Rhizobia inoculation to mitigate nitrous oxide (N2O) emissions from UK grasslands and herbal leys under intercropping systems.

N₂O is a potent greenhouse gas, with ~300 times the warming potential of carbon dioxide.  The current trajectory for N₂O emissions follows the highest warming RCP8.5 scenario, with agriculture accounting for ~70% of global emissions.  As demand for food and livestock feed is expected to increase, mitigation measures which reduce agricultural N₂O emissions and simultaneously increase nitrogen use efficiency (NUE) are urgently required to limit warming below the 2°C target set by the Paris Agreement.

The promotion of biological nitrogen fixation (BNF) in crop and forage systems via the incorporation of legumes has been advocated as a N₂O mitigation strategy because it reduces synthetic N fertiliser application and increases NUE.  However, novel strategies suggest that in addition to BNF, manipulation of the soil microbiota could hold the key to N₂O mitigation.  Soybean studies have successfully identified strains of symbiotic N-fixing rhizobia which can reduce N₂O because they possess the gene encoding for nitrous oxide reductase (nosZ).  The potential of N₂O-reducing (NosZ+) rhizobia inoculums could therefore be critical to agricultural N₂O emission mitigation; however, few studies have explored other legume-rhizobia associations for NosZ+ strains.  Most notable is the complete lack of research on permanent grassland ecosystems, which cover 40% of global land surface and account for 54% of global N₂O emissions.

This study aims to investigate the potential of clover-rhizobia associations to mitigate N₂O emissions from UK grasslands and herbal leys under intercropping systems.  Soils from five different land uses were sampled from FarmED (agroecology demonstration farm) and Pudlicote Farm in the Cotswolds, UK: unfertilised permanent pasture, unfertilised clover/grass sward, herbal ley (1^st and 5^th year) and conventionally farmed winter wheat.  Native rhizobia present in the soil samples were selected by the growth and nodulation of Red Clover (Trifolium pratense) plants.  Rhizobia extracted from the harvested root nodules were cultured on yeast mannitol agar to isolate individual strains.  Strains then underwent gDNA extraction and whole-genome sequencing using the Illumina NovoSeq X platform to determine the presence of the nosZ gene.  Biogeochemical analysis of the soils was related to the presence/absence of the nosZ gene to infer potential genotype environmental controls.

Finally, identified NosZ+ strains will undergo a phenotype assessment using a soil-plant-atmosphere mesocosm experiment, whereby N₂O emissions from clover plants inoculated with NosZ+ strains will be monitored. Control strains; Rhizobium leguminosarum bv.trifolii T117 (nosZ+) and T132 (nosZ-) were obtained from the MIAE collection (INRAE, France) and will be tested alongside Bradyrhizobium diazoefficiens G49 (nosZ+) (soybean specific strain) and the identified native strains. The overall aim of the study is to create a rhizobia inoculum able to reduce N₂O emissions when included in the intercropping sequence of leys and pastures, thus contributing to Net Zero global strategies.