Aserse, Aregu Amsalu
(Helsingin yliopisto, 2013)
Nitrogen is one of the major essential nutrients for plant growth along with phosphorus and potassium. Some specialized bacterial and archaeal species are able to fix atmospheric N2 into NH3, and that is subsequently converted into plant usable form of nitrogen, NH4+ or NO3-. The biological nitrogen fixation (BNF) process that occurs by the symbiotic interaction of leguminous plants and certain bacterial species (commonly known as rhizobia) is the main source of biological nitrogen input into the soil and therefore plays an important role in maintaining the sustainability of ecosystem services. Due to the fixed N they get from symbiosis, legume species grow better than other plants in nutrient poor, degraded soils. Thereby leguminous trees and shrubs restore degraded farmland and soil fertility by increasing the content of nitrogen and organic carbon in the soil. The versatile leguminous trees and shrubs, such as Erythrina brucei, Crotalaria spp., and Indigofera spp., can be used as forage for cattle and applied as intercrops or fallow crops in low-input agriculture. The usefulness of these legumes can be boosted by inoculating them with effective symbiotic nitrogen-fixing rhizobia. The yield of food legumes such as common bean and soybean can also partly be increased through the use of efficient rhizobial inoculants. Thus, detailed information about the indigenous rhizobia nodulating local food and woody legumes is essential for selecting good inoculant strains. Therefore, this thesis deals with diversity and phylogeny of 143 bacterial isolates obtained from root nodules of E. brucei, Crotalaria spp., Indigofera spp., common bean and soybean growing in different sites in Ethiopia.
Taxonomy of the root nodule bacteria was studied using multilocus sequence analyses (MLSA) of the core genes 16S rRNA, recA, rpoB, and glnII. Phylogeny of nodulation (nodA, nodC, nodK/Y) and nitrogen-fixation (nifH) genes of the rhizobia were also studied. The whole genome based AFLP fingerprinting technique was used to study the diversity of the strains within the species. Based on MLSA and AFLP fingerprinting analyses combined with nodulation test result, twenty-five strains belonging to the Rhizobium leguminosarum complex (Rhizobium phaseoli, Rhizobium etli, Rhizobium leguminosarum and a novel Rhizobium taxa) were found to be true common bean nodulating rhizobia in Ethiopia (Paper I). Fifty-six strains isolated from root nodules of E. brucei, Crotalaria spp., Indigofera spp., and soybean (Glycine max) were mainly identified as genetically very diverse slow-growing Bradyrhizobium species, being distributed into fifteen phylogenetic groups under Bradyrhizobium japonicum and Bradyrhizobium elkanii super clades. The majority of these strains represented undescribed Bradyrhizobium genospecies. Two unique lineages which most likely represent novel Ethiopian Bradyrhizobium species were discovered among the collections (Paper II). In addition to Bradyrhizobium species, a few Rhizobium species (six strains) were found to sporadically nodulate E. brucei, Indigofera spp., and common bean. Fifty-six non-symbiotic endophytic bacterial strains representing diverse Gram-negative and Gram-positive bacterial genera were also isolated from nodules of E. brucei, Crotalaria spp., Indigofera spp., soybean and common bean (Paper I, III). Among the non-symbiotic bacteria, five strains obtained from nodules of Crotalaria spp. and E. brucei represented a putative novel Rhizobium species (Paper III).
Phylogenetically the nodA genes of all Ethiopian Bradyrhizobium species belonged to the cosmopolitan nodA clade III.3, which includes nodA genes from Bradyrhizobium species nodulating diverse legume hosts in sub-Saharan Africa. The nifH and nodY/K gene phylogenies of the Ethiopian Bradyrhizobium strains were generally consistent with the nodA gene phylogeny, supporting the monophyletic origin of the symbiotic genes in Bradyrhizobium (Paper II). The symbiotic gene phylogenies of Bradyrhizobium species were somewhat consistent to their housekeeping gene phylogenies. Nevertheless, the symbiotic gene phylogenies of different Rhizobium species (Paper I and III) were fairly similar regardless of their taxonomic background, suggesting that, in contrast to the core genome of the species, the symbiotic genes required for nodulation and nitrogen fixation might have a common origin in Rhizobium, indicative of horizontal gene transfer among these rhizobia.
The nodulation test results showed that most rhizobial species were effective in nitrogen fixation on their respective host plants. Non-nodulating, endophytic bacterial strains representing seven genera, namely Agrobacterium, Burkholderia, Paenibacillus, Pantoea, Pseudomonas, Rhizobium and Serratia, were found to colonize nodules of Crotalaria incana and common bean when co-inoculated with symbiotic rhizobia. In addition, the majority of nodule endophytic bacterial strains and the sporadic symbionts showed several plant growth promoting activities, which indicate their potential role in improving plant growth.