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Viability of ascospores in previously frozen herbarium material
Wendy Untereiner,
01-10-2021 19:28
I'm curious to know if any member of this community has attempted to obtain cultues from ascospores using herbarium specimens that have been previously frozen. I ask because I'm considering requesting the loan of recently collected specimens that were frozen for 7 days on two searate occasions at -40 F (= 4 C).
I suppose the answer may hinge on whether these Fungi normally experience periods of freezing followed by warmer temperatures during which ascospores are discharged and germinate. Many Ascomycota operate in this manner during the fall, winter and spring in North America. If these Fungi are adapted to intermittent freezing in situ. then I'd expect that mature ascospores from frozen herbarium specimens would be capable of germinating.
I'd be pleased to hear from anyone who has attempted to culture this type of material.
Thank you.
Wendy
Andrew N. Miller,
01-10-2021 19:52
Re : Viability of ascospores in previously frozen herbarium material
We were successful in germinating conidia (and maybe some ascospores) of soil fungi that were frozen as soil samples at -20C and -80C for 1 month, 6 months and 1 year.
Cheers,
Andy
Cheers,
Andy
Wendy Untereiner,
01-10-2021 19:55
Re : Viability of ascospores in previously frozen herbarium material
Thanks Andy.
Hans-Otto Baral,
01-10-2021 20:28
Re : Viability of ascospores in previously frozen herbarium material
Hi Wendy and Andy
I have no personal oservations about germination triggering, but I suspect that it makes a difference if spores are frozen when hydrated or when dry. I suspect that freezing of hydrated spores may kill them in some species, while other species easily survive the procedure.
For experiments I suggest to test both ways. In the soil I suspect that spores are generally more or less hydrated during winter.
Zotto
Wendy Untereiner,
01-10-2021 20:56
Re : Viability of ascospores in previously frozen herbarium material
Dear Zotto,
Thank you for your input. Another consideration might be the type of freeer used (dessicating vs non-dessicatining). In any case, I think it's worth trying to germinate ascospores from the two specimens I plan to request on loan. Nothing ventured, nothing gained.
With kind regards.
Wendy
Thank you for your input. Another consideration might be the type of freeer used (dessicating vs non-dessicatining). In any case, I think it's worth trying to germinate ascospores from the two specimens I plan to request on loan. Nothing ventured, nothing gained.
With kind regards.
Wendy
Mario Filippa,
05-10-2021 00:23
Re : Viability of ascospores in previously frozen herbarium material
Well,
I'm not so sure that ascospores from exiccata are necessarily "dried", i.e. without any water inside.
For the species with bigger spores at least, it is possible to take a look at the "dried" spore-print without add any liquid on the glass and many spores may look more or less like the "hydrated" ones. If not, they hardly regain their original size when rehydrated and maybe they are died (broken walls?).
I suspect that as long as "dried" spores are alive, their walls may keep some water inside.
But that may be highly species-specific of course, many species could well germinate from really "dried" ascospores.
Regards
Mario
Hans-Otto Baral,
05-10-2021 09:03
Re : Viability of ascospores in previously frozen herbarium material
Hi Mario
I see no reason to accept your view. Do you have any data supporting this? Spore walls are water-permeable, which is essential for their water uptake. Therefore they lose all their free water under dry condition and achieve an equilibrium with the surrounding air. This is independent on whether they are alive or dead.
I have written a chapter in my paper about VT from 1992 (Mycotaxon). Two examples: Sarcoscypha spores woith their thick walls collapse in the herbarium, although they survive several years there. Xylariales spores do not collapse due to their rigid walls but form DBBs inside, a gaseous phase of unclear composition and pressure. The spore walls has no means to avoid water molecules evaporating.
My concern of my post was that dried fungi which survive drying withstand 100°C or more in the stoven (our experiment with Orbilias) whereas hydrated fungi of course get killed.
Zotto
Mario Filippa,
05-10-2021 14:18
Re : Viability of ascospores in previously frozen herbarium material
No data Zotto,
just impressions and "suspect", I'm sure my bad English did not express the tone of doubt in my sentences.
No intention to refute your results, I deeply respect your work.
And of course that's going a little off-topic so I go silent :)
Ciao
Mario
Hans-Otto Baral,
05-10-2021 16:05
Re : Viability of ascospores in previously frozen herbarium material
No, your contribution has to do with Wendy's question. That the degree of hydration of a cell strongly influences the tolerance against drought, heat or cold, we have proven, and I suspect hydration also influences the triggering of germination. A well-known example are the Tardigrada which survive harsh conditions in the dry state (cryptobiosis), and nothing else is the case with the survival of fungal spores.
Jason Karakehian,
17-10-2021 01:29
Re : Viability of ascospores in previously frozen herbarium material
I have seen colleagues culture from Morchella herbarium specimens. I think it depends on the group of fungi that you want to work with. You can always request the loan and examine the spores in water to see if they are alive. You can use Zotto's 1992 paper and Karakehian et al. 2021 https://asianjournalofmycology.org/pdf/AJOM_4_2_1.pdf to help determine if the spores are living or dead in your water mount microscopy preparations. The asci may not discharge spores, but the ascospores within them may still be alive. You can prod an apothecium across growth media supplemented with antibiotics with a sterile probe, trying to break up the apothecium a little as you go along. Then monitor this plate to see if any ascospores or ascospores still within the asci germinate. Then you'll have to transfer these to fresh media. The process is in our paper.
Also, I just tried an experiment transferring a small amount of culture tissue that I had frozen at -20 for about 2 years dry in an Eppendorf tube. I thought it was dead for certain, but it seems to be growing out now after a month of sitting on the growth medium.
I think that fungi are very resilient. I hope that you get lucky and can grow them out!