Rhizobia were isolated from nodules off a stand ofLotus corniculatus established with a single inoculant strain, ICMP3153, 7 years earlier in an area devoid of naturalized Rhizobium loti. The isolates showed diversity in growth rate, Spe I fingerprint of genomic DNA, and hybridization pattern to genomic DNA probes. The 19%o of isolates that grew at the same rate as strain ICMP3153 were the only isolates that had the same fingerprint as strain ICMP3153. Sequencing of part of the 16S rRNA gene of several diverse isolates confirmed that they were not derived from the inoculant strain. Nevertheless, all non-ICMP3153 strains gave EcoRI and Spe I hybridization patterns identical to ICMP3153 when hybridized to nodulation gene cosmids. Hybridization of digests generated by the very rare cutting enzyme Swa I revealed that the symbiotic DNA region (at least 105 kb) was chromosomally integrated in the strains. The results suggest that the diverse strains arose by transfer of chromosomal symbiotic genes from ICMP3153 to nonsymbiotic rhizobia in the environment.

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Results from oversowing trials in the tussock grasslands have shown that nodulation problems can limit the establishment of Caucasian clover (Trifolium ambiguum). They also strongly suggest that previously reported difficulties with establishment of Caucasian clover, in areas where white clover (T. repens) had established sucessfully, were likely to be due to inoculation failures due to low numbers of rhizobia on the seed, or in the case of tetraploid cultivars, to the use of an unsatisfactory strain. With the tetraploid cultivar Treeline, inoculation with the recommended strain ICMP4074b resulted in low levels of effective seedling nodulation even with very high levels of rhizobia/seed at sowing (149 000). Treeline seedling nodulation was increased when inoculated with strain ICMP4073b, the approved strain for hexaploid cultivars, and the strain was effective at symbiotic nitrogen fixation. Effective seedling nodulation of both tetraploid Treeline and hexaploid Monaro cultivars inoculated with ICMP4073b increased as the number of rhizobia on the seed at sowing was increased. Low levels of seedling nodulation occurred when populations of rhizobia on the seed were in the range likely to be present when seed is slurry inoculated, or inoculated and pelleted and stored for several days before oversowing. To maximise effective nodulation, seed should be inoculated at least 5 times the recommended rate, with the incorporation of gum arabic in the slurry or pellet and oversown within one day. Keywords: Caucasian clover, establishment, inoculation, nodulation, rhizobia, Trifolium ambiguum

Four alternative legume species, Caucasian clover (Trifolium ambiguum), zigzag clover (T. medium), crown vetch (Coronilla varia), and birdsfoot trefoil (Lotus corniculatus), were compared with white clover (T. repens) on four tussock grassland sites. Seed was inoculated and pelleted, and then oversown at a rate equivalent to 5 kg/ha viable seed. Germination, nodulation and survival of white clover, Monaro Caucasian clover, and zigzag clover were similar on the two lowest sites (600 and 830 m). However, low rates of zigzag clover germination occurred on the two higher sites (1090 and 1100 m). Germination of crown vetch appeared to be prevented or delayed by the low temperatures, and no plants established on any site. Low nodulation occurred in Treeline Caucasian clover and birdsfoot trefoil. Further research is required to select more suitable strains of rhizobia for both these legumes. The results provide information for estimating likely plant densities of these new cultivars when oversown in tussock grassland environments. Keywords Coronifla varia, establishment, germination, inoculation, Lotus corniculatus, nodulation, Trifoliumambiguum, Trifolium medium, Trifolium repens