Molecular analysis of root-associated diazotrophs in important plants from Southern Africa and South America

Abstract

Biological nitrogen fixation (BNF) is a process exclusively carried out by a group of prokaryotes known as diazotrophs that possess the enzyme nitrogenase. This enzyme converts Nitrogen (N2) to ammonia (NH3), which can then be assimilated by plants for growth. The study of active diazotrophic bacterial communities in natural environments has been carried out using both culture-dependent and culture-independent techniques. This thesis focuses on the active nitrogen-fixing bacteria associated with the roots of non-legume and legume plants, specifically sugarcane (a principal crop for sugar and biofuel production) and the South African legume tree, mopane (used as animal feed and for domestic purposes in the region). The presence of the nifH gene, which encodes for the iron protein of the nitrogenase complex, has been widely used as a functional and phylogenetic marker to indicate nitrogen fixation in the environment. In order to improve nifH gene detection, the effect of locked nucleic acids (LNAs) substituted primers in RNA extracts from roots of rice and sugarcane was studied by direct reverse transcription polymerase chain reaction (RT-PCR). It was found that the use of an LNA modified RT primer increases the sensitivity and efficiency of RT-PCR reactions and therefore allowed the detection of nifH transcripts where DNA primers alone failed to produce RT-PCR products. RT-PCR was then used to detect active diazotrophs in sugarcane root samples from Africa and America. Denaturing gradient gel electrophoresis profiles showed a low diversity of diazotrophs in all samples locations. A major nifH phylotype was found to have a high DNA sequence identity (93.9 99.6%) with the partial nifH sequence from Rhizobium rosettiformans, which belongs to a genus typically found in legume plants. The prevalence of this globally distributed nifH phylotype indicates a tight interaction between the corresponding nitrogen fixing bacteria and their host. In mopane samples, root nodules were not detected. However, in some samples the lateral roots showed an outgrowth-like protuberance. Their root-associated bacterial isolates belonged to Actinobacteria, Firmicutes and Proteobacteria. The nifH phylotypes found are related to Rhizobiales, Spirochaetes, Firmicutes, Bacteroidetes and delta-Proteobacteria. These were different from the phylotypes found by the 16S rRNA analysis, which are mainly dominated by Actinobacteria-like sequences. Additionally, the isolation and characterization of putatively beneficial bacteria from traditional crops (sorghum, pearl millet and maize) grown by subsistence farmers in the Kavango region of Namibia was performed. Gram-positive bacteria (Firmicutes and Actinobacteria) showed a higher diversity than gram-negative bacteria (Proteobacteria). Plant-growth promoting characteristics were tested in vitro and revealed promising candidates with multiple beneficial properties. This collection of native microorganisms has the potential for application as inoculants adapted to regional conditions. In conclusion, this PhD work shows that the application of a nifH-mRNA based approach can provide insights into the active diazotrophic communities of mopane and sugarcane, plants that were studied for the first time by molecular approaches. It was found that sugarcane plants are mainly associated with one diazotroph, extending the typically root-nodule-associated rhizobia to this graminaceous plant. On the contrary, the diazotrophs associated with mopane are related not only to Rhizobiales, increasing the spectrum of nitrogen fixers in this legume plant. Further studies are required to isolate these bacteria and test their N2 fixation capacity in planta. For sugarcane in particular, it is important to determine whether the N2 fixation occurs only in the root and/or the shoot and to quantify the relative contribution of each part to the total amount of N2 fixed.