Root attachment assays used to study the ability of rhizobia to form biofilms on Trifolium repens . (A-C, E-G) Confocal laser scanning microscopy of propidium iodide-stained roots inoculated with gfp-tagged ANU843 and its derivatives showing biofilm formation along the root surface at different magnifications. (D) Number of colony-forming units (cfu) per gram of root tissue after washing and sonicating the roots. Each datum point is the average of at least 9 determinations. Error bars indicate the standard error from the mean. Root biofilms with either wild-type or celC mutant bacteria were harvested 72 h post-inoculation. Fluorescence (H-J) and phase-contrast (K) microcopy show root hair colonization in detail. The wild-type strain (A, E, H) forms three-dimensional biofilms that cover both root surface and root hairs forming distinct “caps” on the tip (H). In contrast, ANU843ΔC2 (B, F, I) establishes aggregates that cover the root irregularly and forms a thicker cap on the root hairs (I) whereas ANU843C2+ (C, G, J) appears to coat the root surface without cap formation (J). Nevertheless, sufficient adhesion of individual bacteria occurs on the tip to produce the hot (hole on the tip) phenotype (K).