Root adhesion test
The adhesion of rhizobial cells to root surface (here used as a proxy of
biofilm formation on roots) was estimated as described previously . Five
days-old seedlings were transferred in 1.5 ml tubes containing Fahraeus
supplemented with 50% v/v spent medium and bacterial culture at
OD600 0.1. After 48h, roots of similar length were
washed in 500 μl of 0.9% NaCl to remove loosely adherent cells by
vortexing for 10 s in 500 μl of 0.9% NaCl. Then roots were transferred
to 500 µl of fresh 0.9% NaCl and vortexed for 30 s to collect bacterial
cells that are strongly adherent to the root surface. The quantity of
bacterial cells was evaluated by qPCR on rpoE1 gene following an
already established method . For each condition, three biological
replicates were performed, with three seedlings each. Differences were
evaluated by one-way ANOVA Tukey pairwise contrast.