Soil Sampling and DNA Extraction

The above ground or below ground biomass of R. pseudostellariae become significantly different after 5 months of planting (Figure ​Figure1A1A), according to our previously study (Wu et al., 2016b). Therefore, soil samples were randomly collected from five different points at each field on April 22nd, 2015. Additionally, we harvested the plants for yield determination on July 2nd, 2015 (Figure ​Figure1B1B).

(A) Photographs of above and below ground components of R. pseudostellariae under 1-year, 2-year, and 3-year consecutive monoculture. (B) Yield of R. pseudostellariae under 1-year (FP), 2-year (SP) and 3-year consecutive monoculture (TP).

Soil samples were collected after digging the plant samples. Firstly, the loosely adhering soil was shaken off, then scraping the soil that was still attached to the root as rhizosphere soil. DNA was immediately extracted from 0.5 g soil sample per treatment using Biofast Soil Genomic DNA Extraction Kit (BioFlux, Hangzhou, China) according to the manufacturer’s protocols. We further determined the DNA concentration using Nanodrop 2000C Spectrophotometer (Thermo Scientific, United States) and then diluted it to 20 ng μL^-1.