Identification and characterisation of bacterial endophytes in Miscanthus

Endophytes are described as microorganisms capable of establishing a mutualistic relationship within a plant without causing disease. They are considered to be endemic within Gramineous plants and are frequently linked to plant benefits such as nitrogen fixation and salinity tolerance as well as 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. Brazilian sugarcane varieties demonstrate significant nitrogen fixation; producing abundant biomass yields with minimal fertilizer requirements and nitrogen fixing bacteria are also known to be widespread in wild rice species and in pioneer plants growing in unfavourable environments.Miscanthus is emerging as a major bioenergy crop and increasing yield in this hitherto unimproved plant is a major target for breeders in order to improve land use efficiency. Miscanthus is closely related to sugarcane and is native to South East Asia where it often grows in areas where the nitrogen content of the soil is very low; interestingly Miscanthus is reported to contain a wide range of endophytic bacteria including novel nitrogen-fixing clostridia. An endophytic association conferring nitrogen fixation to Miscanthus would be highly attractive for the sustainability of the crop as high biomass yields could be achieved over long periods of time with little or no nitrogen fertilizer requirement.The aim of the project is to determine whether an endophyte population confers any advantage to the Miscanthus host in terms of increased biomass under low nitrogen or saline conditions. IBERS curates a large genotypic collection of Miscanthus including a large number of accessions selected primarily for high biomass yields from a wide geographical distribution, a number of which were collected from saline locations. This collection will be used to determine the diversity of endophytes in Miscanthus and to further establish whether these populations are genotype specific or geographically & environmentally influenced.The majority of bacterial endophytes are recalcitrant to culture, many are anaerobic and others exist as mixed communities. The most reliable way to characterise the endophytes of a plant is to employ a molecular approach using the 16S rRNA gene to identify the bacteria present in a plant tissue. Further microscopic study will be required to establish bacterial numbers and localisation within the plant. Both approaches will be performed in 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 should be carried out under different environmental conditions to determine any differences in growth in these plants. Where culturing of specific endophytes or endophyte communities is possible, endophyte-free plants should be inoculated and likewise compared with uninnoculated plants in growth studies. Summary: Use culture-independent 16S rDNA sequences and microscopy to determine endophytic diversity in Miscanthus genotypes.Determine specificity between endophytes and host plantsCompare rhizome- and seed-collected plants.Determine any agronomic advantage conferred by endophyte: N fixation, salt tolerance etc.