electronic supplementary material

sediments, sec 4 • sediment-ecology interactions • research article

Properties of root exudates and rhizosphere sediment of Bruguiera gymnorrhiza (L.)

Received: 8 December 2015 / Accepted: 25 August 2016

© Springer-Verlag Berlin Heidelberg 2016

Responsible editor: Haihan Zhang

Beibei Liu1,4 • Xinyu Liu1,2 • Shanshan Huo3 • Xin Chen1,4 • Lin Wu1,4 • Miao Chen1,4 • Kaibo Zhou1,4 • Qinfen Li1,4 • Lixu Peng1,4

1 Institute of Environmental and Plant Protection/Environmental Impact Assessment and Risk Analysis Center, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan, China

2 College of Environment and Plant Protection, Hainan University, Haikou, Hainan, China

3 Environmental Safety Supervision and Inspection Centre for Genetically Modified Plants and Microorganisms used in Plants, Ministry of Agriculture, Haikou, Hainan, China

4 Agricultural Environmental Science Observation and Experiment Station, Ministry of Agriculture, Danzhou, Hainan, China

* Lixu Peng

* Beibei Liu

Table S1 Recovery of spiked organic compounds and LMWOAs in sediment by ethyl acetate extraction

Organic compounds / Recovery (%) / LMWOAs / Recovery (%)
Hydrocarbons / 83.6 - 92.4 / Oxalic acid / 90.2 - 107
Esters / 83.8 - 105 / Malic acid / 89.3 - 98.50
Phenols / 85.4 - 97.1 / Malic acid / 91.7 - 105
Aromas / 86.7 - 103 / Fumaric acid / 88.20 - 98.6

Table S2 Measurement methods of enzyme activities in sediment

Enzyme / Measurement of activity
Invertase / Determine the production rate of glucose from sucrose (125 mM/20mM-phosphate buffer) with the incubation at 37 °C for 24h.
Urease / Determine the hydrolysis of urea in citrate buffer (pH 6.7) with the incubation at 37 °C for 24 h.
Dehydrogenase / Determine the reduction of 2, 3, 5-triphenyl tetrazolium chloride (TTC) to 1, 3, 5-triphenyl formazan (TPF) with the incubation at 30 °C for 6 h.
Polyphenol oxidase / Determine the oxidation of pyrogallol with the incubation at 30 °C for 3 h.

Fig. S1 GC-MS spectrum of B. gymnorrhiza root exudates in hydroponic collection solution

Method of 16s rDNA analysis

Total genome DNA from samples was extracted using CTAB/SDS method. DNA concentration and purity was monitored on 1% agarose gels. According to the concentration, DNA was diluted to 1ng/μL using sterile water. The V4 region of the bacterial 16S rDNA gene was amplified with the primers 515f and barcoded-806r. All PCR reactions were carried out with Phusion® High-Fidelity PCR Master Mix (New England Biolabs). Mix same volume of 1X loading buffer (contained SYB green) with PCR products and operate electrophoresis on 2% agarose gel for detection. Samples with bright main strip between 400-450bp were chosen for further experiments. PCR products was mixed in equidensity ratios. Then, mixture PCR products was purified with Qiagen Gel Extraction Kit(Qiagen, Germany).

Sequencing libraries were generated using TruSeq® DNA PCR-Free Sample Preparation Kit (Illumina, USA) following manufacturer's recommendations and index codes were added. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. At last, the library was sequenced on an IlluminaHiSeq2500 platform and 250 bp paired-end reads were generated. Raw sequences were processed in QIIME 1.7.0. Sequences were quality trimmed and clustered into operational taxonomic units (OTUs) at a 97% identity threshold using uclust. Representative sequences from individual OTUs were then aligned against the Greengenes core set using PyNAST Taxonomic assignment was carried out with the RDP Classifier.

OTUs abundance information was normalized using a standard of sequence number corresponding to the sample with the least sequences. Subsequent analysis of alpha diversity was performed basing on this output normalized data.