STRUCTURE AND FUNCTION OF FUNGI IN ANTARCTIC LAKE ICE … · RIGHT-CLICK on the poster background and select LAYOUT to see the column options available for this template. The poster

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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