DIRECT IN-SITU 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 invo lves the elaboration of extracellular microfibrils extending from the bacteria and contacting the root surface at multiple sites. We investi gated the nature of these extracellular microfibrils associated in sit u with Rhizobium leguminosarum bv. trifolii colonized on the root epid ermal surface of its legume host, white clover (Trifolium repens L.). Scanning electron microscopy of seedling roots inoculated with the wil d-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 t o 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 pu rified from Streptomyces sp. strain A20, shown to exhibit high substra te specificity for beta-1,4-glucans, and used in enzyme cytochemistry to investigate the nature of these extracellular microfibrils. Combine d scanning electron microscopy and computer-assisted image analysis in dicated that the extracellular microfibrils associated with attached b acteria were degraded by a brief exposure to the purified cellulase bu t not by a broad-spectrum protease. These results provide direct in si tu evidence of the cellulosic nature of the extracellular microfibrils associated with cells of R. leguminosarum bv. trifolii that have colo nized the root environment of its legume host, white clover.