Using phytohormones and genotype selection to enhance biological nitrogen fixation in soybean

Kempster, Robert and Dodd, Ian and Rufino, Mariana (2021) Using phytohormones and genotype selection to enhance biological nitrogen fixation in soybean. PhD thesis, Lancaster University.

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Abstract

Soybean is the most important plant-based protein source and may be vital to sustainable agricultural intensification, required to support an ever-growing global population. Soybean, however, has a high nitrogen (N) demand which often limits yield. Biological nitrogen fixation (BNF), through symbiosis with rhizobia, is a natural process through which legumes can derive much of their N requirement, with many environmental benefits over chemical fertiliser application. Enhancement of BNF therefore offers an opportunity to sustainably increase yield. Nodules are root organs that form following legume perception of nod factors (NF) produced by compatible rhizobia species, leading to expression of NF response genes, including ENOD40. They are the sites of BNF, providing a highly controlled optimal environment for rhizobia to fix N. Primarily, work on symbiosis has focused on the signalling events that coordinate nodule initiation and development, with a key role of phytohormones tightly controlling this process. However, the effect of varying nodule morphologies on BNF capacity is little studied. This thesis uses two approaches, genotypic variation and phytohormone application, to enhance BNF and seeks to understand if differences in nodule traits (such as size, number or distribution) can explain differences in BNF. To better quantify nodule traits, a novel image analysis protocol was developed. To determine whether genotypic variation in nodule traits could explain differences in BNF capacity, six soybean genotypes with contrasting BNF capacities (based on previous literature) were studied. Although BNF and nodule traits varied between genotypes by up to 60%, there was no simple relationship between nodule formation and BNF as hypothesised. Instead, both genotype and growth stage interacted to affect BNF capacity, with different nodule traits influencing BNF to different extents across the soybean genotypes when measured at different stages. Nodule traits (nodule area or diameter) derived from novel image analysis techniques were better correlated with BNF than traditional assessment methods (nodule number or weight). To establish if phytohormone application could enhance BNF, three positive regulators (cytokinin, auxin and gibberellic acid) were applied at various concentrations via different application methods (seed coat, seed soak, root application and foliar spray). Of the phytohormones tested, only cytokinin (kinetin) gave promising results and was most effective when applied as a seed soak treatment (10-9 mol L-1). This treatment increased total nodule area (32%), doubling BNF. Interestingly, cytokinin treatments led to the nodules being more tightly clustered near the root crown (81% decrease in distance from root crown) suggesting that early nodule development was promoted. Indeed, cytokinin seed treatment increased the expression of ENOD40 by 52% after 72 hours. This suggests that cytokinin seed soaking primes the seeds by promoting the symbiotic pathway. As both genotype and cytokinin application changed nodule development and BNF in controlled environments, their agronomic potential was assessed in field trials in Argentina with early and conventional sowing dates. Low root zone temperature is a key limiting factor of nodule formation but is little studied despite growing interest in European grown soybean and early sowing dates elsewhere, where crops often experience cool growing conditions. Cytokinin treatment had limited positive effects on BNF but did not increase yield or total N uptake. Genotypic differences in yield were not explained by overall BNF across the growth period and were better associated with differences in soil N uptake, with 21% increase in higher yielding DM50I17. Interestingly, DM50I17 showed increased early nodule development (18% increase in nodule number) and correspondingly greater (52%) early BNF which might have improved yield through greater canopy N accumulation (9%), available for remobilisation. These field trials indicate BNF is an important N source during cold conditions, maintaining N supply leading to more consistent yield. In contrast to previous literature, soil N uptake was more sensitive to low root zone temperature than BNF. Taken together, this thesis is the first detailed examination of whether nodule traits affect BNF in soybean. It explored the potential of genotype selection and phytohormone application to enhance BNF by altering the number or size of nodules. Differences in nodule morphologies, particularly nodules with 4 mm diameter, influence BNF with increased nodule development positively correlated with BNF. Cytokinin seed priming enhances root ENOD40 expression and thus nodule formation and BNF. However, future work should further examine the effect of cytokinin priming on the symbiotic pathway to fully understand the mechanism(s) behind this treatment. Equally, greater understanding of changes in endogenous phytohormone concentration across genotypes and following cytokinin treatment may explain nodule variation seen in this study. Although genotype selection and phytohormone treatments did not always enhance BNF sufficiently to increase soybean yield, cytokinin application altered important nodule traits that may provide greater N (and yield) benefits in specific agronomic circumstances with limited N inputs.