Project Description/Abstract

• Manipulating fungal species diversity, testing effects on enzyme production and decomposition rate.

Methods

Site

Lab experiment, with fungi from Delta Junction, AK (see site description in (Treseder et. al. 2004 Fires Forests Fungi, Ecol. Mon.)

Experimental Design

Isolation of fungal species from soil and woody debris

Overview

If so, you will need antibiotics. But you need to know what you are looking for in order to select for it and kill everything else, e.g., I used benomyl and phenyl-phenol to kill all except basidiomycetes. If you want to get some bad-ass wood-rot fungi, these are the things to use together and separate. If you just want to isolate tissue from mushrooms or from a log that has been rotted through with a fungi, that will be much easier, and can be done without anti-biotics.

MYA and MYA+

• • For happy, healthy fungi (and contaminants if you are not careful)
  • I pour these thick and let fungi grow on them for a long time.
• MYA+Benomyl and / or 2-Phenyl-Phenol :
  • For "rescue" of higher basidiomycetes.
  • Pour these thin, and transfer to MYA ASAP

For 1L of MYA

• 20g agar
• 5g Malt extract
• 5g yeast extract, (is this the "same" as yeast?)

This is a low-sugar diet. Could probably go lower, but this puts the balance more in favor of slow growers than, say, 20g Malt. I use 20g agar because I like the consistency.

To transfer a sterile culture or to isolate from a fresh fruitbody, this is all you need. But, if there is danger of contamination - e.g. with tiny, slimy mushrooms that are pulled out of the freezer after 6 months, or from soil slurries, or from litter, sticks, etc, you should use the following **note, must filter-sterilize these after autoclaving

For MYA+

see Carey and Hull 1989 [1] for details

*0= TOXIC CHEMICALS =0*

• 8mg/L Benomyl
• 6mg/L 2-phenyl-phenol
  • dissolve Benomyl and phenyl-phenol in 50 ml EtOH, add prior to autoclaving at 121°C for 20 min

Litter γ-irradiation (sterilization)

With assistance from Dr. Leslie Redpath, UCI Radiation Oncology

• 50g aspen and 50g spruce litter
• dry for 24h at 60C
• grind 30s in coffee grinder
• fill 50ml Tubes with dry ground litter
• (irradiator details?)
• set at position 2 where the central dose-rate is 8.0 Gy/min.
• Exposure time was 3750 min.
• Dose approx. 3.0 Mrad at center and 2.5 Mrad at edge.

Microcosm Innoculation

Litter preparation: ground, homogenized, and sterilized partially decomposed Aspen litter by exposure to 2.5-3.0 Mrad of γ-irradiation in a self-sheilded gamma irradiator with a CS-137 source (Shepherd and Associates, Mark I).

40 ml glass vials with septa Microcosm Added 1.00 g of 30-60 grit sand to each of sixty microcosms.

autoclaved sand + tube at 120∘C for 40 minutes.

Next, added 50 mg of γ-irradiated Aspen litter to each microcsom.

CO2 measurement

CO₂ production in each microcosm and calculated total yield as mg C respired per g litter during the course of the experiemnt, 122 days.

Enzyme Assay [1]

Data

Goal: Available Dec 2009

Statistics

Goal: Available Dec 2009

1. Carey J.K. and A.V. Hull 1989 A selective medium for the isolation of wood-rotting basidiomycetes International Biodeterioration 25:5 373-376] [carey1989smi]

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