2.2 Field experiment, sampling and DNA extraction
The field experiment was performed in a plot system located in Suixi County, Zhanjiang City of Guangdong Province (21°14′23′′N, 110°4′28′′E) to evaluate the effect of clothianidin with/without fertilizer on the structure of microbial communities in the rhizosphere and in bulk soil. When the sugarcane seedlings grew with 3~5 leaves, we stated the field experiment including four treatments SPF, SP, SF, and SA with 100 g of each spreading into the soil close to sugarcane per plot (7.4 × 1.8 m in size), and a thin layer of soil applied to cover. Each treatment consisted of 4 plots and one meter between each plot, as well as a blank control.
After four weeks, rhizosphere soil and bulk samples were collected from sugarcane plots. For the rhizosphere samples, the complete root systems of 3~5 plants per plot were combined as a composite sample after removing loosely adhering soil by vigorous shaking. 20 composite samples deposited in sterile zip-lock bags were carried to the lab with ice. Roots with soil on the surface were washed with a sterile 1 × PBS (phosphate-buffered saline) solution (Solarbio Life Sciences, China), and then centrifuged at 8,000 × g for 10 min at 4°C. The resulting pellet, defined as the sample containing the rhizosphere-enriched microbial communities, was frozen in liquid nitrogen and stored at -80°C.
DNA extraction was performed using a soil DNA isolation kit (Tiangen Biotech, Beijing, China). DNA concentration and purification were quantified by Nanodrop 2000 as well as checked on 1% agarose gels for quality. The average DNA yield was 2-5 μg DNA with the concentration of 10-50 ng/μL. DNA concentrations were additionally estimated using a Qubit 2.0 fluorometer (Thermo Fisher Scientific, USA).