lettuce not used for microbiota transplantation (F), and laboratory-grown plants sampled 48 h after being transplanted with a fresh (direct) T1_2011 field community (T_Fr) or a glycerol-preserved T1_2011 field community (T_Gly). One of the communities from the “C” treatment was designated the outgroup. The phylogenetic tree was visualized and modified (to display as a cladogram and to add color to the branches) in FigTree version 1.4.1 (http://tree.bio.ed.ac.uk/).
- FIG 4
The effect of E. coli O157:H7 on phyllosphere bacteria. Cryopreserved microbiota from the T1_2011 or T2_2011 field plants collected from the field trial in 2011 was applied to laboratory-grown lettuce prior to E. coli O157:H7 inoculation; microbiota from these plants are referred to as T1_Ec and T2_Ec and compared to microbiota of laboratory-grown plant controls also inoculated with E. coli O157:H7 (Control_Ec). All DNA sequences classified as Escherichia were removed prior to analysis to show the changes in the diversity of the other bacteria present. (A) Principal coordinate analysis using the weighted UniFrac metric comparing the phyllosphere communities on T1_Ec, T2_Ec, and Control_Ec plants 4 days posttransplantation (open symbols) to the corresponding transplanted plants or untreated controls 2 days posttransplantation without any exposure to E. coli O157:H7, which are referred to as T1, T2, and Control (closed symbols). (B) The relative abundances of bacterial genera in the phyllosphere of the field plants and the laboratory-grown plants with the applied field communities with and without the E. coli O157:H7 inoculant. The numbers directly above the time point information for the E. coli-inoculated plants correspond to the plant numbers in Table S1 in the supplemental material.
Supplemental Material
Figure S1
Description of the Romaine lettuce plants used in this study. For each experiment, the cultivar, the age of plant when harvested, the soil that was used, and the figures showing results associated with those plants are provided. The top two boxes represent the field plants collected, and the other boxes describe the laboratory-grown lettuce. Download Figure S1, TIF file, 12.6 MB.
Copyright © 2014 Williams and Marco
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S2
Proportions of bacterial phyla on laboratory- and field-grown lettuce. (A and B) Bacterial phyla on field-grown (A) and laboratory-grown (B) lettuce. The average bacterial distributions on 6 plants are shown in both panels. (C) Average relative proportions ± standard errors of the top 17 genera on laboratory and field-grown plants. An asterisk indicates significance by Student’s t test (**, P ≤ 0.01; *, P ≤ 0.05). Download Figure S2, TIF file, 12.9 MB.
Copyright © 2014 Williams and Marco
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S3
Bacterial diversity and abundance on field-grown, laboratory-acclimated, and laboratory-grown lettuce. (A) Bacteria were enumerated on TSA for 8 laboratory-acclimated plants (Acclimated), 8 age-matched field plants (Field), and 6 laboratory-grown (Laboratory) plants. Whiskers show the minimum and maximum values for each group of plants. An asterisk indicates significance by Student’s t test (*, P ≤ 0.05). (B) Average ± standard error of the observed phylogenetic diversity, a measure of the overall diversity within a community, on 6 plants for each group. An asterisk indicates significance by Mann-Whitney direction test (*, P ≤ 0.05). (C) Relative proportions of sequences classified at the phylum level for each growth environment (n = 6). The four dominant phyla are shown, and the remaining phyla are indicated as “Other.” (D) Classification of the sequences at the class level. The average bacterial distribution on 6 plants per group is shown. The top 6 classes are shown, and the remaining classes are indicated as “Other.” Download Figure S3, TIF file, 16 MB.
Copyright © 2014 Williams and Marco
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S4
Proportions of bacterial families on field plants used for transfer experiments. The community composition of the four field plants used in the transfer experiments was determined by pyrosequencing and classification of the sequences at the family level. The T1_2011 and T2_2011 plants were collected from an agricultural field in the Salinas Valley, CA, USA, in June 2011. T1_2012 and T2_2012 plants were collected from the same field in September 2012. Download Figure S4, TIF file, 8.9 MB.
Copyright © 2014 Williams and Marco
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S5
Phylogenetic diversities on lettuce from the field and laboratory-grown lettuce with or without a transplanted field microbiota. The averages ± standard errors of the phylogenetic diversities (PD) are shown for field-grown plants from the 2012 field and for laboratory-grown plants with and without a field community inoculation from T_2012 plants. The PD measure for the transplanted laboratory plants constitutes the average of the PD for plants collected at 2 and 7 d.p.i. An asterisk indicates significance by Mann-Whitney directional test (*, P ≤ 0.05). Download Figure S5, TIF file, 11.3 MB.
Copyright © 2014 Williams and Marco
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S6
Bacterial diversity in the phyllosphere after whole-community transplantation. Log-transformed relative proportions of the 16S rRNA gene sequences classified at the genus level. The averages ± standard errors of bacterial proportions are shown (n = 3). (A) Proportions of bacterial genera on plants after the fresh transfer of phyllosphere microbiota from lettuce collected from the field in early 2011 (T1_2011 and T2_2011) to laboratory-grown plants and on uninoculated lettuce plant controls. (B) Proportions of bacterial genera after cryopreserved cell transfer of the T1_2011 microbiota and on uninoculated lettuce plant controls. (C) Proportions of bacterial genera on plants after the fresh transfer of phyllosphere microbiota from lettuce collected from the field in late 2012 (T1_2012 and T2_2012) to laboratory-grown plants and on uninoculated lettuce plant controls. Download Figure S6, TIF file, 12.4 MB.
Copyright © 2014 Williams and Marco
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S7
Escherichia coli O157:H7 abundance on Romaine lettuce. Two days prior to inoculation of E. coli O157:H7, laboratory-grown Romaine lettuce was inoculated with the frozen communities from T1_2011 or T2_2011. The control group contained plants that had been submerged in buffer supplemented with glycerol. Escherichia coli O157:H7 cell numbers were enumerated on TSA supplemented with rifampin 0 and 2 h after E. coli O157:H7 inoculation (hpi) and 2 and 7 days postinoculation (d.p.i.). The detection limit for E. coli O157:H7 was approximately log 1.5 CFU/g plant. Download Figure S7, TIF file, 9.6 MB.
Copyright © 2014 Williams and Marco
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Table S1
Abundances of Escherichia coli sequences as determined by culturing and pyrosequencing. Table S1, DOCX file, 0.1 MB.
Copyright © 2014 Williams and Marco
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Additional Files
Supplementary Data
Supplementary Data
Files in this Data Supplement:
- Figure sf01, TIF - Figure sf01, TIF
- Figure sf02, TIF - Figure sf02, TIF
- Figure sf03, TIF - Figure sf03, TIF
- Figure sf04, TIF - Figure sf04, TIF
- Figure sf05, TIF - Figure sf05, TIF
- Figure sf06, TIF - Figure sf06, TIF
- Figure sf07, TIF - Figure sf07, TIF
- Table st1, DOCX - Table st1, DOCX
