Title: Impacts of bulk soil microbial community structure on rhizosphere microbiomes
Journal: Plant and Soil, 2015
Matthew G. Bakker1*, Jacqueline M. Chaparro2, Daniel K. Manter3, and Jorge M. Vivanco2
1
United States Department of Agriculture
Agricultural Research Service
National Laboratory for Agriculture and the Environment
Ames, IA 50011 USA
2
Department of Horticulture & Landscape Architecture
Colorado State University
Fort Collins, CO 80523-1173 USA
3
United States Department of Agriculture
Agricultural Research Service
Soil-Plant-Nutrient Research Group
Fort Collins, CO 80526 USA
* Corresponding author
Matt.Bakker@ars.usda.gov
Phone: 1-515-294-9419; Fax: 1-515-294-8125
Supplementary Methods
Root exudate collection and analysis
To test whether Z. mays cultivars P9714XR and 35F40 differed in root exudation, exudates were
collected in an axenic hydroponic system. Seeds of each cultivar were surface sterilized by
shaking for 2 min in 70% ethanol, 25 min in 0.5% NaOCl with 0.01% Tween 20, and rinsing
four times with sterile distilled water. Seeds were transferred to petri plates containing nutrient
agar. After incubation for 2 days, seeds showing no evidence of contamination were transferred
to sterile glass culture tubes (25 X 250 mm; 1 seed per tube) containing 2.5 mL of Hoagland’s
nutrient solution (pH 5.8). Tubes were covered with translucent caps and arranged on a light
table with a bank of four fluorescent bulbs (approximately 45 µmol m-2 s-1) lowered as close as
possible to the top of the tubes. The base of the light table was a reciprocal shaker, which was
operated at 100 rpm. Plants were grown with a photoperiod of 16 h light/8 h dark at 25 ± 2 °C.
After three days, an additional 7 mL of sterile Hoagland’s nutrient solution was added to each
tube. Tubes were culled if seedlings grew abnormally (e.g., shoots growing downward). After 7
days in the culture tube, the growth medium was collected. A sample of hydroponic solution
from each tube was spotted onto nutrient agar and any tubes showing microbial contamination
were discarded. Exudates were washed off of plant roots by spraying the root system with
deionized water. The combined sample of rinsate and hydroponic solution was frozen and
lyophilized.
Lyophilized product was dissolved in 1 mL of 80% methanol / 20% water (v/v), which had been
spiked with ribitol (10 µg mL-1) as an internal standard. One sample from each Z. mays genotype
was prepared without the internal standard, to verify that ribitol was not an abundant component
of the exudates. Each sample was filtered (nylon membrane, 0.2 µm pore size, 13 mm diameter)
and the filter was washed with 500 µL of 80% methanol. Samples were frozen prior to
derivitization.
For derivitization, samples were completely dried under a stream of nitrogen gas. Each sample
was treated with 55 µL of pyridine containing 16 mg/mL of methoxyamine hydrochloride, mixed
by vortexing, incubated at 37 °C for one hour, vortexed again, and incubated for an additional
hour. Samples were cooled to room temperature and then 65 uL of N-methyl-Ntrimethylsilyltrifluoroacetamide with 1% trimethylchlorosilane (MSTFA + 1% TMCS) was
added. Vials were capped tightly, vortexed and incubated at 37 °C for 1 hour. Samples were
transferred to 1.5 mL microcentrifuge tubes and centrifuged at 12,000 RPM for 5 minutes. One
hundred μL of supernatant was transferred to a new tube, which was centrifuged again at 12,000
RPM for 5 minutes. Eighty μL of supernatant was transferred to a 150 µL glass insert in a GCMS autosampler vial.
Metabolites were detected at the Proteomics and Metabolomics Facility at Colorado State
University, using a Trace GC Ultra coupled to a Thermo DSQ II mass spectrometer (Thermo
Scientific). Each sample was run in two technical replicates, in an order randomized separately
for each block. For each sample, 1 μL was injected in a 1:10 split ratio. Separation occurred
using a 30 m TG-5MS column (Thermo Scientific, 0.25 mm i.d., 0.25 μm film thickness) with a
1.2 mL/min helium gas flow rate, and the program consisted of 80 ºC for 30 sec, a ramp of 15 ºC
per min to 330 ºC, and an 8 min hold. Masses between 50-650 m/z were scanned at 5 scans/sec
after electron impact ionization. For each sample, raw data files were converted to .cdf format,
and a matrix of molecular features as defined by retention time and mass (m/z) was generated
using XCMS software in R (Smith et al. 2006) for feature detection and alignment. Raw peak
areas were normalized to total ion signal, outlier injections were detected based on total signal
and PC1 of principal component analysis, and the mean area of each chromatographic peak was
calculated among technical replicate injections (n=2). Peak areas were subjected to principal
component analysis to visualize potential differences in exudation profile among cultivars.
References
Smith CA, Want EJ, O'Maille G, Abagyan R, Siuzdak G (2006) XCMS: Processing mass
spectrometry data for metabolite profiling using nonlinear peak alignment, matching, and
identification. Analyt Chem 78:779-787. doi:10.1021/ac051437y
Supplementary Figures
Supplementary Figure S1 - Contrasts of soil edaphic properties after three different resource
amendments, for each soil. A) Soil pH; B) Soil carbon content; C) Soil nitrogen content. Mean ±
SE values are shown. * indicates a significant change relative to the initial soil (p < 0.01,
ANOVA with Dunnett's test).