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The permanently ice covered lakes of Antarctica's Dry Valleys
region harbor a diverse group of microbes that live in unique liquid
water habitats. In particular, the lake ice hosts microbial
communities that form functional ecosystems. My research focuses
on the functional role of fungi in the permanent ice covers of lakes
in the McMurdo Dry Valleys. Laboratory cultures obtained from
ice cores taken from two lakes were tested for growth
characteristics under various temperature and nutrient regimes.
Partial ITS- DNA sequencing was used to screen for functional
genes and to identify novel fungal types unique to this region of
Antarctica. Our results show that axenic cultures were successfully
obtained from the permanent ice cover of the lake ice. Temperature
experiments revealed that these organisms were psychrotolerant,
but grew most rapidly at 20°C. In addition, the isolated organisms
possess antifungal activity that has not been previously reported
from fungi isolated from Antarctic lake ice. Results from the study
will be the first to address the structure and function of fungi in
these sub-zero habitats and the potential for eukaryotic life to exist
in icy worlds beyond Earth.
ABSTRACT
Overarching Objective:
To study the structure, function and distribution of fungi in the lake
ice ecosystem of the McMurdo (MCM) Dry Valley lakes.
Hypotheses:
1. Fungi are present in the lake ice and are viable as spores and
mycelium
2. Fungi in the lake ice are Psychrophilic
3. Fungi in the lake ice produce bioactive volatile compounds
OBJECTIVES AND HYPOTHESES
Field Sampling:
Samples were collected during the 2012- 2013 field season from several lakes in Taylor
Valley (Figure 1).
Decontamination Protocol:
Ice cores were decontaminated using the protocol of Christner et al., 2005 in a Class 1000
Cold Clean lab (Figure 2)
Isolation and Culturing :
Fungal mats were isolated and cultivated on Potato Dextrose Agar (PDA) medium.
Phylogenetic identification:
1. DNA extraction using DNeasy Plant Mini Kit (Qiagen)
2. Phylogenetic analysis was carried out by the acquisition of the ITS- 5.8 S ribosomal gene sequence
3. The ITS regions of the fungus were amplified with the universal ITS primers, ITS1F and ITS4 using PCR (White et al. 1990)
4. Sequencing was performed by Functional Biosciences (Madison, WI)
5. Sequences were organized and queried against the NCBI database using an in house program (SeqTrace)
Growth Rates:
The isolated cultures grown on PDA plates were tested for growth
characteristics at several different temperatures
Antimicrobial Activity of Fungi:
Dual Culture assay
An agar block of a 15-day old culture of fungus was
placed in the center of PDA plate and the pathogen was inoculated
at 2 cm juxtaposed to the pathogen and tested for bioactivity (Figure 3)
Gas test
An agar strip 2.5 wide was completely removed from
the mid-portion of a Petri plate of PDA. Then, the test organism was inoculated and grown on one side while
The pathogen was placed on to the agar half-moon strip on the opposite side of the plate (Figure 4)
METHODS
Figure 6 demonstrates the dependence
of growth rate on temperature
At 0°C, the growth is the slowest and
most rapid at 20°C
RESULTS
CONCLUSIONS
1. Axenic cultures were successfully obtained and identified
from the permanent lake-ice cover:
• Tetracladium sp. from Lake Chad
• Mucorales sp. from East Lake Bonney
• Unknown from East Lake Bonney
2. Temperature experiments revealed that the isolated
organisms were psychrotolerant. Growth rate was greatest at
20°C
3. The isolated organism Tetracladium sp. possess antifungal
activity against known plant pathogens that has not been
previously reported from fungi isolated from Antarctic lake
ice
REFERENCES
White TJ, Bruns T, Taylor JW (1990) Amplification of direct sequencing of fungal
ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ
(eds) PCR protocols: a guide to methods and applications. Academic Press, San Diego, pp
315–324
Christner BC, Mikucki JC, Foreman CM, J Denson, JC Priscu (2005) Glacial cores:a model
system for developing extraterrestrial decontamination protocols. Icarus 174.2
ACKNOWLEDGEMENTS The authors would like to acknowledge the Undergraduate Scholars Program, NSF’s Division
of Polar Programs, NSF- LTER Program, PHI Helicopters Inc. for air support in the Dry
Valleys, as well as all of the members of the 2012-2013 field team.
1Land Resources and Environmental Sciences and 2 Department of Plant Sciences Montana State University, Bozeman, MT 59717, USA
Priyanka S. Kudalkar1 , John Priscu1, Gary Strobel2 and Cathy Cripps2
STRUCTURE AND FUNCTION OF FUNGI IN ANTARCTIC LAKE ICE
STUDY SITE
Figure 1: Location of the MCM Dry Valleys (77°S, 163 °E ) and the study lakes in the Taylor Valley
km
0 5 N
Lake Chad
Lake Bonney
Figure 2: Processing Ice Samples in the Subzero facilities (Picture by: Alex Michaud )
Figure 3: A 9cm wide petri dish plated with the fungus and pathogens placed in periphery
Figure 4: A 9cm wide petri dish plated with the fungus and the pathogen to check its bioactivity (Picture by Dr. Gary Strobel)
Pythium ultimum Phytophthora cinnamoni Sclerotinia sclerotiorum Botrytis cinera
Tetracladium sp. 17.46 91.89 96 70
Mucorales sp. 0 0 0 0
Unknown 0 0 0 0
Isolates
Inhibition (%) after 48 h exposure with test organisms
Figure 7: Effects of the bioactive compounds produced by the isolates on fungal pathogens
Figure 6: Effect of temperature on the radial growth of fungal isolates identified as A .Tetracladium sp., B. Mucorales sp. and C. East Lake Bonney isolate on PDA medium.
Figure 5: Petri plates supporting the growth of mycelial colonies of 10 day old axenic cultures
The isolates exhibited antagonistic properties against the
most detrimental pathogens
A B C
SIGNIFICANCE Isolating and characterizing fungi in the icy ecosystem of the dry
valleys provides a better understanding of the component
members of the ecosystem that have been not studied previously.
These fungi from the polar region are capable of inhibiting the
growth of detrimental pathogens. This could have several
promising applications in the field of medicine and agriculture. Figure 5 demonstrates isolation and
characterization of fungi from the lake ice was
achieved
Based on the morphology and DNA analysis it
was confirmed that these isolates were axenic
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
0 5 10 15 20 25 30
Gro
wth
Rat
e (
cm/d
ay)
Temperature (°C)
Growth Rates Tetracladium sp.
Mucorales sp.
East Lake Bonney isolate