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Darklight

Outrunning Trichoderma when isolating from the wild?

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I'm isolating some local woodloving species onto agar and Trichoderma contam is a constant hassle with one of them.

 

Trouble is the little buggers don't show up as discernable until they sporulate. And the mycelia I am working with is slow to start.

 

What are your best options for working against Tricoderma spp on agar? I couldn't find anything specific on it.

 

My best options so far are

 

  • Serial dilution- a huge pain in the arse, because Trich is sporulating once it's visible, so the spores go into solution, and there are gazillions of them. Plus a good serial dilution run takes heeeeeeeaps of containers. Over a month. Minimum a hundred.

 

  • Subculturing ahead- taking only the tiniest fragment of the leading edge, culturing it on, repeat

 

Cardboard slopes haven't worked, as I'm uncertain of this species' ability to grow on cardboard. Still waiting at +10 days to see signs of mycelia. And I've seen Trich grow on cardboard heaps of times. The mycelia would have to grow much faster than the Trich to outrun it and I don't think that's going to happen

 

Is there an agar type media selective against Trichoderma I could try?

 

 

 

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use wet card perhaps?, the trich is definitely slower on that substrate ,,, and can be out run by the odd operation to remove the white myc

and re-add that to fresh card ;) , then wet it I guess

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... works as a pocket fae pet too :3 - especially useful if you encounter a stint of homelessness to preserve the faeries for safe keeping/later down the line..

 

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sorry i was too quick to answer without reading the full post , sorry Darklight ... : /

 

wish you luck and fast, smooth passage to where ya wanna be

Edited by ☽Ţ ҉ĥϋηϠ₡яღ☯ॐ€ðяئॐ♡Pϟiℓℴϟℴ

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use wet card perhaps?, the trich is definitely slower on that substrate ,,, and can be out run by the odd operation to remove the white myc

and re-add that to fresh card ;) , then wet it I guess

attachicon.gif2016-04-17 23.06.00.jpgattachicon.gif2016-04-17 23.06.27.jpgattachicon.gif2016-04-17 23.06.30.jpg

... works as a pocket fae pet too :3 - especially useful if you encounter a stint of homelessness to preserve the faeries for safe keeping/later down the line..

 

I do like the pocket fae idea, I hope you didn't have to go thru that to find it out tho :)

 

I'm thinking selective media trial. Research is showing the putative isolate could survive on a high pH media, and Trichoderma favours lower. ( Edit: more checking proves this could be wrong, I may try it anyhow )

 

Will keep a few colonies back tho, just in case the publication is wrong.

 

Other option could be a low nitrogen media, maybe even a water media.

Edited by Darklight
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what works for me is h202 and sub culturing on agar plates. Every other technique is hit and miss,

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Have you done much experimentation with temperatures Darklight?. You may find changing a few degrees that the new temp has less effect on mycelia growth rates than it does on the occurance of the dreaded "Trichoderma", I'm guessing a lowering of the temps would be the way to go but I'm no expert. Good luck mate. 

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11 hours ago, Zen Peddler said:

what works for me is h202 and sub culturing on agar plates. Every other technique is hit and miss,

 

Ha! Brilliant, why didn't I think of that BM? I'd dismissed it in favour of antibiotics at an earlier stage cos H2O2 has a tendency to  reduce growth in that species significantly for a few weeks. But it might just work now I have the species more-or-less isolated away from everything but the Trich.

If Trich can't set viable spore it might slow it down and the mycelia have a chance to recover.

 

Is odd, I've never found Trich *inside* a specimen, so I wasn't expecting it. Worth a shot anyhow. I'll run a side-by-side.

 

I have two candidates for the mycelia to be the target species, so will need to fruit and test them both

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You shouldn't find Trich sp. inside stipe or cap tissue, but you will regularly find pseudomonas which can be easily cleaned up with amp. I also wipe down my specimens with h2o2 and tear them open rather than cutting. This is important because often contaminants can be introduced into the the tissue by the knife as it passes through the 'dirty' surface layer.

 

Also, where is the contamination popping up? If at the inoculation site then this would be consistent with specimen contam, but if elsewhere then maybe your plate wasn't sealed?

 

With love,

 

Mimz

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You shouldn't find Trich sp. inside stipe or cap tissue, but you will regularly find pseudomonas which can be easily cleaned up with amp. I also wipe down my specimens with h2o2 and tear them open rather than cutting. This is important because often contaminants can be introduced into the the tissue by the knife as it passes through the 'dirty' surface layer.

 

Also, where is the contamination popping up? If at the inoculation site then this would be consistent with specimen contam, but if elsewhere then maybe your plate wasn't sealed?

 

With love,

 

Mimz

 

Ta Mimz, I hadn't thought about tearing instead of cutting. The mushroom itself is quite fine and not sure it's possible with this species but I will keep it in mind for future isolations. I do frequently sterilise the scalpel with a bead steriliser, but not always between slices, and I know it's an issue

 

This species hates H2O2. I've pretty much established that lol, a negative result is also a result. I've seen a pure culture of a closely related species re-establish after a 5 week lag on H2O2 spawn, but in the face of actual isolation from a small section, with competitors, I think it's not likely to work. Might try it with a small number of mycelia subcultures once I have a few- will be interesting to see how far Trich vegetative cells can run without spawning too. And if there are no Trich spore I can re-run serial dilutions

 

Contam is from inoculation point, it's of specimen origin. Bacto contam was present but outrun with antibiotics.

 

Dunked the latest acquisitions in a 70% ETOH soak 30 seconds before isolation, waited 5 min to see if it had killed them, they were still solid

 

One thing with cardboard slopes I'll do next time is to use cardboard from three different sources. Trouble with cardboard, even the rough looking stuff, is you rarely know if it's been impregnated with anything, or recycled from that. I use a flame test - burn a bit with a lighter- to check for coloured flame or fire retardants, but one of the batches of cardboard I used for cardboard slopes came out of the autoclave with distinct green stripes- made me think of copper- possibly a fungal retardant.

 

I *finally* got a spore print on alfoil. Tricksy little hobbitsses it is. Can try it on a range of media via serial dilution this way as well

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