Colecta de Hongos para estudios de ADN

Recomendación de Karen Hughes (University of Tennessee)

The most effective way to collect both plant and fungal materials is to dry them gently using silica gel beads. I use Dri Splendor from Miracle Coatings, Anaheim California 92806 which I bought on the internet. The beads are easier to remove from the tissue than silica sand and the silica must be removed because it sometimes will interfere with PCR amplifications. I suggest using either lock-top 2 ml microfuge tubes or a small screw capped microvial to put the silica gel in. A piece of fungal tissue about 0.5 square is removed from the specimen and placed on top of the silica gel and the tube is closed. I keep the tubes at -80 until I use them for DNA. Caution – if you don’t use a screw-top cap or locking cap, when you take the tubes out of the freezer, the top will pop open as it warms and you can loose the sample. My colleagues who are collecting plant material put several leaves in a ziplock plastic bag with silica gel.

This should work for all fungi.

In some cases, it might be necessary to try to get a culture because of the possibility of contamination with another fungus. Fungi that are flat on the bottoms of logs and endophytes (fungi within plants) might fall into this category. To culture fungi that produce spores, we take a small amount of petroleum jelly (Vaseline) and put it on the lid of a small 16mm Petri dish. We stick a small piece of fungus on the lid with the spore-forming surface down and the put the lid over a bottom plate containing malt agar. The plate is slanted at a 45-70 degree angle overnight and in the morning, we examine the plate to see if spores have dropped. If they have, we remove the tissue, seal the plate with parafilm and watch for growth. It’s sometimes necessary to go back in and remove contaminants. I don’t work with endophytes but I believe that a piece of plant stem is surface sterilized in about 10% Clorox bleach and then sectioned using sterile technique. The sections are placed on malt agar medium and observed for growth.

DNA cloning is necessary when a fungus is heterozygous for more than one insertion-deletion event. Some genera of fungi are worse than others in terms of needing cloning. Some “zygomycete” fungi are coenocytic and present special problems but for the most part, these will not be fungi that you collect.

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