Characterization of Endophytic Cellulose Degrading Fungi at the Sevilleta LTER siteZachary T. Gossage1, Carolyn Weber2, Cheryl Kuske2, and Andrea Porras-Alfaro1,3

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 SummaryVery 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.

Culture Origin Isolation Date Isolation LocationID (based on

BLAST results) GenBankHit E-Value Bit Score Identities %

P-12 Root of Nama carnosum 6/20/2008 Sevilleta – NM Fusarium EU750688.1 0 1114 100

P-40 Stem of Cryptantha sp. 6/20/2008 Sevilleta – NM Fusarium EU750688.1 0 1109 99

P-49Leaf of Selinocarpus lanceolatus 6/24/2008 Sevilleta – NM Fusarium GU566205.1 0 1064 99

P-50 Root of Nama hispidum 6/24/2008 Sevilleta – NM Monosporascus AM167935.1 0 819 91

P-56Leaf of Sporobolus wrightii 6/24/2008 Sevilleta – NM Fusarium FJ904916.1 0 1077 100

P-62Leaf of Selinocarpus lanceolatus 6/24/2008 Sevilleta – NM Fusarium EU750688.1 0 1114 100

P-76 Root of Mentzelia sp. 6/24/2008 Sevilleta – NM Fusarium GU566205.1 0 1075 100

P-80Leaf of Sporobolus nealleyi 6/27/2008 Sevilleta – NM Paraphoma HM242215.1 0 942 99

MG-128 Root of Nama carnosum Oct 2008 Sevilleta – NM Chaetomium EU040239 0 803 93

MG-129 Root of Mentzelia sp. Oct 2008 Sevilleta – NM Myrothecium EF423537 e- 136 492 94

MG-136 Root of Mentzelia sp. Oct 2008 Sevilleta – NM Nectria AJ557830 0 946 96

MG-154 Leaf of Calylophus Oct 2008 Sevilleta – NM Phoma EU144366 0 1063 99

MG-156 Leaf of Calylophus Oct 2008 Sevilleta – NM Preussia DQ865095 0 930 99

MG-162Root of Nerysyrenia lanci Oct 2008 Sevilleta – NM Monosporascus EU144435 0 783 94

MG-167Leaf of Nerysyrenia lanci Oct 2008 Sevilleta – NM Preussia EF419922 0 755 98

MG-175Root of Nerysyrenia lanci Oct 2008 Sevilleta – NM Aspergillus EF669970 0 852 99

ZG-5 Soil sample 5/21/10 Duke Forest – NC Hypocreales HQ630960.1 0 1199 99

ZG-16 Soil sample 5/21/10 Duke Forest – NC Hypocreales HQ630960.1 0 1199 99

ZG-31 Soil sample 5/25/10 Duke Forest – NC Hypocreales GU566243.1 0 1199 99

ZG-41 Soil sample 5/27/10 Duke Forest – NC Hypocreales HM176572.1 0 1199 99

Future Work

Cellobiohydrolases (CBHs) SequencingAfter 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 ProcessCBH 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, 1982. Clearing zones were visible after a significant incubation period (1-2 months).

Initial Testing

Degradation of cellulose by fungi

Cont

rols

Cellu

lose

http://plantbio.berkeley.edu/~bruns/picts/results/its-map.GIF

ITS Primer Map

18S rDNA ITS 1 5.8S rDNA ITS 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 GenesCBH gene sequences were aligned using ClustalW and Jalview to visualize conservation of the gene among different isolates.

AcknowledgmentsThis project was funded by LANL-DOE, WIU and NSF Sevilleta Grants