Screening Test for AHL-Producing Strains

N-acyl-homoserine lactones screen was detected using biosensors according to Chu et al. (2011) methods. Briefly, A136 and KYC55 were grown overnight at 28°C in 5 ml of LB medium. The reactivated rhizosphere isolates was cultured overnight at 28°C in 96-well plates. Seventy-five microliter of 5 × diluted A136 overnight culture were added to 75 μl of samples (activated rhizosphere isolates) in a 96-well plate and incubated at 28°C with constant shaking at 150 rpm before X-Gal (final concentration of 40 μg/ml) was added to each well. Plates were incubated overnight at 28°C and the strain with production of blue color after 24–48 h incubation was recorded as an AHL-producing strain. P. aeruginosa PAO1 was used as the positive control and E. coli DH5α as the negative control.

The potential AHL activity of the isolates was tested by well diffusion agar plate assays as described elsewhere (Zhu et al., 2001; Lv et al., 2016). Briefly, 5 ml overnight reporter strain culture was mixed with 50 μl X-Gal in 50 ml LB media containing 1% agar when the temperature of the LB was about 45°C. The mixture was plated and allowed to solidify before sterile filter paper circles (0.5 cm diameter) were attached to the LB surface at regular intervals. The putative AHL-positive bacterial strains identified in the preliminary screen were pipetted onto the filter paper. Positive AHL production was recorded as visible blue pigmentation after overnight incubation at 28°C. AHL production active against A136 was confirmed by streaking the test-isolate on LB agar plates supplemented with 40 μg/ml X-Gal. To detect if any of the isolates produced extracellular factors that could hydrolyze X-Gal and thus give a false-positive readout, isolates that produced a blue coloration in the patch test were retested on a plate containing only agar with X-Gal. Isolates producing blue coloration on these plates were considered false-positive results in the A136 or KYC55 assay and were scored as negative.