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.