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

Abstract

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.