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SUPPLEMENTARY INFORMATION FOR
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Nicotiana roots recruit rare rhizosphere taxa as major root-inhabiting microbes
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Muhammad Saleem1, Audrey D. Law1 and Luke A. Moe1*
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Muhammad Saleem: m.saleem@uky.edu; Audrey Law: audrey.law@uky.edu; Luke Moe:
luke.moe@uky.edu
Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY, USA, 40546-0312
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Materials and Methods
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Plant growth and root sampling
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Two commercial burley tobacco (Nicotiana tabacum) varieties (KT204LC and NCBH129LC)
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were grown at the University of Kentucky Spindletop Farm (Lexington, KY) in the summer of
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2013 using standard agronomic practices (Pearce et al., 2015). Briefly, seeds were germinated in
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a dedicated greenhouse and seedlings were transplanted to field plots in June 2013. The plants
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were grown in a plot that was used for burley tobacco in the previous growing season. At
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tobacco harvest, the vegetative portion of the plants was removed for curing, and the rhizosphere
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and root samples (n=6) were taken from six plants of each variety. The root and associated soil
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was removed using a spading fork. After gently shaking the roots to remove loosely adhered soil,
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the soil remaining attached to the root (rhizosphere soil) was physically removed by hand, using
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sterile latex gloves, into a sterile bag. The remaining root mass was put into a sterile bag and all
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samples were subsequently stored at -80C until processing. Soil data is listed in Table S1.
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Isolation of bacteria from plant roots
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Roots were washed with distilled water to eliminate any remaining soil. The larger roots linked
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with the stem were removed and are considered primary roots. Roots emerging from the primary
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roots were removed and are considered secondary roots. Fine roots were collected from
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secondary roots. Cutting of the roots was done using a sterilized blade. Bacteria were isolated
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from plant tissues following a previously established method (Ikeda et al., 2009) with some
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modifications. About 20 g of fresh root composite sample was taken from each root portion
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(main root, secondary roots and fine roots). The root samples were cut into small pieces and
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added to to a 1 L blender jar (Kinematica Microtron MB 550) with 400 ml BCE buffer (50 mM
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Tris-HCl, pH 7.5, 1% Triton X-100, 2 mM 2-mercaptoethanol added right before blending).
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Samples were blended for 2 min at full speed, followed by 5 min incubation on ice, for a total of
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three cycles. The homogenized samples were filtered through a sterile Mira cloth using a
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Buchner funnel. The resulting filtrate was centrifuged at 500  g for 5 min at 10C. The
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supernatant was centrifuged again at 5,500  g for 20 min at 10C. The supernatant was then
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discarded and the pellet resuspended in 250 ml BCE, and centrifuged at 10,000  g for 10 min at
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10C, for a total of two times. The final pellet was resuspended in 6 ml Tris Buffer (50 mM, pH
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7.5). The suspension was gently overlayed onto 4 ml nycodenz (8g nycodenz in 10 ml Tris
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buffer) in a 15 ml glass tube. The glass tube was centrifuged at 10,000  g for 40 min at 10C. A
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band containing the bacterial cells formed at the interface of buffer and nycodenz was removed
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with a pipette and transferred into a 2 ml microcentrifuge tube with an equal volume of deionized
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sterile water. After centrifugation at 21,130  g for 5 min, the supernatant was removed and the
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pellet was stored at -20C.
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Genomic DNA purification
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Genomic DNA purification followed the protocol of Wilson (2001). The frozen microbial
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pellets isolated from plant roots were dissolved in 567 l of TE buffer, 30 l of 10% SDS and 3
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l of proteinase K (20mg/ml) were added, and the mixture was incubated at 37C for one hour.
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100 l of 5 M NaOH and 80 l of a CTAB/NaCl solution (41 g NaCl and 100 g CTAB per liter
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of solution) was added before another incubation for 10 min at 65C. An equal volume of
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chloroform/isoamyl alcohol (24:1) was used to extract the lysate, followed by extraction with an
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equal volume of phenol/chloroform/isoamyl alcohol (25:24:1). Nucleic acids were precipitated
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using 0.6 volumes of isopropanol. The precipitate was centrifuged at 12,000  g for 25 min at
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4C, and the pellet was rinsed twice with 1 ml of 70% ethanol. The DNA pellet was dissolved in
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TE buffer and quantified using a Qubit fluorometer. DNA from the rhizosphere soil samples was
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extracted using the PowerSoil® DNA Isolation Kit according to manufacturer’s instructions. The
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V4 region of the 16S ribosomal RNA gene was amplified and sequenced on an Illumina MiSeq
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machine following the protocol of Kozich et al. (2013). MiSeq sequencing was performed at the
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University of Kentucky Advanced Genetic Technology Center and at the Host Microbiome
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Initiative (HMI) facility (http://medicine.umich.edu/medschool/research/research-strengths/host-
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microbiome-initiative) at the University of Michigan, Ann Arbor, MI, USA.
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Bioinformatics and statistical analysis
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MiSeq community sequence data was processed in mothur (http://www.mothur.org/) according
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to the MiSeq SOP as of September 2014 (http://www.mothur.org/wiki/MiSeq_SOP) (Kozich et
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al. 2013). Briefly, paired-end reads were assembled into contigs, sequences were filtered for
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length, ambiguous bases, and homopolymer regions, and aligned to the SILVA reference
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alignment (release 102) to remove sequences that do not align to the V4 region of the 16S rRNA
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gene. Pre-clustering to merge highly similar (2 bp or less mismatch) sequences was followed by
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the removal of chimeras. Sequences were classified based on the RDP reference version 9 and
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16S rRNA sequences derived from mitochondrial and chloroplast DNA as well as unidentified
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sequences were removed. The average number of remaining sequences per sample was 13,478
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(range 2,598–39,474). We randomly selected 2,500 sequences from each sample, and binned
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them to OTUs based on sequence similarity with a 97% similarity cut-off.
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Since many of the bacterial taxa were rare, data was normalized to 995 OTUs to reduce stress for
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performing Non-Metric Multidimensional Scaling ordination (NMDS) in PC-ORD 6 (Figure
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S1). The NMDS was applied to assess the beta-diversity based on the Bray–Curtis measure of
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dissimilarity (Bray and Curtis, 1957; Walke et al., 2014) in OTUs relative abundances across
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rhizosphere and root samples. To test the selection of dominant and rare taxa by the rhizosphere
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and root system, we performed Student's t-test and linear regression. In this particular analysis,
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the top rhizosphere OTUs (with abundance greater than 10) were sorted, and their abundance
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was compared across fine roots, secondary roots and primary roots using Student's t-tests (Figure
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2a). Similarly, the top fine roots OTUs (n>10) were sorted, and their abundance was compared
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across rhizosphere, secondary and primary roots using Student's t-tests (Figure 2b). The top
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secondary root OTUs (n>10) were sorted, and their abundance was compared across rhizosphere,
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fine roots and primary roots using Student's t-tests (Figure 2c). The top primary root OTUs
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(n>10) were sorted, and their abundance was compared across rhizosphere, fine roots and
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secondary roots using Student's t-tests (Figure 2d). Both Student's t-test and linear regression
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were used to test whether the abundance of dominant OTUs differs across fine roots, secondary
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and primary roots. Wherever the results were the same in terms of statistical significance, we
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present only linear regression results. In cases in which both tests did not reveal any differences,
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the OTUs were defined as generalist colonizers (listed in separate excel file in Supplementary
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Material) of different root portions, and the converse is true for specialist colonizers (Figure S2-
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3).
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References
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Pearce B, Bailey A, Walker E, eds. 2015. Burley and dark tobacco production guide. University
of Kentucky Extension Publication.
http://www2.ca.uky.edu/agc/pubs/id/id160/id160.pdf (Accessed May 5, 2015).
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Walke JB, Becker MH, Loftus SC, House LL, Cormier G, Jensen RV, et al. (2014). Amphibian
skin may select for rare environmental microbes. ISME J 8:2207–2217.
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Bray JR, Curtis JT. (1957). An ordination of the upland forest communities of Southern
Wisconsin. Ecolog Monogr 27:325–349.
Ikeda S, Kaneko T, Okubo T, Rallos LEE, Eda S, Mitsui H, et al. (2009). Development of a
bacterial cell enrichment method and its application to the community analysis in soybean stems.
Microb Ecol 58:703–714.
Kozich JJ, Westcott SL, Baxter NT, Highlander SK, Schloss PD. (2013). Development of a dualindex sequencing strategy and curation pipeline for analyzing amplicon sequence data on the
MiSeq Illumina sequencing platform. Appl Environ Microbiol 79:5112–5120.
Wilson K. (2001). Preparation of genomic DNA from bacteria. In: Current protocols in
molecular biology, John Wiley & Sons, Inc.
http://onlinelibrary.wiley.com/doi/10.1002/0471142727.mb0204s56/abstract (Accessed January
20, 2015).
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Supplementary Tables
Table S1. Analysis of rhizosphere soil
Location
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P(lb/ac) K(lb/ac) pH
Analysis Data
Buffer
C(lb/ac)
pH
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M(lb/ac) ZN(lb/ac)
Spindletop
farm
516
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5.28 6.62
4890
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4.1
Table S2. Composition of rhizosphere and root microbiome in both varieties.
KT 204LC
Bacterial phyla (%)
Acidobacteria
Actinobacteria
Armatimonadetes
Bacteroidetes
Chlamydiae
Chloroflexi
Firmicutes
Gemmatimonadetes
Nitrospira
Planctomycetes
Proteobacteria
unclassified
Verrucomicrobia
TM7(100)
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Rhizosphere
23.38
8.513333333
0.813333333
5.506666667
0.046666667
0.486666667
2.7
1.946666667
0.746666667
2.806666667
21.94
21.39333333
9.486666667
0.02
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NCBH 129LC
Fine roots
Secondary roots Primary roots
Rhizosphere Fine roots
Secondary roots Primary roots
2.86
2.12
2.05
20.752
1.89
2.496
2.1066667
7.106666667
3.056
3.52
9.928
7.31
5.616
5.4266667
0.173333333
0.136
0.3
0.784
0.13
0.168
0.2
4.733333333
4.84
4.41
5.112
2.89
5.088
4.96
0.02
0
0
0.392
0.16
0.048
0.0266667
0.046666667
0.008
0.02
0.344
0.08
0.024
0.0133333
0.48
0.24
0.69
2.488
1.49
0.864
0.5733333
0.04
0.024
0.07
2.832
0.06
0.064
0.04
0.006666667
0.024
0
0.976
0.01
0.016
0
2.153333333
1.376
2.25
2.088
3.51
2.696
2.12
74.34
83.04
79.59
23.52
73.25
75.784
77.173333
4.04
2.184
2.6
22.416
4.85
3.584
3.92
3.853333333
2.888
4.48
8.392
4.29
3.488
3.4
0.046666667
0
0.01
0.024
0.07
0.032
0.04
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Supplementary Figures
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between microbial communities of rhizosphere and root samples collected from two Nicotiana
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varieties. Overall, the ordination axes explain about 95% of the variance in the dissimilarities
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among microbial communities of different habitats. Each point represents one sample.
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Figure S2.
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Abundance of specialist OTUs across root architectural traits gradient in the Nicotiana variety
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KT204LC. Significance for specialists is shown by p-values. Each box represents specialist
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OTUs from a different phylum (a, Proteobacteria; b, Verrucomicrobia; c, Actinobacteria; d,
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Bacteroidetes).
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Figure S3.
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Abundance of specialist OTUs across root architectural traits gradient in the Nicotiana variety
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NCBH129LC. Significance for specialists is shown by p-values. Each box represents specialist
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OTUs from a different phylum (a, Proteobacteria; b, Verrucomicrobia; c, Bacteroidetes; d,
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Planctomycetes).
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Figure S4
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Dominant OTUs (abundance >10) in the rhizosphere and their abundance in different root
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portions of KT204LC.
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Figure S5
Figure S1.
Nonmetric multidimensional scaling (NMDS) ordination based on Bray–Curtis distances
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Dominant OTUs (abundance >10) in the rhizosphere and their abundance in different root
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portions of NCBH129LC.
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Figure S6
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Dominant OTUs (abundance >10) in the fine roots and their abundance in rhizosphere and other
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root portions of KT204LC.
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Figure S7
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Dominant OTUs (abundance >10) in the fine roots and their abundance in the rhizosphere and
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other root portions of NCBH129LC.
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Figure S8
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Dominant OTUs (abundance >10) in the secondary roots and their abundance in the rhizosphere
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and other root portions of KT204LC.
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Figure S9
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Dominant OTUs (abundance >10) in the secondary roots and their abundance in the rhizosphere
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and other root portions of NCBH129LC.
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Figure S10
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Dominant OTUs (abundance >10) in the primary roots and their abundance in the rhizosphere
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and other root portions of KT204LC.
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Figure S11
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Dominant OTUs (abundance >10) in the primary roots and their abundance in the rhizosphere
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and other root portions of NCBH129LC.
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Figure S12
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Average abundance of top ten OTUs in the roots of KT 204LC. Lowercase letters indicate
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statistical significant differences among treatments. The significance was determined using
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ANOVA followed by Student's t-test.
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Figure S13
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Average abundance of top ten OTUs in the roots of NCBH 129LC. Lowercase letters indicate
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statistical significant differences among treatments. The significance was determined using
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ANOVA followed by Student's t-test.