Nodulation of the N2-fixing legume narrow-leafed lupin (Lupinus angustifolius L.) : soil inoculation methods, root nodule development and molecular identification of rhizobia
Abstract: The following paper is a study on the interaction between Lupinus angustifolius L. and N2-fixing nodulating bacteria in selected Swedish soils. Bacteria being able to induce N2-fixing symbiotic root nodules on legume plants are generally called rhizobia. The main objectives were to investigate (1) the presence of rhizobia compatible with two cultivars of L. angustifolius, Bora and Galant in an agricultural soil in Skåne and a soil in Uppsala where the perennial lupin L. polyphyllus is growing, (2) the development of lupin root nodules, (3) taxonomic identity of the nodulating rhizobia using DNA sequencing and (4) effects of various pre-treatments of the inoculum as well as seed treatments on the nodule formation in L. angustifolius. Two experiments were conducted by growing plants in controlled light and temperature during May to August 2010, in the growth rooms at the Department of Plant Biology and Forest Genetics, SLU. The Skåne agricultural soil and the Uppsala perennial lupin soil induced nodules in both cultivars. The number of nodules per plant using a seed dipping inoculation technique was similar for both soils. However, in the experiment where soil suspension was added as inoculum to the pot the number of nodules was lower for the perennial soil suggesting that mineral nitrogen or another soil factor inhibited nodule formation. Using a method where seeds were inoculated by dipping into the soil suspension, the effect of drying soil in room temperature before use as inoculum and the effect of time after germination on nodule formation were investigated. Fresh soil induced a higher number of nodules in L. angustifolius than the dry soil indicating a decline of the infective population size or diversity in the dry soil. Seeds sterilized immediately prior to inoculation and seeds germinated for one day gave plants with higher nodule number compared to seeds germinated for two days. Sequences of a part of the 16S-23S internal transcribed spacer showed that the nitrogen fixing rhizobium of both soils had highest identity to Bradyrhizobium sp in the B. japonicum group. However, a phylogenetic analysis showed good separation of the two clusters formed by the rhizobia of the two soils suggesting that the Bradyrhizobium sp in the Skåne agricultural soils is genetically different from that in the Uppsala perennial lupin soil.
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