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Invisiblemicololo2
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Registered: 11/05/05
Posts: 388
Loc: Québec, Canada
Need Help for Cloning and More...
    #5926647 - 08/02/06 07:54 PM (17 years, 5 months ago)

For Boletus Edulis:
1. As every time I've tried to have a mycelium from that mush. and failed, I need your suggestions on how I could do to have success.
As some mushs. mycélium has colors EX: Nameko turns orange in the petri dish, White Elm brown etc.. Wich color has Boletus mycelium?

2. Soon (When temp. will fall) I'll get better Boletus . If I can get a mycelium I'm willing to share with some of you, who would have help me to find a way to get a mycelium, some tissus of that mycelium to find a transfer substrate that can grip on rootlets.
For Chanterelles:
1. I've found young Chanterelles sooner today. I've made plates with OMBY (Oatmeal Brewer's yeast)agar medium at PH 7-7.5. The Chanterelles were soaked in H2O2 for about 2 minutes (wishing it's a good way to start). If I can get a mycelium I'll show here.
I need suggestions to find a transfer substrate that could grip on rootlets. Here to, I'm willing to share some tissus to find that substrate.

Maybe some of you knows all of that, if so please tell me. If not it could be interesting to work and share together to find that substrate.
Thanks to all who has interest in that project


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Offlinefalcon
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Re: Need Help for Cloning and More... [Re: micololo2]
    #5926946 - 08/02/06 09:34 PM (17 years, 5 months ago)

Interesting project Micololo,

Boletus subcaerulescens turns brown, as do two other species of Boletes. I don't know the species of either of the other two. One was a close relative of B. bicolor.

The mycelium of chanterelles should be white.

Last year I tried sawdust as inoculate for roots with both species and had no luck so far.

Chantrelles will form pale stroma on substrates in jars when it very warm.
I had this happen on coffee grounds and grain.

I haven't done anything with these two species this year, would like to see how your work progresses.


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Invisiblemicololo2
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Registered: 11/05/05
Posts: 388
Loc: Québec, Canada
Re: Need Help for Cloning and More... [Re: falcon]
    #5927846 - 08/03/06 05:17 AM (17 years, 5 months ago)

What kind of sawdust did you used?
Very warm? 25, 30, 35 Celcius?
Did you clone the mushrooms or start with spores?
Many thanks Falcon


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Offlinefalcon
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Re: Need Help for Cloning and More... [Re: micololo2]
    #5927898 - 08/03/06 06:24 AM (17 years, 5 months ago)

I used very fine oak sawdust.
The stroma show up at about 30 Celcius.
I cloned the mushrooms. I did start one culture from spores of B. subcaerulescens to see if they would grow.


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Invisiblemicololo2
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Registered: 11/05/05
Posts: 388
Loc: Québec, Canada
Re: Need Help for Cloning and More... [Re: falcon]
    #5928186 - 08/03/06 10:16 AM (17 years, 5 months ago)

Thanks
I do have that sawdust, if I can get a mycelium I'll try it. But I think another form of substrate would be better to get a chance to the mycelium to stick on the rootlets.
Later + about your B.subcaerulescens


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InvisibleCorporal Kielbasa

Registered: 05/29/04
Posts: 17,235
Re: Need Help for Cloning and More... [Re: micololo2]
    #5932520 - 08/04/06 07:38 PM (17 years, 5 months ago)

Are you planing on spawning the ground with said substrate? Or do you plan on inoculating saplings then transfering to the ground?


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Invisiblemicololo2
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Loc: Québec, Canada
Re: Need Help for Cloning and More... [Re: Corporal Kielbasa]
    #5935119 - 08/05/06 05:33 PM (17 years, 5 months ago)

I'm planing on spawning the rootlets of the 20 years white and black spruces with some kind of substrate that can grip on those rootlets.


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OfflineFeelers
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Re: Need Help for Cloning and More... [Re: micololo2]
    #5935549 - 08/05/06 10:01 PM (17 years, 5 months ago)

I was thinking planting trees that are grown in say a bucket of spawn would be a good way to go - a little bit more intensive than just a straight inoculation, but thats pretty much what you're going for anyway. :smile:


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InvisibleCorporal Kielbasa

Registered: 05/29/04
Posts: 17,235
Re: Need Help for Cloning and More... [Re: micololo2]
    #5936746 - 08/06/06 11:54 AM (17 years, 5 months ago)

By planting small inoculated trees along the existing larger colony i think would be a good way. As soon as the roots come in contact with the larger trees they bridge the gap and the mushroom continues its way onward into the future. Saplings cost .50 to 1.50 per tree.


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Invisiblemicololo2
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Posts: 388
Loc: Québec, Canada
Re: Need Help for Cloning and More... [Re: Corporal Kielbasa]
    #5937148 - 08/06/06 02:09 PM (17 years, 5 months ago)

Quote:

Corporal Kielbasa said:
By planting small inoculated trees along the existing larger colony i think would be a good way. As soon as the roots come in contact with the larger trees they bridge the gap and the mushroom continues its way onward into the future. Saplings cost .50 to 1.50 per tree.



I don't think that could work because spruce do not support shade, and in a tree plantation there is a lot of shade. So the little trees will die.


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Invisiblemicololo2
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Registered: 11/05/05
Posts: 388
Loc: Québec, Canada
Re: Need Help for Cloning and More... [Re: micololo2]
    #5937745 - 08/06/06 05:21 PM (17 years, 5 months ago)

Here some horrible results. The chanterelles I've tried to clone. Contam, contam, contam. Take a look on the photos.
I don't know why I can easily clone a wood lover while I can't clone a mychorryzal mushroom. Could you tell me why? What am I doing wrong?


Cob web


Here what I call a wax looking contam


Wax contam with probably cobweb.

Does someone knows how long it takes to see mycelium in the plate with Chanterelle and Boletus Edulis?

Does someone have pics of those mycelium?

Thanks for your help and interest


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OfflineFeelers
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Re: Need Help for Cloning and More... [Re: micololo2]
    #5938259 - 08/06/06 08:32 PM (17 years, 5 months ago)

Is that with peroxidated agar also?


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Offlinefalcon
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Re: Need Help for Cloning and More... [Re: micololo2]
    #5938493 - 08/06/06 09:34 PM (17 years, 5 months ago)

Does someone have pics of those mycelium?




These are chanterelles on brown rice. I don't think I have a good picture of the mycelium on agar, all my agar was in jars. The mycelium on rice looks pretty much like it does on agar, on agar it was not as vigorous about growing off the media.

It takes about ten days to see mycelium with both species.

Make sure there are no fly larva hole at all in the mushroom you are going to clone. The larva usually burrow up from the bottom of the mushroom so if you cut your mushroom at the base you can see the larva holes, if it has any don't use it.

Use as tiny of a piece as you can cut to do the clone a 2mm cube is good. Do a lot of plates to start. Like 5 to 7 or more. Use just one small piece per plate.

I'll look around for pictures of boletus on agar. Most of the pictures I have the agar is in jars and the distortion ruins the image.


--------------------
Keep growing.


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InvisibleSpeeker

Registered: 02/11/04
Posts: 877
Re: Need Help for Cloning and More... [Re: micololo2]
    #5938823 - 08/06/06 10:40 PM (17 years, 5 months ago)

>What am I doing wrong?
you are just using wrong medium for those species..
and even with the good one it still might be very difficult..

have you read the "Cantharellus thesis"
http://www-mykopat.slu.se/Newwebsite/mycorrhiza/kantarellfiler/texter/thes.phtml

Quote:

All tissue samples were surrounded or embedded in mucus of bacterial origin within a few days (Paper IV). Growth did not start until after a 17 to 53-day-long lag period (35 days on average). Growth from tissues was only observed on MS, and only if activated charcoal had been embedded in the agar.
<snip>
No growth was observed on 2% malt agar. All 36 tissue samples directly inoculated on MFM died owing to fatal bacterial infections. Repeated trials were never successful.






:goodluck:


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InvisibleSemilanceata
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Registered: 05/26/03
Posts: 841
Loc: República Federal Íbera
Re: Need Help for Cloning and More... [Re: Speeker]
    #5939313 - 08/07/06 12:01 AM (17 years, 5 months ago)

What is activated charcoal? I know what charcoal is but don´t understand what activated charcoal means.
Thanks.


--------------------
Sr_Setahongo


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OfflineFeelers
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Re: Need Help for Cloning and More... [Re: Semilanceata]
    #5939591 - 08/07/06 01:32 AM (17 years, 5 months ago)

Activated carbon is basically coal that has been cooked at a very high temperature and has huge surface to mass ratio. I think one teaspoon of activated carbon has the surface area of 5 football feilds (or something like that).

Because of its huge surface area it's very absorbant. I think it absorbes metabolic waste products in the media, and things such as growth inhibitors.
http://en.wikipedia.org/wiki/Activated_Carbon


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Invisiblemicololo2
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Registered: 11/05/05
Posts: 388
Loc: Québec, Canada
Re: Need Help for Cloning and More... [Re: micololo2]
    #5939971 - 08/07/06 07:06 AM (17 years, 5 months ago)

I'm confused. Falcon said that he has seen mycelium growing after 10 days while Speeker found in articles that it grows after an average of 35 days.

Falcon: Are you able to send me a sample of your mycelium tissues. I'll give you what you want in exchange. If so, could you give me the secrets of your success?

Speeker: Many thanks for all the info. you find that make those threads so interesting. Because I'm building a new Shiitake growing house-trench (I'll show you picture of it)and English is not my native language, I do not have the time to read all the info., but be sure I will.

This morning, my friend found 2 new baby chanterelles for me. Here they are. Could you give me a good agar recipe to start a new mycelium?



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InvisibleSpeeker

Registered: 02/11/04
Posts: 877
Re: Need Help for Cloning and More... [Re: micololo2]
    #5940886 - 08/07/06 01:43 PM (17 years, 5 months ago)

Here's one mentioned in that thesis:
http://en.wikipedia.org/wiki/Murashige_and_Skoog_medium


Here's another .. [used at the Technical Research Centre of Finland (VTT)]

Quote:


CANTHARELLUS MEDIUM

Glucose 4.0 g
Na-succinate 1.35 g
NH4Cl 0.58 g
KH2PO4 0.2 g
MgSO4 × H2O 0.1 g
CaCl2 × H2O 26.5 mg
NaCl 20.0 mg
EDTA 9.3 mg
FeSO4 × 7 H2O 6.95 mg
MnSO4 × H2O 0.845 mg
H3BO4 0.31 mg
ZnSO4 × 7 H2O 1.44 mg
KI 0.415 mg
Na2MoO4 1.21 g
CuSO4 × 5 H2O 0.125 g
CoCl2 × 6 H2O 0.12 g
m-Inositol 10.0 mg
Nicotinic acid 0.1 mg
p-Aminobenzoic acid 0.1 mg
Pyridoxine hydrochloride 0.1 mg
Calsium pantothenate 0.1 mg
Thiamine hydrochloride 0.1 mg
Riboflavin 0.1 mg
Biotin 0.025 mg
Agar 12.0 g
Distilled water to 1.0 l

Add onto the agar a piece of fresh tomato root near the inoculum.





good ol' tomato root trick there..  :grin:


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Offlinefalcon
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Re: Need Help for Cloning and More... [Re: micololo2]
    #5941396 - 08/07/06 04:50 PM (17 years, 5 months ago)

I don't have the culture any more.

I use PDYA agar. I haven't used antibiotics when trying to clone a mycorrhyzal mushroom.

My recipe is a little different from the normal one.
I cook about 4.5 Kg of peeled potatos in an 8 liter Aluminum pot with enough water to just cover them. When the potatos are done I strain the potatos out.
This leaves me with 3 liters of broth.

I freeze 325ml of the potato broth.
Then I thaw it. Then strain out the solids. When you freeze the broth, the solids, I guess mostly starch clumps and is easily strained with a fine wire stainer. I then add enough water to the broth to make 500ml.

To the 500 ml of broth I add about 1/2 gram nutrayeast and 2grams of fructose
and 10 grams of powdered agar. I heat this in a stainless steel pot so that the agar is well disolved and then pour about one ounce into 8 ounce vented jars and pressure cook at 15psi for 15 to 20 minutes.

Most recipes call for .3 Kg of potatos and you finish with 1 liter of media.

I use 4.5 Kg if I turned all of it into media I would end up with about 5 liters or 6 liters of media.

Edit, I changed the 8 Kg to 4.5 Kg, I use about 10lbs. of potatos.


Edited by falcon (08/07/06 09:49 PM)


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Invisiblemicololo2
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Registered: 11/05/05
Posts: 388
Loc: Québec, Canada
Re: Need Help for Cloning and More... [Re: falcon]
    #5941856 - 08/07/06 07:22 PM (17 years, 5 months ago)

Thanks Falcon, nice of you.

In the thesis I've read this:
The long period of establishment, slow growth and dispersal mainly via vegetative growth makes C. cibarius a late colonizer in forest plantations without contact with older trees. The youngest tree I found associated with fruit bodies of C. cibarius was a 28-year-old Picea abies (Paper V). Fungi associated with older forests are sometimes called "late-stage fungi" (Newton 1992).

Well my tree plantations where we find the chaterelles are at the oldest 20 years. I've planted those trees. It looks like chanterelles can grow with white spruces too.

Thanks again for that document, I've learned a lot with it. I now will have a new approach.


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