Surface sterilization and isolation of culturable Bacteria:
One gram soil sample from LARS, SARS, and BS was serially diluted in 9
ml sterile deionized water. Similarly, 1 g of sterilized roots were
crushed using a mortar and pestle and serially diluted in 9 ml sterile
deionized water to isolate endophytes. 100 μ l homogenate from
each dilution (up to 10-6 for soil samples and
10-4 for root samples) were plated in triplicate on
four different culture media. Nutrient agar (NA) (general-purpose
media), King’s B agar (KB) (selective media), Pikovskaya’s agar (PKA)
(selective media), Jensen’s agar (JA) (selective media). The growth of
colonies on the selective media, i.e., PKA, JA, KB, was assumed to
reflect the functional traits of the isolates. For example, PKA
bacterial isolates with phosphate solubilization function, JA
nitrogen-fixing activity, and KB pseudomonas bacteria, respectively.
Agar plates were incubated at 28 ºC and observed for bacterial growth
for 48 hours. The number of colony-forming units (cfu) from each
replicate was counted under a colony counter and reported in log cfu
g-1 soil or roots. Isolates were grouped based on
their unique colony morphologies such as colour, shape, size, etc.
Randomly selected isolates of different morphologically distinct groups
were further cultured individually on agar plates for pure colonies and
purified for the further 16s RNA gene-based identified.