Direct in siut identification of cellulose microfibrils associated with Rhizobium leguminosarum biovar trifolii attached to the root epidermis of white clover

Firm attachment of Rhizobium species to the legume root epidermis involves the elaboration of extracellular microfibrils extending from the bacteria and contacting the root surface at multiple sites. We investigated the nature of these extracellular microfibrils associated in situ with Rhizobium leguminosarum bv. trifolii colonized on the root epidermal surface of its legume host, white clover (Trifoiium repens L.). Scanning electron microscopy of seedling roots inoculated with the wild-type strain ANU843 showed that these extracellular microfibrils were associated with the bacteria attached not only to root hairs but also to the non-root-hair epidermis and the external environment under the influence of the developing root. Polystyrene microspheres adsorbed to the root surface did not accumulate similar microfibrils, ruling out their formation by nonspecific deposition of mucigel or self-assembly of rhizoplane fibrils of plant origin. An isozyme of cellulase was purified from Streptomyces sp. strain A20, shown to exhibit high substrate specificity for β-1,4-glucans, and used in enzyme cytochemistry to investigate the nature of these extracellular microfibrils. Combined scanning electron microscopy and computer-assisted image analysis indicated that the extracellular microfibrils associated with attached bacteria were degraded by a brief exposure to the purified cellulase but not by a broad-spectrum protease. These results provide direct in situ evidence of the cellulosic nature of the extracellular microfibrils associated with cells of R. leguminosarum bv. trifolii that have colonized the root environment of its legume host, white clover.Key words: Rhizobium, clover, cellulose microfibrils, enzyme cytochemistry, surface ecology, rhizoplane.