DNA Extraction From Fungal Plate

Please record all extractions correctly in the database. See database protocols for isolations for more information. Below is the current method:

General Ex-N-Amp protocol:

1. Prepare strip tubes with 40uL of Sigma Aldrich extraction solution. This can be found in aliquots in the shared box in the door of the small freezer.
2. Scrape approximately 10uL of hyphae from fungal colony using a sterile pipette tip (20uL works best and is not used for much else) or scalpel.
3. Add fungal material to extraction solution and vortex.
4. Run Ex-N-A protocol on a thermocycler (96C for 30 minutes).
5. Spin down with high rpm.
6. Pipet off the top 25uL: that’s your extract. It is often safe to dilute this a bit if you will need more than 25 uL, but this is not standard procedure.

Note: If BSA is needed, we have a stock of 2% molecular grade BSA in our freezer. This should be made into aliquots before using for samples. This is not standard, and only used in cases where it increases DNA amplification success.

OLD: DNA Extraction from mycangia

Modified Sigma Ex-N-Amp protocol:

1. add contents of mycangium (if dissectable) or the body part with mycangium (for example, mandibular mycangium) to 10uL of XNA Extraction solution in a PCR tube
2. mash the sample with a melted pipette tip
3. run Ex-N-A protocol on a thermocycler (96C for 10 minutes)
4. add 10uL of 3% BSA. DON’T ADD THE STANDARD DILUTION SOLUTION FROM SIGMA! It decreases yield sugnificantly.
5. spin down with high rpm
6. pipet off the top 10uL, and dilute with 10uL of water: that’s your extract

Nutrient media for abundant mycangial fungi

Additional media formulations can be found in the media table in the isolations database. Please add new media formulations to the table in the database.

• PDA: 39g PDA dried media from BD-Difco, 1L water, 10ml of Streptomycin (10,000 I.U./ML) & Penicillin (10,000 MCG/ML) mixture. To our experience, this produces richer cultures of mycangial fungi than other media. It’s also rather acidic and so prevents growth of many bacteria even without the antibiotics. Can add 5g of agar for harder media.
• YMEA: (4 g yeast extract, 10 g malt extract, 4 g dextrose, 15 g agar, 1L water), 10ml of Streptomycin (10,000 I.U./ML) & Penicillin (10,000 MCG/ML) mixture per 1L of media. Fungi grow slower than on PDA.
• MEA: same as YMEA, without the yeast extract.
• PYDA: 15g PDA, 10g Agar, 2g Yeast Extract, 1L Water.
• MYEA: 15g MEA, 10g Agar, 2g Yeast Extract, 1L Water.

Antibiotics

DO NOT AUTOCLAVE AGAR MEDIA WITH ANTIBIOTICS IN IT! Add when media has cooled down to touch.

Streptomycin/Penicillin: catalog #516106, supplied in powder. Mix into liquid media at a concentration of 100-200 ppm. For PDA, 100 mg/L is equivalent to 100ppm.

Add cycloheximide for Ophiostomatales, including Raffaelea (but not Ambrosiella). Kolarik & Hulcr (2008) used 0.1 mg/L cycloheximide to select for ophiostomatoid mycangial fungi, people use betweeen 0.1-0.5mb/L. We are using 0.5 mg/L cycloheximide in media.

Excessive growth of yeast (often on YMEA) can be prevented by slapping a chunk of agar on top of the inoculum.

All media containing antibiotics should be kept in darkness and cold temperature, to prevent degradation.

Sabouraud Dextrose media

Simple medium for testing utilization of various carbon sources.

Diana Six added 10g yeast extract to isolate Ambrosiella beaveri.

Minimal medium for Aspergillus, per liter

Barratt et al., 1965 Genetics 52:233-246

Adjust pH to ca 6.5 (usually requires 1 ml of 1 N NaOH). Can prepare 2OX stock solution of the above salts.

Note: cultures of pathogenic fungi may need to be inoculated in plant material, and then subcultured on media prior to use in experiments. This increases the likelihood that the fungus will express pathogenic traits.

Spore suspension preparation

1. Pick a culture with substantial and active growth (3-5 weeks for most Raffaelea).
2. Add 2 mL of sterile DI water to plate
3. Using a plate spreader, apply modest pressure and homogenize water with fungal culture.
4. Tilt the plate and gather the water and fungi together at the bottom end of the plate. Then transfer the sample to a 2 mL microcentrifuge tube using a pipette.
5. Dilute about 200-500 µL (depending on fungus) of the original spore suspension to 2 mL of sterile DI water.
6. Use this dilution to determine spore concentration using hemocytometer (see separate protocol) and create desired spore concentration.
7. Although often an extraneous detail, you may want to aim for 100,000 spores/100 µL.

Inoculation procedure

1. Use an electric drill/screwdriver with 3/32″ or 7/64″ bit for mimicking a beetle gallery.
2. First score a point of inoculation about a half inch below an active branch node.
3. Point the drill downwards at about a 45 degree drilling angle at the inoculation point. Hold the drill bit steady with your thumb and forefinger, cradling the tree trunk using the same hand.
4. While still guiding the drill bit with your fingers, quickly and steadily drill about a half inch deep into the tree, enough to hold 50 uL of the spore suspension.
5. Pipette spore suspension into the tree.
6. Parafilm the trunk of the tree, surrounding and sealing the point of inoculation.
7. Later, plate some of your spore suspension to ensure that the inoculum contained viable fungus material.