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Offlinerifampicin
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Registered: 06/09/23
Posts: 5
Last seen: 2 months, 29 days
Successful isolation of Claviceps purpurea/Ergot * 2
    #28353278 - 06/09/23 01:58 PM (11 months, 3 days ago)

So here I am finally making a post on this forum! I've been a long time lurker, but I for once feel like I have something worthy to add upon the hundreds of thousands of threads on this forum.

As the title says, I have been able to isolate the highly sought after Claviceps purpurea. I won't lie, having access to a professional lab helped a lot, but I don't believe it to be impossible to someone in an amateur setting, as long as they know what they are doing.

I can confirm that this is Claviceps purpurea. The sclerotia were harvested in 2021 and 2022 on 6 different locations. I ended up using only 2 strains collected in 2022, but whatever. They were stored in the refrigerator for later use. Still have the other strains that I will plate later to have more genetic diversity to screen from.

The outside of the sclerotia was cleaned with 5% bleach and a few drops of tween20 (any detergent will do) for 5 minutes in order to limit the amount of contamination brought onto agar, the detergent helping to reach small crevices. The sclerotia where then soaked in distilled water for 3 hours and then cut in disks with a sterile scalpel blade.

At first, I placed this disks on YEB medium, but that didn't prove very effective, ergot doesn't like this medium that much so I then switched to PDA, which yielded the beautiful fluffy mycelium that you can see on the 2 photos. The plates you can see on these photos are the result of the third transfer of clean mycelium onto fresh plate, in order to get any bacteria/spores out of the culture

https://anonfiles.com/N3rbUfucz0/Culture_1jpg_jpg

https://anonfiles.com/MarbUau2z6/Culture_2_jpg

All medium contained Rifampicin and Streptomycin at a concentration of 100 µM both. I will try using other antibiotics in the future as well, just for the sake of testing. I still got a few contamination by molds, strangely mostly penicillium.

I am now waiting on a UV lamp to do some basic screening. Afterwards, that's the long game of mutagenesis, screening, mutagenesis, screening.... I plan on mutating with both UV and diethyl sulfate to accelerate the results.

Edited by rifampicin (06/10/23 08:33 AM)

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OfflineBuster_Brown
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Re: Successful isolation of Claviceps purpurea/Ergot [Re: rifampicin]
    #28354025 - 06/10/23 04:47 AM (11 months, 2 days ago)

Nice.

:howyoudoing:

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InvisibleCreonAntigone
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Registered: 05/30/21
Posts: 2,971
Re: Successful isolation of Claviceps purpurea/Ergot [Re: Buster_Brown]
    #28354358 - 06/10/23 10:42 AM (11 months, 2 days ago)

Do you want more strains?

I read a study - still trying to find it, I'll update if I do - about how ergot actually has many parasites that parasitize it - and hence that cultures which appear axigenic have many sub-species of lower molds and such living inside it. I wonder if there is a way to tell if there is mutualistic bacteria or molds in the culture.

What are your plans for the culture?

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Offlinerifampicin
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Registered: 06/09/23
Posts: 5
Last seen: 2 months, 29 days
Re: Successful isolation of Claviceps purpurea/Ergot [Re: CreonAntigone]
    #28354543 - 06/10/23 02:02 PM (11 months, 2 days ago)

Yeah I'm trying to add more strains to my collection. I'm waiting to get a UV lamp because I love the color blue, and I want to make an aquarium that would glow of a beautiful blue :wink:
So I'm trying to get more genetics to screen from.

I guess it would be possible by sequencing the ribosomal RNA and seeing what species are present in the sample. I haven't seen any abnormal growth after the second transfer, so if the partner doesn't grow out of ergot's mycelium I don't think I have anything. However it might be constrained to the inside of the mycelial mass, who knows! First time I hear about it, if you find anything I would love to read about it

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InvisibleCreonAntigone
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Registered: 05/30/21
Posts: 2,971
Re: Successful isolation of Claviceps purpurea/Ergot [Re: rifampicin]
    #28354686 - 06/10/23 04:38 PM (11 months, 2 days ago)

I found the study, take a look: the word they use is mycoflora.

Effect of the mycoflora of ergot (Claviceps purpurea)sclerotia on their viability (2010)M. Ondřej, B. Cagaš, E. Ondráčková


Quote:

Twenty fungal species were isolated from sclerotia of ergot (Claviceps purpurea) originating from rye. In in vitro tests with the anamorphic stage of ergot (known as Sphacelia segetum) high mycoparasitic activity was exhibited only by two of them – Clonostachys rosea and Trichoderma harzianum, moderate mycoparasitic activity was detected in Ulocladium sp., Clonostachys catenulata, Trichoderma hamatum, and Trichothecium roseum. In in vitro tests of mycoparasitic activity with sclerotia of the fungus Sclerotinia sclerotiorum their rapid and total destruction was brought about only by the fungi Clonostachys rosea and Trichoderma harzianum. The viability of overwintering ergot sclerotia in the soil was influenced by their placement, age, depth of placement and treatment with a conidial suspension of the fungus Clonostachys rosea. The highest viability values were reported in fresh one-year-old sclerotia. In two-year-old sclerotia their viability was reduced and their sensitivity to microbial activity was higher. The sclerotia aged 3 years or more did not germinate and were all microbiologically degraded in the soil. More than 80% of degradation of sclerotia which were 1 to 2 years old was caused by the fungus Clonostachys rosea. The degradation of sclerotia aged 3 to 4 years was brought about by bacteria, soil edafauna (mites, nematodes) and fungi of the genera Trichoderma, Fusarium, Clonostachys, etc.





Their method involved no surface sterilization - that might impact the ability of one to see these mycoparasites living within ergot. That being said, in their method they tested these isolated species to see if they actually were parasites, and they found that they were. So essentially the germinated fungi weren't just there by chance but actually invaded the ergot in a sense (but possibly mutualistically?)

Quote:

Isolation of fungi from ergot sclerotia. The isolation of mycoflora contaminating ergot sclerotia was carried out from 114 ergot sclerotia coming from the soil surface (collected 14 days after the harvest of rye) and from 72 ergot sclerotia placed
in the soil during winter (depth 5–15 cm, placed in mesh bags with the pore size 0.5 cm). Sclerotia were washed with tap water and after drying they were placed on the surface of nutrient agar
(Czapek-Dox) – 5 sclerotia per dish. After 10 days of incubation (20 ± 3°C) the number of fungal colonies around sclerotia was evaluated. The detection of individual fungi was realised microscopically
after isolation and obtaining pure cultures. All fungi produced conidia spontaneously.




So I wonder if these parasites help ergot because they produce their own conidia, weaken the plant, then ergot jumps in? And these fungi invade ergot too, but by that time the plant has already been taken over, thus acting as 'mutualist parasites'.

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OfflineMushalla
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Registered: 01/07/23
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Loc: Massachusetts
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Re: Successful isolation of Claviceps purpurea/Ergot [Re: CreonAntigone]
    #28354801 - 06/10/23 07:01 PM (11 months, 2 days ago)

Wish I could see those pics…. I’m intrigued.
:popcorn:

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Offlinerifampicin
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Registered: 06/09/23
Posts: 5
Last seen: 2 months, 29 days
Re: Successful isolation of Claviceps purpurea/Ergot [Re: CreonAntigone]
    #28355131 - 06/11/23 02:04 AM (11 months, 1 day ago)

I think that what this article says is more that when the sclerotia fall to the ground, they get colonized/invaded by other fungi, thus impacting viability.
Like the use of the term "mycoparasitic activity" does make me feel that this is not a beneficial association for the fungi!

Still, that's interesting to know! I'll try to see if the mycelium of the mycoparasites could maybe correspond more to the contams I got on my plates

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Offlinerifampicin
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Registered: 06/09/23
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Last seen: 2 months, 29 days
Re: Successful isolation of Claviceps purpurea/Ergot [Re: rifampicin] * 3
    #28356865 - 06/12/23 10:32 AM (11 months, 11 hours ago)

The mycelium under UV light! Notice the blue glow on the center/older mycelium


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