Characterisation of grain legume rhizobia for the potential development of inoculants with an improved shelf-life

Abstract

Nitrogen (N) is a limiting element for plants; however, the use of synthetic N fertilisers in agriculture has increased crop production and yield. Importantly, a significant proportion of chemical fertilisers applied to soils will not be taken up by the roots of crops, but lost to the environment via run-off into waterways, or denitrification by soil bacteria. Legumes are plants that can transform atmospheric di-N into ammonia through a symbiotic association with rhizobia, a group of N-fixing bacteria, in root organs called nodules. Natural populations of rhizobia often exhibit below optimal N-fixation or nodulation, although so-called 'elite’ strains with optimal abilities can be applied as inoculants. Although inoculants can be formulated with crop-compatible elite strains of rhizobia, their shelf-life is often compromised by high rates of cell die-off caused mainly by desiccation, which is an environmental stress that rhizobia are not good at withstanding. Therefore, there is a need to identify novel rhizobial strains that are able to tolerate desiccation stress. Recent evidence has suggested that strains isolated from areas with higher water deficit can better tolerate desiccation than those from wetter locations. Therefore, the overarching aim of this project was to isolate and characterise novel rhizobia strains from a semi-arid environment and assess their tolerance to desiccation for their potential use in inoculants for grain legumes. In addition, this project also evaluated the impact of agricultural land management on natural soil populations of rhizobia. Over 80 strains were isolated from soil from a semi-arid area of Spain using pea as a trapping plant. After a series of glasshouse and growth room experiments two strains were tested in field trials during two consecutive seasons where they showed a similar performance to strains from commercially available inoculants (used as positive control strains). Desiccation tolerance of strains isolated from Spain was tested and compared in vitro against strains from a wetter environment. The strains isolated from the semi-arid region showed 1.55-fold increased tolerance to desiccation. The genomes of 70 strains were sequenced and characterised, and a genome-wide association study on desiccation tolerance revealed that genes involved with regulating the concentration of solutes in the cytoplasm, and the protection and stabilisation of genetic material, were involved in the tolerance to this environmental stress. Finally, it was found that a change in land management and the presence of legumes in the crop rotation increased nodulating rhizobia in soil by 15 and 30 % respectively over a period of 4 years. This project has successfully isolated strains with comparable symbiotic performance to standard commercial strains that show improved tolerance to desiccation, which makes them potentially superior for use in commercial inoculants with longer shelf-lives. Furthermore, this project has demonstrated that the reintroduction of a legume host after long absences produces an at least 4-year lasting effect that increases the proportion of nodulating rhizobia in soil year-on-year.