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.