Functional and community investigation of nodule endophytes for the selection of phosphate solubilizing rhizobial inoculants for clover : A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Lincoln University

Abstract

Nitrogen (N) and phosphorus (P) are limiting nutrients for plant growth and are the largest fertilizer inputs in agriculture. Phosphorus fertilizer has a low efficiency of use with only 5-30% utilized by the plant, the rest forming metal complexes and becoming unavailable to plants. In New Zealand, most pastures contain Trifolium repens (white clover) and T. subterraneum (subterranean clover) grown in mixture with forage grasses. Clovers are nodulated with Rhizobium spp. to fix nitrogen. These bacteria can also release organic acids, phytases and phosphatases to make P soluble and plant available. The goal of this thesis was to understand the potential of P solubilizing rhizobia (PSR) to perform dual functions of P solubilization (PS) and N fixation and determine the magnitude of growth benefit to white clover. To achieve this, the study was divided into four objectives: 1) to assess the populations of P solubilizing bacteria inhabiting clover nodule, 2) to profile the nodule bacterial community in different clover hosts and study how its affected by different soil P environments, 3) to increase the mechanistic understanding of PS and assess N fixation by PSR in vitro and 4) to assess growth promotion of clover by PSR. A total of 1,915 nodule inhabiting bacteria were collected from white and subterranean clovers, grown in soils with different P fertilization histories. A subset (1,260) of these bacteria were used to determine that hydroxyapatite (HA) was most suitable substrate for in vitro screening. Of the 79 nodule bacteria that could solubilize P, 32% were rhizobia, 29% Enterobacter spp. and 17% Pseudomonas spp.. More PSB were found in subterranean clover (58) nodules than the white clover (21) nodules. Subterranean clover nodules also had more non-rhizobial PSB (44) compared with white clover nodules (10), suggesting that there may be an active recruitment by subterranean clover. PSB had a higher frequency in soils with high levels and duration of P fertilization, suggesting that the soil bacteria that could solubilize more P may have been selected. 16S rRNA gene amplicons sequencing (Illumina HiSeq) was used to characterise the bacterial communities of nodules, roots and rhizosphere of white and subterranean clovers, grown in different P fertilization histories. Bacterial communities were different in the two clover hosts and in the three P treated soils with 15 - 20% of nodule reads being non- rhizobial. More gammaproteobacteria, especially Pseudomonas and Enterobacter spp. were found in subterranean clover nodules. Nodule and root communities of subterranean clover were more affected by P fertilization than white clover. Root and nodule communities were more similar to each other than rhizosphere communities, suggesting that non-rhizobia entered nodules through the roots. P solubilization was quantified in liquid media and a decrease in pH was the main mechanism behind PS. Enterobacter and Pseudomonas had higher PS activity than Rhizobium spp.. The PS activity was unaffected by the presence of soluble P, suggesting that this activity may be a by-product of some other metabolic activity. Glucose was essential for the PS activity and only a few Enterobacter spp. could solubilize hydroxyapatite utilizing mannitol, indicating that these strains may have additional or alternate mechanisms for PS. Only 11 PSR could nodulate white clover, therefore, these strains were selected for further experiments. Of these, six increased (p<0.05) clover growth compared with the current commercial inoculant (TA1) while performing dual functions. These six were further assessed in a glasshouse experiment in soils with a background rhizobial population of 104 CFU per g. When added along with hydroxyapatite (1 g/kg), three strains increased plant growth compared with TA1 by 21-35%. These three strains were recommended for field experiments and potential commercialization. Overall, this study successfully investigated clover nodule bacterial community that could solubilize P and studied the effect of P fertilization on this community. This study also increased the mechanistic understanding of PS activity. Finally, this study defined that dual function rhizobia could improve clover growth.