Identification and characterisation of bacterial endophytes in miscanthus

The hypotheses we aim to test in this project are (1) whether an endophyte population confers any advantage to the Miscanthus host particularly in terms of increased biomass under low nitrogen or in saline conditions, (2) whether endophyte populations are genotype specific, and (3) whether specific endophytes are associated with geographical region or environmental stress. BACKGROUND Endophytes are micro-organisms capable of establishing a mutualistic relationship within a plant without causing disease. They are endemic within Gramineous plants and are frequently linked to nitrogen fixation, abiotic and biotic stress tolerance including salinity and drought tolerance, and pathogen suppression. Endophytic bacteria are either seed borne or colonise the host plant post germination. Biological nitrogen fixation is unique to Bacteria and Archaea and so plants must form associations with nitrogen fixing organisms in order to benefit from the process. Rhizobial endosymbionts living within the root nodules of legumes are the most well known of these interactions, but this is not the only example of nitrogen fixing bacteria living symbiotically within living plant tissues. Miscanthus often grows in areas where the nitrogen content of the soil is very low and is reported to contain a wide range of endophytic bacteria including nitrogen-fixing Clostridia. Such endophytes have not been heavily exploited in food and feed crops because they often produce toxins, not an issue for energy crops. APPROACH IBERS curates a unique genotypic collection of Miscanthus including a large number of accessions collected primarily for high biomass yield from a wide geographical distribution. The majority of bacterial endophytes are recalcitrant to culture, many are anaerobic and others exist as mixed communities. We propose the most reliable way to initially characterise the endophytes of a plant is therefore to employ a molecular approach such as 16S rRNA sequencing or TRFLP (terminal restriction FLP-digest 16S, label 5' end) to identify the bacteria present. Microscopic studies will be used to establish bacterial numbers and localisation within the plant. Both approaches will be performed across a range of genotypes in order to make comparisons between accessions of different genetic backgrounds and geographic locations to determine which of these factors has the greater influence on endophytic communities. Further comparison of plants generated from rhizome collected from the wild with those generated from seed will enable differentiation between seed borne and colonising types of endophytes. Growth studies between genetically similar plants with and without endophytes will be carried out under different environmental conditions to determine any differences in biomass yield or other phenotypes. This work will exploit and may help explain agronomic differences between genotypes which are observed in field trials being set up by Ceres in the UK and worldwide. Where culturing of specific endophytes is possible, endophyte-free plants will be inoculated and compared with uninnoculated plants under controlled conditions. Clonal genotypes planted at multiple sites within UK and worldwide will also be sampled and characterised by molecular methods to determine the stability of endophytes under different environmental conditions over time. TRAINING The training of the next generation of researchers is key. Within IBERS we aim to deliver distinctive and excellent training in research that meets student needs yet builds upon our particular research environment spanning the bioenergy research pipeline. IBERS offers unique opportunities to new researchers because of its broad, cross-disciplinary mission, its emphasis on outputs that are driven by science policy and that are relevant to industry, and its integrated research agenda covering basic, strategic and applied science linked through to teaching, knowledge transfer and extension activities.