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    Investigation of genetic changes in inoculant strains of Rhizobium trifolii isolated from the soil : a thesis presented in fulfillment of the requirements for the degree of Master of Science in Microbiology at Massey University
    (Massey University, 1985) O'Hara, Michael John
    Information about the fate of plant inoculating strains of Rhizobium trifolii entering the soil environment is incomplete. It is known that inoculating strains must compete with existing adapted strains, if such are present. It is not known whether or not the introduced strains can adapt to soil conditions. Strains of the white clover (Trifolium repens) symbiont, R. trifolii, were isolated from plants growing as a result of sowing virgin soil with bacteria-coated seed. Rhizobium bacteria were isolated from one nodule on each randomly chosen plant at two and then six months after sowing. Three different methods were used to type the isolated strains because of the importance of distinguishing between derivatives of the inoculant (R. trifolii #2668) and adapted rhizobia immigrating from adjacent pastures. Gel diffusion identification of antigens showed that all strains reacted positively to anti-2668 serum, although the response was not identical for all strains. The determination of intrinsic antibiotic resistance patterns showed that low level resistances were accumulating in a non-random manner as time progressed. Initial isolates showed the same pattern as 2668. Restriction endonuclease analysis of the isolated strains showed them all to have a high degree of similarity to 2668, with a few being identical in pattern. This was despite alterations in numbers and sizes of plasmids (as compared to those in 2668) seen in these isolates. A nif gene probe of a plasmid profile showed that several strains had alterations in the size and number of bands which would hybridize, as compared to 2668. The field isolated strains had gained the ability to produce a broad range bacteriocin-like inhibitor. Conjugation experiments between R. trifolii #0/18 and E. coli HB101 showed that this inhibitor was transferable to and expressable by the E. coli, strain. This suggests the existence of a broad host range replicon in the field isolates which either carries or mobilizes this function.
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    Analysis of genomic rearrangement and plasmid conjugation of an inoculant strain of Rhizobium leguminosarum bv. trifolii : a thesis presented in partial fulfillment of the degree of Doctor of Philosophy
    (Massey University, 1989) O'Hara, Michael J
    Variation of plasmid profile is the hallmark of Rhizobium lequminosarum bv trifolii strains isolated from the nodules of pasture plants. Previous attempts to demonstrate that these variant types were derived from the original inoculant were inconclusive. Subsequent laboratory based simulations revealed that the broad host range plasmid RP4 was capable of generating stable alterations in the plasmid and total genomic DNA profile. This variation involved an apparent loss of pSym, with concurrent loss of the nod and nif genes, but these strains produced nodules on white clover plants. The strains recovered from the nodules, while not identical, were clearly derived from the pSym¯ strain. A plasmid closely corresponding in size to pSym was detected in the nodule re-isolates and repeated trials of this experiment involving antibiotically marked (apparently) sym¯ strains confirmed this observation. This left the conclusion that the DNA was still there but in a form which was difficult to detect by conventional DNA hybridization procedures, a result which is not totally without precedent (Downs and Roth, 1987). The second portion of this project involved an investigation of the transmissability of plasmids from the inoculant strain 2668. The strain was marked with Tn5 with the expectation that some of the movable DNA pieces would carry a Tn5 insert and could thus be selected, for. A transmissable symbiotic plasmid was detected, as had been previously observed in other rhizobia by Johnston et al (1978). The plasmid was shown to be transferable, in an altered form, to soil microorganisms of unidentified genera, to a sym¯strain of R. lequminosarum bv trifolii, to its original parent 2668 and to E. coli. In all strains, with the exception of E. coli the nodulation genes were functional, producing normal looking nodules (on the outside) and in many strains nitrogen was also fixed, though not generally as well as by the parent 2668. The significance of a self-transmissable broad host range symbiotic plasmid is discussed in the context of the microbial ecology Of rhizobia.