Transcript Novelcelluloseposter32012

Characterization of Endophytic Cellulose Degrading Fungi at the Sevilleta LTER site
Zachary 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
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).
ITS Primer Map
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).
Culture
Degradation of cellulose by fungi
Controls
Identities %
EU750688.1
0
1114
100
P-40
Stem of Cryptantha sp.
Leaf of Selinocarpus
lanceolatus
6/20/2008
Sevilleta – NM
Fusarium
EU750688.1
0
1109
99
6/24/2008
Sevilleta – NM
Fusarium
GU566205.1
0
1064
99
Root of Nama hispidum
Leaf of Sporobolus
wrightii
Leaf of Selinocarpus
lanceolatus
6/24/2008
Sevilleta – NM
Monosporascus
AM167935.1
0
819
91
6/24/2008
Sevilleta – NM
Fusarium
FJ904916.1
0
1077
100
6/24/2008
Sevilleta – NM
Fusarium
EU750688.1
0
1114
100
Root of Mentzelia sp.
Leaf of Sporobolus
nealleyi
6/24/2008
Sevilleta – NM
Fusarium
GU566205.1
0
1075
100
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
Root of Nerysyrenia
MG-162 lanci
Leaf of Nerysyrenia
MG-167 lanci
Root of Nerysyrenia
MG-175 lanci
Oct 2008
Sevilleta – NM
Preussia
DQ865095
0
930
99
Oct 2008
Sevilleta – NM
Monosporascus
EU144435
0
783
94
Oct 2008
Sevilleta – NM
Preussia
EF419922
0
755
98
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
P-80
CBH Protein Alignment
About the Process
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).
Alignment of ITS Sequences
18S rDNA
ITS 1
Alignment of CBH Genes
CBH gene sequences were aligned using ClustalW and Jalview to visualize conservation of the gene among different
isolates.
5.8S rDNA
http://plantbio.berkeley.edu/~bruns/picts/resu
lts/its-map.GIF
Acknowledgments
This project was funded by LANL-DOE,
WIU and NSF Sevilleta Grants
Bit Score
Fusarium
P-76
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.
E-Value
Sevilleta – NM
P-62
Cellobiohydrolases (CBHs) Sequencing
GenBankHit
6/20/2008
P-56
Fungi were identified using ITS primers. At
least eight clones were sequenced to verify
purity of isolates. BLAST was used for
preliminary identification.
Isolation Date Isolation Location
Root of Nama carnosum
P-50
Identification
Origin
ID (based on
BLAST results)
P-12
P-49
Cellulose
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.
Plant –associated fungi as a source of
novel enzymes
Data Summary
Initial Testing
ITS Sequences
ITS sequences of all CBH isolates were compiled with
ClustalW and Jalview for visualization conserved and
unconserved regions of ITS.
Future Work
CBH data will be deposited in FunGene:
a functional gene database.
ITS 2
28S rDNA