Epsilon Archive for Student Projects

Abstract

Bacteria play several important ecological functions in soil and in relation to plants. Legumes, such as the recently introduced grain legume narrow-leafed lupin (Lupinus angustifolius L.), develop N2-fixing root nodules in response to infection by soil bacteria generally called rhizobia but can also interact with other soil bacteria. To investigate the identities and diversities of root nodule bacterial communities, seedlings of two cultivars of narrow-leafed lupin (Galant and Bora) were inoculated with a range of soil samples. The samples were collected from fields at three locations where four different farming practices were applied. They were described as organic farming, as integrated conventional and organic farming, a crop demonstration site and a low-intensive agricultural field having a natural population of Lupinus polyphyllus, a wild relative of L. angustifolius. In the experiment, half the numbers of seedlings were in addition to soil also inoculated with a pure culture of Bradyrhizobium sp. to ensure root nodule formation. Seven weeks after inoculation, bacteria were extracted from the rhizosphere of young roots, the exterior of root nodules (nodulesphere) and the interior of surface-sterilized root nodules. The extracts were used for extraction of DNA and for isolation of bacteria into pure culture. Community diversity analysis was carried out using terminal restriction fragment length polymorphism (T-RFLP) analysis of the 16S rRNA gene. Bacterial isolates were identified using PCR of the 16S rRNA gene and the 16S-23S-internal transcribed spacer region. All soil samples were found to lack compatible nodule-inducing Bradyrhizobium strains except the soil with L. polyphyllus, while all seedlings inoculated with the Bradyrhizobium isolate formed root nodules. The T-RFLP analysis showed that a certain restriction fragment, corresponding to Bradyrhizobium, dominated in the nodules, which is expected since no nodules were formed unless plants were inoculated with Bradyrhizobium. Additional restriction fragments represented other bacteria in the communities, which suggest that there exist diverse bacterial communities in and associated with the root nodules. Of the three root zones, the nodulesphere was found to have the most diverse bacterial communities and varied among soils. Some of the bacterial isolates that were identified were found to belong to species that are considered to be plant-growth promoting bacteria. The functional roles of the members of these bacterial communities need further investigation.