Sugarcane growth promotion and defense protein production. The
micropropagated sugarcane seedlings (varieties SP80-1842 and SP80-
3240) were transferred to plastic pots containing the organic substrate
PlantMax Horticultura (Eucatex). The transferred plants were first acclimated
in a humid chamber at 28°C for 7 days. The bacterial wild-type 33.1
and tagged 33.1::pNKGFP strains were inoculated into the substrate (108 CFU/plant), and the pots were then transferred to a greenhouse at 28°C.
The substrate of the control pots was inoculated with PBS buffer that did
not contain any bacterial cells. The dry mass, related defense protein production,
and bacterial cross-colonization of the plants were evaluated at
30 DAI.
Forty plants from each treatment were sampled, washed, and divided
into root and aerial parts. The vegetative tissues were weighed and dried at
55°C for 5 days to reach a constant weight.
Enzymatic activity was assessed using separate total protein extractions
from the leaves and roots as described by Ferreira-Filho et al.
(26). The amount of protein present in the supernatant was measured
using the Bradford procedure (10), with bovine serum albumin (BSA)
as the standard. Four replicates were used for the measurements of
enzymatic activity.
The endoglucanase and chitinase activities from the crude plant extract
were assayed using Remazol brilliant blue carboxymethylcellulose
(RBB-CMC) and carboxymethyl-chitin-Remazol brilliant violet (CMchitin-
RBV) from Loewe Biochemica GmbH (Sauerlach, Germany), respectively.
The enzyme activity was measured as the absorbance per milliliter
of the substrate reaction per hour (29).