I was wondering for how much time does a mycelium freshly isolated from plant tissue continue to be "similar to what it used to be" in nature. I can see that morphological changes frequently occur when transferring from plate to plate so in short, my question is: can I keep using a mycelium for a long time, making sure it still is the same? Should I re-isolate it from time to time? And, are there some techniques to prevent changes to happen?
Hi, using sub_culturing from the same isolate many times can cause mutation in some types of pathogens. What you can do is use the same culture to re-infect the plant host and after the disease, symptoms appear you can re- isolate the pathogen again.
Good luck
There are many answers to this question, depending on what the genus is. You need to store the isolate in such a way that it is not actively growing, and therefore is not mutating or sectoring. When I work with Oomycetes that have resting structures (chlamydospores or oospores), I can keep them for years on inoculated sterile ryeseed in water. For fungi that form microsclerotia, I store them on agar topped with cellophane; microsclerotia are produced on the cellophane and the cellophane can be peeled off when the microsclerotia are needed. They can last for years this way. Some people store cultures in test tubes topped with mineral oil. A colleague of mine stores his Trichoderma cultures on infected wheat bran. Rhizoctonia (which is only mycelium) can be stored in infested seed in liquid nitrogen.
Thank you both for your answers. I'll try the "re-infect and re-isolate" technique and I'll try to find the best way to store my collection. I work with basidiomycetes that do not produce (easily) spores or other reproductive structures on plate so I think I'll try to store them on glycerol at -80°C and use them for a set amount of experiments before re-isolation. Thanks again for your answers!
Dear Alessndro,
It depends on the type of fungus. Generally, you must not preserve the fungus with this method for a long time. You can use another methods for preserving the fungi for a long time such as glycerol 10%, lyophilization, liquid nitrogen.
Regards
This also depends on the type of medium used. Many plant pathogens mutate readily on rich media, and at a lower rate on lower nutrient media, and some other fungi do so as well. See for example recommendations for Fusarium, where SNA shows little mutation, while potato dextrose agar (PDA) shows a high rate of mutation (Leslie and Summerell 2006, etc.). We have seen similar results for some basidiomycetes, with a much lower mutation rate on water agar or a dilute plant decoction agar than PDA. There were a number of papers on this in the 1950s and 60s with recommendations on good media for reducing cultural changes for a number of fungi.
If the organism has not changed too much, passaging on plants is a good method, but may not work if too much virulence has been lost. If it is a foliar pathogen or endophyte, using a surface-disinfected detached leaf can reduce issues with other organisms that may interfere with re-isolation.
For the Basidiomycota we work with, colonized plant tissue (especially sterile seed, that has been moistened and autoclaved twice, 24 hours apart) has worked well for long term storage. We can store these at -20, -80, or for some in liquid nitrogen vapor for up to 10 years. This also can help for getting samples out to work with. We generally shake out 2-3 seeds from a container onto a plate each time we want to get some freshly growing material. The rest can go back into long term storage. We generally keep a working set, which can go in and out of cold storage, and a long-term storage set that stays in the cold as viability generally goes down each time you warm and refreeze the isolate.
Oui il peut avoir des changements car s'ils sont repiqués et conservés ils peuvent muter c'est le cas de certains oomycétes tel que phytophthora
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