This material is based upon work supported by the National Science Foundation under several grants. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation.
Laboulbeniales: Methods
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Pyxidiophora and other sticky-spored ascomycetes associated with arthropods: Isolation from nature and culture.
Isolation of  spores from nature:
Dung is a good substrate on which to find insect-dispersed ascomycetes. Place the dung on the surface of 1/2 cornmeal or other agar medium low in carbon source, and moisten it slightly.  Examine the dung surface in the moist chambers daily because insect-dispersed fungi often come up early and it is easiest to transfer spores when they are clear of  neighboring fungi.  Use a cover slip shard to transfer the sticky spores of these fungi.  If you break a cover slip into pieces, often you can get several triangular pieces (isoceles triangle with two angles about 40-60 degrees). Hold the triangle in fine forceps by the wide bottom.  Use a high powered disecting microscope (about 50-80X) to observe as you touch the narrow end of the triangular piece of glass to the sticky spore mass. Just touch it lightly and some spores should stick.  The magnification needs to be high so that you can see stray hyphae and spores that might have been carried to the perithecium and spores of interest.

Culture medium:
Half strength corn meal is often a good culture medium.  Very gently place the cover glass triangle (spore side down) on the agar.  Put the spore area near the edge of the plate so that it is in position to be observed under the 40X lens of the compound microscope by changing from the X10 lens--not too near the edge, but not to far in --you need to experiment. The dish should not need to be moved, so that finding the exact spot at higher magnification is not a problem.

Monitor the cultures immediately after transfer of the spores to see what was transfered (if anything) and then EVERY morning to check for spore germination. This way you can check to be sure what spore is germinating even if there are contaminants.  Write notes all over the plates as you check them so you will know if spores were transfered or if contaminants are present.  To save space put about 5-7 triangles on each plate, and once one has a spore that begins to germinate and is clean, transfer it to its own plate.  You might also transfer some contaminated spores as well (see below, mycoparasitism).

Mycoparasitism:
Some of the fungi associated with ascomycetes for dispersal may be mycoparasites.  You can get a hint of this if you fail to get germination or if the spore germinates and the germ tube stops growing soon.  If you are lucky, sometime you might transfer the host fungus along with the spore of interest in this process.  For this reason you should watch the germination behavior of contaminated spores as well as the clean ones.

Having spores on glass to transfer them is the real advantage, because you can be certain of what was transfered by looking through the glass.

Reference: 
            Blackwell, M., Malloch, D.. 1989.  Pyxidiophora:  Life histories and arthropod associations of two species.  Canadian Journal of Botany 67:2552-2562.

DNA extraction for Laboulbeniales
DNA extraction protocol.--Four to ten thalli are removed aseptically from each of 5 hosts using a sterile micropin and transferred to a small drop (2 µL) of sterile 0.1xTE buffer (10 mM Tris.Cl, 0.1 mM EDTA, pH 8.0) on a clean microscope slide. Thalli from a single host are grouped together, and a 22 mm x 22 mm coverslip is placed over the material. The slide is transferred to a compound microscope, and the fungal thalli are crushed by exerting pressure on the coverslip. The extent of maceration is observed microscopically (Fig. 3). The slide is placed immediately on a bed of dry ice and allowed to freeze. During this time a fresh extract solution containing 18 µL of 0.1x TE and 2 µL of detergent (1% Triton 100) is prepared. Under the low power of a dissecting microscope the cover slip is removed with a sharp razor blade. The fungal material can be seen attached to the microscope slide. At this point 2 µL of the extract solution is pipetted on top of the crushed fungal material. This solution is allowed to freeze and then the bed of dry ice is removed from beneath the microscope slide.  As the extraction solution and fungal material begin to defrost they are picked up with a micropipettor and transferred back to the extract solution vial. An additional 30 µL of 0.1xTE is then added to bring the final volume up to 50 µL. The vial is then placed in a water bath set at 60 C for 15 min and the crude extract, stored at -20 C prior to PCR amplification.

PCR amplification of  rDNA.--Approximately 1100 bp of  ssu rDNA and  600 bp of lsu rDNA can be amplified using primers NS1 and NS4 and LS1 and LR5 (White et al 1990, Hausner et al 1993, Rehner and Samuels 1995) respectively.  Amplifications are performed in 49.5-µL reactions containing 31.5 µL water, 5 µL 10X PCR buffer, 4 µL 25mM MgCl2 , 2 µL 10mM of dNTPs, 1 µL 10mM each primer, and 5 µL fungal extract.  Negative control reactions include all components except template DNA which is replaced by water. Positive controls use known DNA from other fungi.  Three identical replicates are usually set up for each of the 5 extracts from each species.  Amplifications are performed  using  a Perkin-Elmer Gene Amp 200 system programmed for 1  cycle  pre-PCR  at  98 C for 6 min, and 90 C for 5 min, during which time 0.5 unit Taq  (0.5 µl) is added to each reaction tube.  This is followed  by  45  cycles  of  94  C  for  30  s,  54 C  for  2 min,  with  a ramped increase of 1 C for every  8  s  to  72 C  for 1.5  min,  and  a final  incubation  period  of  72 C  for  7 min.  Amplification products are resolved by electrophoresis through 0.7%  agarose  gels  in  TAE  (2  mM  EDTA,  80 mM Tris-acetate,  pH 8.0)  and  visualized by staining with ethidium bromide. PCR products are purified using a DNA purification kit (Bio-Rad Laboratories, Hercules, California).

References:

[from Weir, A. and Blackwell, M. (2001). Extraction and PCR amplification of DNA from minute ectoparasitic fungi. Mycologia 93: 802-806.]
Laboulbeniales Home Page / Phylogenetic Studies of Laboulbeniales / Methods / Literature / Mycology at LSU
Last Modified: 24 June 2005
Meredith Blackwell
Mycology at LSU