Diversity of rhizobia nodulating the tree legumes Acacia mangium and Paraserianthes falcataria and their interaction with arbuscular mycorrhizal fungi in young seedlings

Acacia mangium and Paraserianzthes falcataria are important leguminous tree species in Indonesia and many other countries. These trees form symbiotic relationships with rhizobia and arbuscular mycorrhizal fungi (AMF). These symbioses enable the trees to grow in adverse soils with low N and P availability, which is typical of tropical soils. Despite the extensive use of A. mangium and P. falcataria, the rhizobia that nodulate these species have not been subjected to a comprehensive systematic analysis, nor have the synergistic benefit of the tripartite symbiosis been fully exploited to improve the growth and nutrient acquisition of the trees. In this study, fifty seven rhizobial strains nodulating either A. mangium (26 strains) or P. falcataria (31 strains), were isolated from diverse geographical areas in Indonesia and were studied for their phenotypic and genotypic relationships. Fifty one strains of non-nodulating root-nodule = bacteria isolated from the root-nodules of A. mangium and P. falcataria were also subjected to the phenotypic characterisation. Five and seven representatives of the rhizobial strains from A. mangium and P. falcataria, respectively, were tested for their effectiveness to promote plant growth and N-fixation when co-inoculated with one of Indonesian isolate of the three species of AMF, Glomus manihotis BEG112, Gigaspora rosea BEG111, and Scutellospora heterogama BEG40. Results of the phenotypic characterisation studies showed that rhizobial strains nodulating A. mangium and P. falcataria exhibited the phenotypic characteristics of Bradyrhizobium and were generally distinct from the non-nodulating root-nodule bacteria. This was confirmed by the results obtained using PCR-RFLP-SSCP analysis on the 57 rhizobial strains and the sequence data of the 16S rRNA gene of representative strains. Furthermore, results from the molecular analysis showed that these strains could be divided into two distinct groups. One group was genotypically close to Bradyrhizobium elkanii and the other to Bradyrhizobium japonicum. Additionally, A. mangium was also nodulated by a strain which was closely related to Mesorhizobium loci. The strains of B. elkanii and B. japonicum nodulating A. mangium and P. falcataria were ubiquitous throughout the Indonesian archipelago. The data from the interaction experiment between AMF, rhizobial strains and P. falcataria showed that rhizobial strains nodulating this species varied in their capacity to promote plant growth and N fixation. These capacities changed considerably when each rhizobial strain was co-inoculated with different species of AMF. Under the conditions used, A. mangium seemed more selective for its AMF and rhizobial symbionts, compared with P. falcataria, showing optimal compatibility only with Gi. rosea BEG111 and B. elkanii strains. This was indicated by a significant improvement in growth and nutrient acquisition of the seedlings when inoculated with these microsymbionts alone or in combination, compared with uninoculated controls or with seedlings inoculated with B. japonicum strains and the other two AMF species in any combination. A novel microcosm system, the Petri-dish microcosm, was developed in this study to investigate the establishment of AMF species from different genera. The results showed that Ac. tuberculata BEG41, G. manihotis BEG112, Gi. rosea BEG111, and Sc. heterogama BEG40 differed considerably in their spore germination, root colonisation, the spread of the extra-radical mycelium (ERM), architecture of the ERM, and spore formation. These observations may suggest differences in their ecological strategy and requirements to complete their life-cycles, which may eventually affect the outcome of their interactions with rhizobial strains and host plants, in terms of nodule formation, N-fixation and plant growth. In conclusion, this work indicates that rhizobia nodulating A. mangium and P. falcataria are phenotypically diverse, and genotypically close to B. elkanii, B. japonicum, and M. loci. These rhizobial 111 strains were also functionally diverse (ability to promote plant growth and N-fixation), and the functional diversity increased when they co-habited roots with AMF. The importance of an appropriate strategy for screening effective combinations of AMF and rhizobial strains for different tree species, and the potential application in nursery production of trees in Indonesia, is also highlighted