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    The expression in soil bacteria of symbiotic genes from Rhizobium leguminosarum biovar trifolii : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University
    (Massey University, 1994) Fenton, Michael
    Rhizobium leguminosarum biovar trifolii strain ICMP2163::Tn5 was able to spontaneously transfer its pSym to the non-nodulating Rhizobium loti soil isolate NR40 in sterile soil microcosms containing Ramiha hill soil or Ashurst silt loam soil at pH 6.0 or higher. In sterile soil microcosms at pH 6.0 containing sterile ryegrass or white clover plants the frequency of NR40 transconjugants was higher than in microcosms containing soil alone. The survival of the parent strains decreased in soil with a pH of 5.5 or less, and no transconjugant NR40 bacteria were detectable. Southern blots of the genomic digests probed with nodA DNA confirmed that transconjugant NR40 contained symbiotic genes. On artificial media strain ICMP2163::Tn5 transferred its symbiotic plasmid, by conjugation, to Sphingobacterium multivorum, an organism that can be found in soil. The transconjugant bacteria were able to nodulate white clover seedlings but were unable to fix nitrogen. Microscopic examination revealed that the root nodule structure, and bacteroid formation, were abnormal. The bacteria occupying the nodules were isolated and the total DNA extracted. The partial 16S RNA gene sequence from a transconjugant derived from a nodule was shown to be identical with that of the recipient S. multivorum. Southern blots of the genomic digests probed with nodA DNA confirmed that the transconjugant contained symbiotic genes. A Caulobacter crescentus sewage isolate was also able to induce a tumour­ like growth on white clover seedlings after receiving the pPN1 co-integrate plasmid from E.coli strain PN200. Eckhardt gel analysis confirmed that the transconjugant Caulobacter carried the R68.45:pSym co-integrate plasmid. Bacteroids were absent but Caulobacter cells were found in the outer two or three layers of the growth and the plant cells in this region had degenerated. Sequence data was obtained for a 260 bp fragment of the 16S rRNA gene from Sphingobacterium multivorum and Caulobacter crescentus corresponding to postions 44 to 360 on the Escherichia coli genome. A distance matrix was constructed showing the relationship between S. multivorum, C. crescentus, Rhizobium, and related bacteria and neighbor-joining was used to construct a tree. From the tree given it is concluded that the ability to carry or express symbiotic genes is not dependant on having a phylogenetic relationship with Rhizobium.
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    Dual mutualistic associations in sainfoin (Onobrychis viciifolia Scop.) : a thesis presented in partial fulfilment of the requirements for the degree of Master of Agricultural Science in Agronomy at Massey University
    (Massey University, 1982) Kon, Kee Fui
    Recent studies established that many legumes, when infected with the appropriate Rhizobium spp. and arbuscular fungi, nodulated better and exhibited greater dinitrogen fixation than plants infected with only the rhizobia. A similar study, therefore, was carried out in a glasshouse using sainfoin (Onobrychis viciifolia Scop.), a legume that is rapidly gaining recognition as a potential forage crop in New Zealand and other parts of the world. Pre-germinated seeds (cv. Fakir) were planted in sterilized soils and incubated with an effective Rhizobium spp. (strain NZP 5301), a mixture of endophytes (Gigaspora magarita Becker & Hall, Glomus fasciculata (Thax. sensu Gerd.) Gerdemann & Trappe and Glomus tenuis (Greenall) Hall), or both eht rhizobia and endophytes. The experiment also included a control, without any inoculation. Endophyte infection, nodulation and dinitrogen fixation, total nitrogen and phosphorus concentrations, and plant growth and development were determined on eleven sequential samplings over about twenty weeks, up to the stage of green inflorescence. Arbuscular mycorrhiza formation did not occur with the first endophyte inoculation, containing Gigaspora magarita Becker & Hall, even after 93 days of growth. This is probably because the inoculum used consisted of a low quantity of viable spores and mycelia. The second inoculation, containing the three endophyte species, produced only a low degree of infection between day 115 and 137, possibly because the extensive root lignification and relatively higher root phosphorus concentration (0.50%) restricted fungal invasion and establishment within the root cortex. Mycorrhiza formation did not increase phosphate uptake, improve nodulation and dinitrogen fixation, or increase plant growth. This is due probably to the already well-developed root systems that were efficiently exploiting the small soil volume within the bags. Rhizobia-inoculated plants produced more nodules, larger nodules and consequently, a greater nodule dry weight than the uninoculated plants. The nodules produced in the inoculated plants were red instead of green as in the uninoculated plants, and exhibited a greater dinitrogen fixation. As a result, these inoculated plants contained a higher concentration of shoot, root and nodule nitrogen, and a greater dry weight accumulation in the shoots and nodules. The shoot and nodule phosphorus concentrations, however, were lower in the rhizobia-inoculated than in the uninoculated plants due to the greater amount of shoot and nodule tissues which caused a dilution effect. These rhizobia effects on nodulation and dinitrogen fixation, nitrogen and phosphorus concentrations, and plant growth and development became more prominent with time. The relatively higher nodule phosphorus concentration when compared with the shoot and root phosphorus concentrations suggests that phosphorus was presumably required in large quantities by the dinitrogen-fixing system.
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    The effects of shading and defoliation on the nodulation and nitrogen fixation of white clover (Trifolium repens L.): a thesis presented in partial fulfilment of the requirements for the degree of Master of Agricultural Science at Massey University
    (Massey University, 1971) Chu, Alexander Cheong Ping
    The ability of the legume-Rhizobium symbiosis to fix atmospheric nitrogen, hence contributing towards the maintenance of soil fertility is well recognised. Although increasing usage of mineral nitrogen is evident in agricultural practices overseas (Watkin, Williams 1970), in New Zealand legume nitrogen is still by far the most important source of nitrogen in grassland farming. Nitrogen fixation in a fertile ryegrass-white clover association has been estimated to yield as much as 550 1b N Per acre per annum (Sears 1953), and it has been emphasised that for maximum benefit to the grass component in a mixed sward the white clover should be periodically defoliated and have ample light for regrowth (Butler, Greenwood &Soper 1959). The present study examines the effects of defoliation and shading on nodulation and nitrogen fixation in white clover.