Characterization of Endophytic Cellulose Degrading Fungi at the Sevilleta LTER site Zachary T. Gossage 1, Carolyn Weber 2, Cheryl Kuske 2, and Andrea Porras-Alfaro 1,3 1. Western Illinois University, 2. Los Alamos National Laboratory, 3. University of New Mexico Abstract CBH data will be deposited in FunGene: a functional gene database. Data Summary Very few microorganisms are currently used in bioethanol production and the discovery of novel species with the capacity to degrade cellulose could impact directly the biofuel industry. Plants could be major reservoirs of cellulose degrading microorganisms because all plants are colonized by diverse communities of endophytic fungi. The main objective of this project was to characterize cellulose degradation by plant associated and soil fungal communities from the SEV LTER. Fungi were isolated from different plant species and plated on two different cellulose media. Cultures were incubated for approximately one month and degradation of cellulose was determined based on media discoloration. The isolates that were positive for cellulose degradation were tested for cellobiohydrolases genes using PCR and sequencing. Approximately 95% of the isolates that degrade cellulose in culture were positive for cellobiohydrolases using direct amplification of CBH genes. The isolates were also sequenced and identified using ITS rDNA primers. Dominant fungi included Ascomycota taxa closely related to Monosporascus, Chaetomium, Phoma and Fusarium. Plant-associated fungi showed great potential as a novel source of cellulose degrading fungi. Fungi were identified using ITS primers. At least eight clones were sequenced to verify purity of isolates. BLAST was used for preliminary identification. Future Work Cellobiohydrolases (CBHs) Sequencing After initial testing, samples were sent to LANL for molecular testing for cellobiohydrolases. CBH genes were amplified using specific primers. There were many isolates shown to degrade cellulose based on the plating technique, however, not all fungi utilize the same genes/enzymes for cellulose degradation. All fungi tested were positive for CBH. Identification Plant –associated fungi as a source of novel enzymes Some of the tested samples were isolated from plant sources. It is known that many bacteria and fungi have plant hosts. It is possible that some of the isolates are plant-pathogens capable to degrade plant cell walls (composed of cellulose). About the Process CBH Protein Alignment The process of degrading cellulose into glucose is a complex process and requires a number of enzymes (cellulase complex). CBHs are exoglucanases, one of the three types of cellulases involved in the complex (Jing 2007). They are involved in the initial step of the degradation process, thought to be the limiting factor and cooperate either in an exo-exo (two CBHs) fashion or exo-endo (involving an endoglucanase) (Liu et. al. 2010). Samples were plated on two modified cellulose media (Egginsand Pugh, 1962 and Otero, Clearing zones were visible after a significant incubation period (1-2 months). Initial Testing Degradation of cellulose by fungi Controls Cellulose lts/its-map.GIF ITS Primer Map 18S rDNAITS 15.8S rDNAITS 2 28S rDNA Alignment of ITS Sequences ITS sequences of all CBH isolates were compiled with ClustalW and Jalview for visualization conserved and unconserved regions of ITS. ITS Sequences Alignment of CBH Genes CBH gene sequences were aligned using ClustalW and Jalview to visualize conservation of the gene among different isolates. Acknowledgments This project was funded by LANL-DOE, WIU and NSF Sevilleta Grants