Ecology of Root Nodule Bacterial Diversity: Implications for Soybean Growth

Diazotrophs supply legumes such as soybean (Glycine max L. Merr) with nitrogen (N) needed for protein synthesis through biological nitrogen fixation (BNF). Through BNF, these bacteria such as Bradyrhizobium that reside in soybean root nodules, convert atmospheric nitrogen (N2) into ammonia (NH3/ NH4), a form that is biologically available for use by the plants, in return for photosynthate carbon from the plant. Abiotic stresses such as drought disrupt BNF and subsequently affects soybean yield. In addition, increasing demand for soybean is leading to supplementing its growth with synthetic N fertilizer. However, fertilizer application is known for its detrimental effects on the environment causing waterways eutrophication contributing to global warming. On the other hand, diazotrophs can supply soybean with up to 90% of N need. As such, improving the understanding and exploiting the relationship between soybean and diazotrophs is key to promoting the sustainable growing of soybean. This dissertation here investigates three main questions. First, how the soybean-diazotrophs respond to changes in water such as rainfall and irrigation. Second, how changes in these bacterial diazotrophs are related to levels of BNF, and N-related soybean molecular markers. Finally, as my colleagues and I found non-diazotrophs in the nodules of some soybean plants, I was curious about the role they are playing inside the nodules in concert with the diazotrophs.

The main hypotheses tested in this dissertation are that root nodule bacterial community (bacteriome) would (1) vary by plant type, (2) respond to changes in water, and (3) be related to BNF. To answer the research questions, I devised the dissertation as follows. In Chapter 2, my colleagues and I used nine commercial cultivars of soybean that vary in drought tolerance and agronomic traits. We show that soybean sometimes, but not always, harbor a consortium of non-nitrogen fixing bacteria belonging to Pseudomonadaceae and Enterobacteriaceae families. However, as expected, nodules diazotrophs rather than non-diazotrophs responded most to changes in soil water status. In chapter 3, I used a collection of 24 genotypes of soybean that vary in their ability to fix nitrogen. The results revealed that the bacteriome diazotroph alpha diversity metrics, phylogenetic richness and evenness, was correlated with changes in BNF. Moreover, few N-related molecular markers were associated with some of the bacteria. However, we have also observed a strong effect of the environment on the diazotroph driven process of BNF (i.e. 39%-75%). For chapter 4, we sequenced three of the Pseudomonas spp. strains that were subsequently recovered again from a diversity of soybean nodules in field trials. I found that one of the strains has the ability to adapt to the nodule's unique hypoxic conditions, supporting Bradyrhizobium nodulation and possibly nodule iron. The results include the draft assembly of the proposed Pseudomonas nodulensis sp. nov. as a novel species of nodule adapted bacteria belonging to the P. fluorescens complex.

The results of this dissertation contribute to the basic knowledge needed to advance sustainable breeding and management of soybean. Nodule diazotrophs are sensitive to water status e.g. drought, and other experiments have shown that the nodule bacteriome is the driver of BNF. Thus, improving the understanding and exploiting the nodule bacteriome will support developing more resilient cultivars of soybean that are efficient in BNF, and tolerant of stress. Identifying and testing diazotrophs and atypical nodule bacteria will provide a platform for developing new inoculants and biofertilizers.