The mechanism(s) involved in the effect of Azospirillum brasilense strain Cd on root susceptibility to nodulation was studied in medic seedlings grown in pouches. The number of nodules above the position of the root-tip mark at the time of inoculation and the position of the uppermost nodule were used as parameters for determining the rate of nodule initiation. Cell-free extracts and culture supernatants prepared from Azospirillum and the cytokinin benzyladenine (10−9 M) significantly increased the number of nodules formed above the root-tip mark when applied together with Rhizobium compared with those formed with Rhizobium alone. The application of indoleacetic acid did not cause an increase in the number of nodules. In the absence of Rhizobium, exposure to Azospirillum at a concentration of 109 cfu/mL or to compounds excreted by the bacteria into the growth medium caused a 40% increase in endogenous ethylene production by the roots. A less concentrated inoculum did not increase ethylene production. Inoculation with Azospirillum significantly increased the specific activity of the enzymes glucose-6-phosphate dehydrogenase, L-phenylalanine ammonia-lyase, and shikimate dehydrogenase compared with roots inoculated with Rhizobium alone. Key words: Azospirillum, Rhizobium, Medicago polymorpha, root morphology, nodule initiation.
Azospirillum brasilense Cd cell concentration of 105–107 colony-forming units (cfu)/mL applied 24 h before Rhizobium (106 cfu/mL), increased nodule formation in the non root hair zone, more than twofold, in pouch-grown Medicagopolymorpha and Macroptilium atropurpureum seedlings, compared with Rhizobium alone. The increase in nodule formation in pouch-grown Trifolium alexandrinum following preinoculation with Azospirillum was 20%. The percentage of nodulated seedlings rose from 0 to 25% when Medicago polymorpha was preinoculated with Azospirillum followed by the application of 10 cfu/mL Rhizobium meliloti, a level which by itself was not sufficient to initiate nodule formation. Acetylene reduction activity in Medicago polymorpha and Macroptilium atropurpureum seedlings after inoculation with Azospirillum–Rhizobium was markedly increased. A possible reason for the increased susceptibility to Rhizobium infection may be that Azospirillum stimulates the formation of a larger number of epidermal cells that differentiate into infectable root hairs.
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