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Supplementary information for
Nicotiana roots recruit rare rhizosphere taxa as major root-inhabiting microbes
Muhammad Saleem1, Audrey D. Law1 and Luke A. Moe1*
1Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY, USA, 40546-0312
Muhammad Saleem: ; Audrey Law: ; Luke Moe:
Materials and Methods
Plant growth and root sampling
Two commercial burleytobacco (Nicotianatabacum) varieties (KT204LC and NCBH129LC) were grown at the University of KentuckySpindletop Farm (Lexington, KY) in the summer of 2013 using standard agronomic practices (Pearce et al., 2015). Briefly, seeds were germinated in a dedicated greenhouse and seedlings were transplanted to field plots in June 2013. The plants were grown in a plot that was used for burley tobacco in the previous growing season. At tobacco harvest, the vegetative portion of the plants was removed for curing, and the rhizosphere and root samples (n=6) were taken from six plants of each variety. The root and associated soil was removed using a spading fork. After gently shaking the roots to remove loosely adhered soil, the soil remaining attached to the root (rhizosphere soil) was physically removed by hand, using sterile latex gloves, into a sterile bag. The remaining root mass was put into a sterile bag and all samples were subsequently stored at -80C until processing. Soil data is listed in Table S1.
Isolationof bacteria from plant roots
Roots were washed with distilled water to eliminate any remaining soil. The larger roots linked with the stem were removed and are considered primary roots. Roots emerging from the primary roots were removed and are considered secondary roots. Fine roots were collected from secondary roots. Cutting of the roots was done using a sterilized blade. Bacteria wereisolatedfrom plant tissuesfollowinga previously established method (Ikeda et al., 2009) with some modifications. About 20g of fresh root compositesample was taken from each root portion (main root, secondary roots and fine roots). The root samples were cut into small pieces and added to to a 1L blender jar (KinematicaMicrotron MB 550) with 400 ml BCE buffer (50 mMTris-HCl, pH 7.5, 1% TritonX-100, 2mM 2-mercaptoethanol added right before blending). Samples were blended for 2 min at full speed, followed by 5 min incubation on ice, for a total of three cycles. The homogenized samples were filtered through a sterile Mira cloth using a Buchner funnel. The resulting filtrate was centrifuged at 500g for 5 min at 10C. The supernatant was centrifuged again at 5,500g for 20 min at 10C. The supernatant was then discarded and the pellet resuspended in 250 ml BCE, andcentrifuged at 10,000g for 10 min at 10C, for a total of two times. The final pellet was resuspended in 6 ml Tris Buffer (50 mM, pH 7.5). The suspension was gently overlayed onto 4 ml nycodenz (8g nycodenz in 10 ml Tris buffer) in a 15 ml glass tube. The glass tube was centrifuged at 10,000 g for 40 min at 10C. A band containing the bacterial cells formed at the interface of buffer and nycodenz was removed with a pipette and transferred into a 2ml microcentrifuge tube with an equal volume of deionized sterile water. After centrifugation at 21,130g for 5 min, the supernatant was removed and thepellet was storedat -20C.
Genomic DNA purification
Genomic DNA purification followed the protocol of Wilson (2001). The frozen microbial pellets isolated from plant rootsweredissolved in 567 l of TE buffer, 30 l of 10% SDS and 3l of proteinase K (20mg/ml)were added,and the mixture was incubated at 37C for one hour. 100 l of 5M NaOH and 80 l of a CTAB/NaCl solution (41 g NaCl and 100 g CTAB per liter of solution) was addedbefore another incubation for 10 min at 65C. An equal volume of chloroform/isoamyl alcohol (24:1) was used to extract the lysate, followed by extraction withan equal volume of phenol/chloroform/isoamyl alcohol (25:24:1). Nucleic acids were precipitated using 0.6 volumes of isopropanol. The precipitate was centrifuged at 12,000g for 25 min at 4C, andthe pellet was rinsed twicewith 1ml of 70% ethanol. The DNA pellet was dissolved in TE buffer and quantified using aQubitfluorometer.DNA from the rhizosphere soil samples was extracted using the PowerSoil® DNA Isolation Kit according to manufacturer’s instructions. The V4 region of the 16S ribosomal RNA gene was amplified and sequenced on an IlluminaMiSeq machine following the protocol of Kozich et al. (2013). MiSeq sequencing was performed at the University of Kentucky Advanced Genetic TechnologyCenter and at theHost Microbiome Initiative (HMI) facility ( at the University of Michigan, Ann Arbor, MI, USA.
Bioinformatics and statistical analysis
MiSeq community sequence data was processed inmothur ( to the MiSeq SOP as of September 2014 ( (Kozich et al. 2013). Briefly, paired-end reads were assembled into contigs, sequences were filtered for length, ambiguous bases, and homopolymer regions, and aligned to the SILVA reference alignment (release 102) to remove sequences that do not align to the V4 region of the 16S rRNAgene. Pre-clustering to merge highly similar (2 bp or less mismatch) sequences was followed by the removal of chimeras. Sequences were classified based on the RDP reference version 9 and 16SrRNA sequences derived from mitochondrial and chloroplast DNA as well as unidentified sequences wereremoved. The average number of remaining sequences per sample was 13,478 (range 2,598–39,474). We randomly selected 2,500 sequences from each sample, and binned them to OTUs based on sequence similarity with a 97% similarity cut-off.
Since many of the bacterial taxa were rare, data was normalized to 995 OTUs to reduce stress for performing Non-Metric Multidimensional Scaling ordination (NMDS) in PC-ORD 6 (Figure S1). The NMDS was applied to assess the beta-diversitybased on the Bray–Curtis measureof dissimilarity(Bray and Curtis, 1957; Walkeet al., 2014) in OTUsrelative abundances across rhizosphere and root samples. To test the selection of dominant and rare taxa by the rhizosphere and root system, we performed Student'st-test and linear regression. In this particular analysis, the top rhizosphere OTUs (with abundance greater than10) were sorted, and their abundance was compared across fine roots, secondary roots and primary roots using Student'st-tests (Figure 2a). Similarly, the top fine roots OTUs (n>10) were sorted, and their abundance was compared across rhizosphere, secondary and primary roots using Student'st-tests (Figure 2b). The top secondary root OTUs (n>10) were sorted, and their abundance was compared across rhizosphere, fine roots and primary roots using Student'st-tests (Figure 2c). The top primary root OTUs (n>10) were sorted, and their abundance was compared across rhizosphere, fine roots and secondary roots using Student'st-tests (Figure 2d). Both Student'st-test and linear regression were used to test whether the abundance of dominant OTUs differs across fine roots, secondary and primary roots. Wherever the results were the same in terms of statistical significance, we present only linear regression results. In cases in which both tests did not reveal any differences, the OTUs were defined as generalist colonizers (listed in separate excel file in Supplementary Material) of different root portions, and theconverse is true for specialist colonizers (Figure S2-3).
References
Bray JR, Curtis JT. (1957). An ordination of the upland forest communities of Southern Wisconsin.EcologMonogr27:325–349.
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.MicrobEcol58:703–714.
Kozich JJ, Westcott SL, Baxter NT, Highlander SK, Schloss PD. (2013). Development of a dual-index sequencing strategy and curationpipeline for analyzingampliconsequence data on the MiSeqIlluminasequencing platform.Appl Environ Microbiol79:5112–5120.
Pearce B, Bailey A, Walker E, eds. 2015. Burley and dark tobacco production guide. University of Kentucky Extension Publication. Accessed May5, 2015).
Walke JB, Becker MH, Loftus SC, House LL, Cormier G, Jensen RV, et al. (2014). Amphibian skin may select for rare environmental microbes. ISME J8:2207–2217.
Wilson K. (2001). Preparation of genomic DNA from bacteria.In:Current protocols in molecular biology, John Wiley & Sons, Inc. (Accessed January 20, 2015).
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Supplementary Tables
Table S1. Analysis of rhizosphere soil
Analysis DataLocation / P(lb/ac) / K(lb/ac) / pH / Buffer pH / C(lb/ac) / M(lb/ac) / ZN(lb/ac)
Spindletop farm / 516 / 209 / 5.28 / 6.62 / 4890 / 245 / 4.1
KT 204LC / NCBH 129LC
Bacterial phyla (%) / Rhizosphere / Fine roots / Secondary roots / Primary roots / Rhizosphere / Fine roots / Secondary roots / Primary roots
Acidobacteria / 23.38 / 2.86 / 2.12 / 2.05 / 20.752 / 1.89 / 2.496 / 2.1066667
Actinobacteria / 8.513333333 / 7.106666667 / 3.056 / 3.52 / 9.928 / 7.31 / 5.616 / 5.4266667
Armatimonadetes / 0.813333333 / 0.173333333 / 0.136 / 0.3 / 0.784 / 0.13 / 0.168 / 0.2
Bacteroidetes / 5.506666667 / 4.733333333 / 4.84 / 4.41 / 5.112 / 2.89 / 5.088 / 4.96
Chlamydiae / 0.046666667 / 0.02 / 0 / 0 / 0.392 / 0.16 / 0.048 / 0.0266667
Chloroflexi / 0.486666667 / 0.046666667 / 0.008 / 0.02 / 0.344 / 0.08 / 0.024 / 0.0133333
Firmicutes / 2.7 / 0.48 / 0.24 / 0.69 / 2.488 / 1.49 / 0.864 / 0.5733333
Gemmatimonadetes / 1.946666667 / 0.04 / 0.024 / 0.07 / 2.832 / 0.06 / 0.064 / 0.04
Nitrospira / 0.746666667 / 0.006666667 / 0.024 / 0 / 0.976 / 0.01 / 0.016 / 0
Planctomycetes / 2.806666667 / 2.153333333 / 1.376 / 2.25 / 2.088 / 3.51 / 2.696 / 2.12
Proteobacteria / 21.94 / 74.34 / 83.04 / 79.59 / 23.52 / 73.25 / 75.784 / 77.173333
unclassified / 21.39333333 / 4.04 / 2.184 / 2.6 / 22.416 / 4.85 / 3.584 / 3.92
Verrucomicrobia / 9.486666667 / 3.853333333 / 2.888 / 4.48 / 8.392 / 4.29 / 3.488 / 3.4
TM7(100) / 0.02 / 0.046666667 / 0 / 0.01 / 0.024 / 0.07 / 0.032 / 0.04
Table S2. Composition of rhizosphere and root microbiomein both varieties.
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Supplementary Figures
Figure S1.
Nonmetric multidimensional scaling (NMDS) ordination based on Bray–Curtis distances between microbial communities of rhizosphere and root samples collected from two Nicotiana varieties. Overall, the ordination axes explain about 95% of the variance in the dissimilarities among microbial communities of different habitats. Each point represents one sample.
Figure S2.
Abundance of specialist OTUs across root architectural traits gradient in the Nicotiana varietyKT204LC.Significance for specialists is shown by p-values. Each box represents specialist OTUs from a different phylum (a, Proteobacteria; b, Verrucomicrobia; c, Actinobacteria; d, Bacteroidetes).
Figure S3.
Abundance of specialist OTUs across root architectural traits gradient in the Nicotianavariety NCBH129LC. Significance for specialists is shown by p-values.Each box represents specialist OTUs from a different phylum (a, Proteobacteria; b, Verrucomicrobia; c, Bacteroidetes; d,Planctomycetes).
Figure S4
Dominant OTUs (abundance >10) in the rhizosphere and their abundance in different root portions of KT204LC.
Figure S5
DominantOTUs (abundance >10) in the rhizosphere and their abundance in different root portions of NCBH129LC.
Figure S6
DominantOTUs (abundance >10) in the fine roots and their abundance in rhizosphere and other root portions of KT204LC.
Figure S7
DominantOTUs (abundance >10)in the fine roots and their abundance in the rhizosphere and other root portions ofNCBH129LC.
Figure S8
DominantOTUs (abundance >10) in the secondary roots and their abundance in the rhizosphere and other root portions of KT204LC.
Figure S9
DominantOTUs (abundance >10) in the secondary roots and their abundance in the rhizosphere and other root portions of NCBH129LC.
Figure S10
DominantOTUs (abundance >10) in the primary roots and their abundance in the rhizosphere and other root portions of KT204LC.
Figure S11
DominantOTUs (abundance >10) in the primary roots and their abundance in the rhizosphere and other root portions of NCBH129LC.
Figure S12
Average abundance of top tenOTUs in the roots ofKT 204LC.Lowercase lettersindicate statistical significant differences among treatments. The significance was determined using ANOVA followed by Student's t-test.
Figure S13
Average abundance of top ten OTUs in the roots of NCBH 129LC.Lowercase lettersindicate statistical significant differences among treatments. The significance was determined using ANOVA followed by Student's t-test.