Sampling procedures and preparation of samples for microbial analysis
Three areas of ̵̴16 m2 each were randomly marked in each of the jhum fields derived from the three fallow ages. In each of the three randomly marked areas, one plant each of maize, arahar, and rice (3 tillers) were randomly selected. These plants were collected with entire roots along with intact adhering soil. The root + soil mix samples were placed in sterile polyethylene bags and transported on ice, and stored at 4 ºC until use.
In single day, the first replicates of each crop from three samples from each of the three fallow periods fields were prepared. Each plant root + adhering soil was separated into three different rhizosphere niches (Fig S1); crop roots, when uprooted from the soil, contained the loosely attached soil (LARS), which was separated by putting the root + adhered soil in a 1L beakers and shaking for 40 minutes for rice samples and 30 minutes for maize and arahar samples. To obtain the strongly adhered rhizosphere soil (SARS), 5 g of roots that already had LARS removed were mixed with 45 ml of sterile distilled water in a centrifuge tube and vortexed vigorously for 10 to 15 minutes until all the associated soil was completely removed. The roots were aseptically removed and the suspension removed as the SARS was divided into two equal parts. One part was used for the determination of soil chemistry whilst the other part was used for bacterial community analysis. To obtain the root interior (RI) microbes, the soil-free roots were cut into 1 cm portions aseptically and surface sterilized by vortexing five times in distilled water for 1 minute each. They were then treated with 70 % ethanol for 30 seconds, followed by 3 % sodium hypochlorite for 10 mins, then thoroughly washed five times with sterile distilled water. Sterility check was carried out by root pieces on the surface of the nutrient agar plates. To obtain bulk soil (BS) samples, root-free soil samples were randomly collected from 0-20 cm depth from each 16-m2area at the same time as plant collection.