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    Drought stress responses of the Medicago truncatula - Ensifer meliloti symbiosis on nodule senescence and nitrogen fixation : a thesis presented in fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Science at Massey University, Palmerston North, New Zealand
    (Massey University, 2018) Dhanushkodi, Ramadoss
    While the use of nitrogen (N) fertilizer has provided many benefits to agriculture, incessant use of it can reduce soil organic matter and fertility resulting in lower crop yields. Legume plants can fix its own N2 through symbiotic nitrogen fixation (SNF) to promote plant growth by developing facultative root organs called nodules. Therefore, increasing the rate of SNF to reduce dependence on N fertilizer is a promising strategy for sustainable legume production. In legume cultivation this symbiotic process confronts two major challenges. First, SNF is suppressed by readily available N in the soil and second, the early senescence of N-fixing nodules can limit any further fixation to occur. Previous reports show that N fertilizer supresses SNF activity in many legumes and supports plant growth better than SNF. Moreover, suppression of SNF can also be induced by drought stress which causes early nodule senescence and subsequent reduced rates of plant growth. This thesis addresses the process of SNF suppression in two Medicago truncatula selected genotypes in response to external N treatment and also delivers a molecular view on the regulation of drought-induced nodule senescence processes.--Shortened abstract
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    A molecular and genetical analysis of symbiotic genes in Lotus rhizobia : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology at Massey University, Palmerston North, New Zealand
    (Massey University, 1984) Chua, Kaw-Yan
    Relative DNA homologies were determined among 24 strains of fast-growing rhizobia and 26 strains of slow-growing rhizobia with particular reference to those which nodulate Lotus species. Two major DNA homology groups were identified which shared less than 10% relative DNA homology. Fast-growing Lotus rhizobia were grouped with related fast-growing strains in DNA homology group I, and these strains are designated as Rhizobium loti. Slow-growing strains from Lotus, (Bradyrhizobium spp. (Lotus)), Glycine max (Bradyrhizobium japonicum), Ornithopus and Lupinus formed DNA homology group II which was further divided into 4 DNA homology subgroups. Bradyrhizobium spp. (Lotus) were shown to be genetically distinct from B. japonicum. Plasmid profiles were determined for strains of R. loti and Bradyrhizobium spp. (Lotus). All R. loti strains contained a single large indigenous plasmid, whereas strains from Bradyrhizobium spp. (Lotus) carried multiple plasmids of molecular weights ranging from 130-280 MDal. A plasmid-cured derivative of R. loti NZP2213 was isolated and found to still form effective nodules on Lotus tenuis, suggesting that nodulation and nitrogen fixation genes are not plasmid-borne in this strain. The functions of the indigenous plasmids carried in Lotus rhizobia are unknown. Symbiotic mutants of R. loti strain NZP2037 were isolated by random Tn5-mutagenesis. Mutants included strains blocked in root hair curling (Hac), nodule initiation (Noi), bacterial release (Bar) and nitrogen fixation (Cof) on Lotus pedunculatus. The nodulation (nod) gene region from R. loti strain NZP2037 was isolated from a pLAFR1-NZP2037 gene library using the cloned Tn5 containing EcoRI fragment from the Nod¯ mutant as a probe. Two cosmids were isolated and were found to complement the NZP2037 Nod¯ mutant. Hybridisation and complementation experiments confirmed that a 7.1 kb EcoRI fragment present in both nod cosmids carried gene sequences involved in nodulation. An EcoRI and Hind III restriction enzyme map of the nod gene region in R. loti NZP2037 was constructed using nod cosmids pPN305 and pPN306. Using the cloned R. loti NZP2037 nod gene region (7.1 kb EcoRI fragment from pPN305) as a hybridisation probe, highly conserved DNA sequences from other strains of R. loti and Bradyrhizobium spp. (Lotus) were identified. The nod gene region from Bradyrhizobium spp. (Lotus) strain NZP2309 was isolated by direct 'in planta' complementation of the R. loti Nod¯ mutant using a NZP2309 pLAFR1 gene library. Comparative physical and genetical studies showed that the R. loti NZP2037 nod gene region isolated shared functional similarities with previously isolated nod gene regions from R. trifolii and R. meliloti despite the fact that only weak DNA homology was observed between the corresponding regions. This suggested that at least some of the nod gene sequences carried on the cloned R. loti nod gene region belong to the highly conserved 'common' nod gene sequence category.
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    Isolation and DNA sequence analysis of a Rhizobium loti gene required for effective nodulation of Lotus pedunculatus : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology at Massey University, Palmerston North, New Zealand
    (Massey University, 1989) Ward, Lawrence James Henry
    A Rhizobium loti gene required for effective nodulation of the host Lotus pedunculatus has been identified by transposon Tn5 mutagenesis. Cosmids from a R. loti gene library which complemented a previously isolated mutant strain, PN239, (Chua et at 1985; J. Bacteriol. 162; 335-343) at this locus were identified by in planta complementation. A physical map of these cosmids was constructed and the site of insertion of the Tn5 was mapped to a 7.5 kb EcoRI fragment common to all cosmids which complemented the mutation. This 7.5 kb EcoRI fragment was subcloned into pBR328 and pLAFR1 and a more detailed physical map constructed. The 7.5 kb EcoRI fragment in pLAFR1 was able to complement the Tn5 mutation when introduced into strain PN239. Further Tn5 mutagenesis of the 7.5 kb EcoRI fragment was carried out in E. coli and the mutations were homogenotised into R. loti NZP2037. Three additional mutations were isolated which caused a Fix- phenotype on Lotus pedunculatus. The Tn5 inserts which caused a Fix- phenotype were mapped to positions adjacent to the position of the original mutation in strain PN239. All other Tn5 insertions isolated in the 7.5 kb EcoRI fragment gave a Fix+ phenotype on Lotus pedunculatus. A region was sequenced which was involved in effective nodulation of Lotus pedunculatus as indicated by the position of the Tn5 insertions. Analysis of the consensus sequence of 2307 bases identified a potential open reading frame (ORF) of 576 base pairs, coding for a putative protein of 21.2 kD. The positions of the Tn5 insertions causing a Fix- phenotype and the adjacent Tn5 insertions which did not affect fixation were determined in the sequence. The position and orientation of the ORF identified was consistent with the sequenced positions of these Tn5 insertions. A fragment containing most of the ORF identified from the sequence was used as a hybridization probe to various strains of rhizobia. Homology was only demonstrated with DNA from other R. loti strains. of loti strains containing Tn5 insertions which were Fix- on Lotus pedunculatus were found to be fully effective on Lotus corniculatus. These observations suggest that the gene characterised in this investigation may be involved in the host specificity of R. loti for Lotus pedunculatus.