Bacterial reisolation. The densities of P. agglomerans in the rhizosphere
and inside the sugarcane tissues were evaluated at 4 and 15 DAI in
the gnotobiotic assay and at 30 DAI in the greenhouse assay. To promote
the detachment of bacteria from the roots, the plant samples were placed
in a new sterile tube containing 2 ml of PBS and agitated at 120 rpm for 1
h. The cell suspension was diluted and plated on 5% tryptic soy broth for
bacterial quantification. The seedlings were washed in running tap water
and surface disinfected using serial rinsing in 70% ethanol and 2% hypochlorite
as described by Araújo et al. (1). To confirm the efficiency of the
disinfection process, aliquots of the sterile distilled water used in the last
washing were spread onto 5% tryptic soy (TS) agar medium and examined
for surface contaminants after a 3-day incubation at 28°C. The surface-
disinfected samples were macerated in a PBS buffer, and the appropriate
dilutions were plated onto 5% TS agar supplemented with
kanamycin (100 g/ml) and benomyl (50 g/ml). The cultivable bacterial
density associated with sugarcane was also evaluated under greenhouse
conditions. For this process, the macerated samples were plated on TS
agar without the selective antibiotic, and the number of tagged strains
among the indigenous community was measured by exposing the plates
to UV light.