Growth of rhizobacteria and co-cultivation with plants
For experiments involving bacterial VCs, bi-compartmented Petri dishes (9 cm diameter) were used. One of the compartments was filled with MSgg (Branda, Gonzalez-Pastor, Ben-Yehuda, Losick, & Kolter, 2001) agar medium, and the other was filled with MS salts agar medium. Four days before plant experiments, all of the Bacillus spp. strains (obtained from China General Microbiology Culture Collection Center:B. amyloliquefaciens SQR9, B. amyloliquefaciens FZB42,B. amyloliquefaciens SCmB, B. megaterium SXwC, B. megaterium D4, B. subtilis GZtD, B. subilis 168) and SQR9 mutants ΔysnE (Shao, Xu, Zhang, Shen, & Zhang, 2014) and ΔalsD (Wu et al., 2018) were grown in Luria-Bertani (LB) liquid medium at 37°C with 170 rpm overnight. The bacterial cells were washed twice by centrifugation for 3 min at 5,000g, and finally resuspended in double-distilled water (DDW). Three drops of 3 μl bacterial suspension (or DDW as a control) was spotted at one side of the bi-compartmented Petri dishes filled with MSgg agar. After 3 d of growth of bacterial strains at 28°C, 3-day-old seedlings (4 plants) were transferred to the adjacent compartment filled with MS salts agar. The plates were sealed with Parafilm and incubated for 6-8 d in a growth chamber at 22°C. At the end of this period, primary root length, lateral root number, and total biomass were recorded.
To evaluate the effect of CO2 level on the LR formation induced by SQR9 VCs, the uncovered bi-compartmented plate containing SQR9 and Arabidopsis was put in a square Petri dish (12 × 12 cm) and two flasks with 4 ml 0.1M Ba(OH)2 solution were placed in both sides of square Petri dish. A filter paper was inserted into each flask to increase the surface for trapping CO2(Ditengou et al., 2015) and the square Petri dish was sealed with Parafilm.