2008 Annual Report 1a.Objectives (from AD-416) To identify endophytic microbes that enhance performance of native grasses. To evaluate performance of native grasses containing novel endophyte combinations in laboratory and field environments. 1b.Approach (from AD-416) 1. Identify individual endophytes inhabiting black grama, sand dropseed, and alkali sacaton, four-wing saltbush, and other species. 2. Cross inoculate black grama and other grass species with identified endophytes to evaluate endophyte effects on early plant vigor. Plant-endopyte combinations exhibiting significantly improved vigor will be selected for longer-term evaluations in greenhouse and field trials. 3. Analyze genetic variability of black grama with respect to reproductive strategies and success, while identifying plant-specific genetic markers. 4. Establishment of field trials and common garden studies to determine success of fungal endophyte transfers. These studies will be planned and conducted in cooperation with the BLM and may include test plots on BLM sites where remediation is desired. 3.Progress Report Endophytes associated with Black grama have been analyzed and described in Plant Science 174: 570-575 (2008). PCR fragments putatively associated with endophytes in sand dropseed, alkali sacaton, and fourwing saltbush have been isolated and await sequencing and analysis. Bioassays have been performed on grasses in vitro using three different endophyte isolates. Results have exhibited extreme variability, indicating a need for method optimization, which is currently underway. Genetic variability of black grama was examined by AFLP. An internal spacer ribosomal gene sequence belonging to Bouteloua eriopoda (black grama grass) has been validated with phylogenetic analysis, which clearly distinguished the grass sequence from sequences associated with endophytes. Field trial sites have been identified on Otero Mesa, and hydrogel-based irrigation strategies for these remote sites have been tested.