Bacterial plant growth promotion mechanisms. (i) IAA production.
The indoleacetic acid (IAA) production of the 33.1 strain was assessed
using the quantitative method developed by Bric et al. (12). The absorbance
(optical density at 520 nm) values obtained were interpolated from
a standard curve to determine the IAA concentration. Two independent
experiments were performed in triplicate.
(ii) Phosphate solubilization. The ability of strain 33.1 to solubilize
inorganic phosphate was evidenced by a halo obtained after cultivation of
the bacteria on culture medium supplemented with two inorganic phosphate
sources, Ca3(PO4)2 and Al(PO4), at 28°C for 72 h, as described by
Verma et al. (80) and Hara and Oliveira (31), respectively. An estimation
of the halo size (cm) divided by the colony size (cm) generated a
solubilization index (SI) that was used to quantify the phosphate solubilization.
(iii) Biological nitrogen fixation (BNF). The ability of the strain to fix
atmospheric nitrogen was assessed as described by Döbereiner et al. (19).
To confirm the ability of the strain to fix atmospheric nitrogen, seven
consecutive streaks were performed in the semisolid nitrogen-free nitrogen-
fixing biomass (Nfb) medium (19). The nitrogenase activity was
measured after 5 days of incubation using an acetylene reduction assay
(33). The chromatography gas analysis was completed at the Post-Harvest
Laboratory, ESALQ-USP. The samples were incubated with 10% acetylene
for 2 h at 28°C. The nitrogenase activity was expressed in nanomoles
of ethylene per hour.
Statistical analysis. The statistical analyses of the data were conducted
using the SAS program (SAS Institute Inc., Cary, NC) and were designed
to account for the random design and subfactorial nature of the qPCR and
reisolation data. To quantify the bacteria, the obtained data were log
transformed to stabilize the variance. The bars in the figures represent the
meansstandard errors of four replicates. The asterisks (* and **) in the
figures indicate significant differences ( 0.05 and 0.01, respectively)
according to Student’s t test.