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331 - 347
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Auxotrophic mutants of Rhizobium trifolii resulting from a single Tn5 mutation were tested for their ability to induce nodule formation and carry out nitrogen fixation, the latter measured as acetylene reduction and plant dry weight, on white clover (Trifolium repens) using a sterile plant assay technique. Mutants requiring a vitamin an amino-acid or a pyrimidine for growth were able to induce nodulation (i.e. posessed a Nod+ phenotype). Nodules formed by P-aminobenzoic acid- and pantothenate- requiring auxotrophs were higly variable in their ability to fix nitrogen in symbiosis whereas the leucine-, methionine- and pyrimidine-deficient mutants fixed nitrogen only very poorly in symbiosis with T. repens. Addition of the necessary compound to the growth medium of the host plant (T. repens) reversed the poor fixation phenotype of these auxotrophs indicating that various compounds are not excluded from Rhizobiwn cells in symbiosis but simply not provided in sufficient quantity by the host plant or the environment to allow the establishment of a normal symbiotic phenotype. Mixed inocula of auxotrophic strains with a poor nitrogen-fixing phenotype gave mixed nodules (ie. nodules occupied by two mutant strains) which could vigorously fix nitrogen. Tests showed that reversion, genetic exchange or contamination were not the cause of the enhanced nitrogen-fixing phenotype of mixed nodules. Mixed nodules occurred at a frequency of at least 9.3% of all tested nodules. An average of 44% of mixed-inoculated white clover plants showed some degree of improvement in symbiotic capacity due to mixed nodules showing that joint symbiotic phenotypes can be produced by a process of cross-feeding in the host plant. The proportions oí Rhizobiwn strains used for inoculation, within a range of Infold, did not greatly affect the frequency of symbiotically improved plants. Prototrophic revenants were shown to occur by both Tn5 loss or transposition resulting in the restoration of a wild-type symbiotic phenotype. Tn5 insertions were shown to stabilize with subculture and purification to the point where reversion could no longer be detected.
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