WP 3: Identifying ‘Elite Rhizobia’ of Clover, and Birdsfoot Trefoil [James Hutton Institute].

The specific plant varieties which are the focus of the NUE-Leg project, have been provided by the work-program Coordinators, Germinal. These include novel varieties which offer a range of improved and advantageous traits (see Work Package 2 for details). The novel varieties span white and red clover (Trifolium repens and T. pratense; respectively), plus Lotus corniculatus L., also known as ‘Birdsfoot Trefoil’. These crop species belong to a family of plants which are collectively termed ‘legumes’, which are characterised by their ability to grow in the absence of a plant nutrient which is critical for growth, specifically, nitrogen.

Legumes such as clover and Birdsfoot Trefoil, are a type of crop termed “forages”, since they are consumed by farmed animals such as cattle, and sheep. These crop types are therefore cultivated for their green biomass, as opposed to the seeds which are harvested from other types of legumes, called the ‘grain legumes’ and include crops such as peas, faba bean, and lupin. Whether cropped legumes serve as forage- or grain-types, they are all characterised by their capacity to form a symbiotic relationship with a specific group of naturally occurring soil bacteria which are referred to collectively as ‘rhizobia’.

Rhizobia span a diverse array of genetically and functionally distinct types, and when the host plant and rhizobial strain are compatible; the rhizobia can stimulate the formation of legume root nodules, within with the rhizobia can carry out a process called ‘biological nitrogen fixation’ (BNF). This process converts (or “fixes”) largely inert atmospheric di-nitrogen gas into biologically useful forms of nitrogen. The energy for that process is provided by the legume in the form of sugar as ‘photosynthate’ (from photosynthesis), supplied to the rhizobia in the root nodules. The nitrogen fixed from this symbiosis is utilised by the legume for growth, and development; so much so that legume growth (biomass) is closely linked to the BNF potential of the legume-rhizobia symbiosis. Due to this unique ability, legumes can show high levels of productivity without the necessity of synthetic nitrogen fertiliser application. Therefore, well managed legumes can help realise more-sustainable cropped systems, though natural (organic) nitrogen-provision and -cycling.

Legumes, and specifically forage legumes such as the clovers and trefoils also present highly nutritious feedstocks for ruminants such as cattle and sheep – though are also beneficial to insects such as pollinators – since their vegetation and flowers are rich in protein, and many other beneficial phytochemicals. In addition, legumes are also effective at improving soil fertility, as a manure. So, considering all these aspects: legumes are highly efficient multi-functional crops that present numerous benefits to the agri-ecosystems, farmed animals, the wider food web, and numerous soil qualities (in addition to plant available nitrogen).

Within the NUE-Leg project, the research team members from the James Hutton Institute and Aberystwyth University have been recovering clover- and lotus-compatible rhizobial strains from culture collections. In addition, they have been ‘trapping’ new rhizobial strains directly from field soils. Preliminary results for the lotus have shown that a wide range genetically diverse rhizobia strains are able to induce the formation of effective root nodules on this host; however when it was grown in field soils, not all plants formed root nodules highlighting that there are locations where there appeared to be no rhizobia capable of nodulating lotus, which would greatly compromise crop performance and yield. In such cases, and for optimum levels of plant production, compatible rhizobia would need to be added (to seed, at sowing), as an ‘inoculant’ to help ensure BNF and good crop yields.

In addition, 190 clover-associated rhizobia have been collected (either recovered from the culture collections or newly trapped from field soil). These have been genetically characterised to identify 51 genetically distinct groups, with representative strains of each group now being characterised further as inoculants for the NUE-Leg project clovers cultivated under nitrogen-fertiliser free conditions in controlled-glasshouse conditions. This research aims to determine which strains show the greatest potential to increase plant growth, such strains may be termed ‘elite rhizobia’.

These potentially elite strains will then be applied as seed inoculants to the novel Lotus and clover crop varieties (and standard commercially available crop varieties, as controls) in field trials. Plant production will then be recorded, and leaf material sampled to determine BNF – that is, the absolute quantities of nitrogen derived from air. Further characterisation of the rhizobia will include sequencing their whole genomes – since such information can be allied to data on crop performance and prevailing growing conditions. This will enable researchers to identify specific genes which possibly underpin the elite potential of these rhizobia, as well as providing information on the adaptation of these strains with respect to environmental conditions – as some strains may show elite in-field performance only in locations with specific pedoclimatic (soil and weather) attributes. Such insight informs the use of specific rhizobia as commercial inoculum for different locations; and optimise the potential for more-sustainable production systems.