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

2Department 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

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 at25 ± 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-N-trimethylsilyltrifluoroacetamide 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 mLmicrocentrifuge tubes and centrifuged at 12,000 RPM for 5 minutes. One hundred μLof supernatant was transferred to a new tube, which was centrifuged again at 12,000 RPM for 5 minutes. Eighty μLof supernatant was transferred to a 150 µL glass insert in a GC-MS 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, 1μL 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).