Mixing spores

[Chiral]

Has anyone ever tried mixing different types of spores in the same syringe..if so was it successful and if not why...?

Was just contemplating weather it's feasible to mix your spore strains and have different types of shrooms on your cakes or in your bulks..

H.

[Hyphal]

Not feasible - they will compete with each other.

[Chiral]

So...compete and whats the outcome of that...? no shrooms or more of one and less than the other...?

H.

[Undergrounder]

The white mycelium won't thatch together, you'll just get lots and lots of different species of mycelium in their own little groups, none of them big enough to fruit.

[Chiral]

ahhh okay that's what I thought...I wasn't sure if the mycelium would knit together or not..

H.

[BlackDragon]

Wont work too well for different species on the same substrate. Competition will be the biggest problem.

Can and will work for like/like species.. but it will form intermediate hybrids. On a small scale this will work. Providing the same species is used for both differing strains, in nutrient rich solution, its the basics of forming hybrids.

Now you cant really control whats going to happen unless you have a powerfull microscope and single out spores, so our hybrids will be totally random. There will be random germination of the spores in nutrient solution(if you decide thats what your after). Depending on what type of similar germinating spore they lie/swim next to wil determine the final outcome. Spores germinate, the rootlets search for there asexual partners, the fusion of the rootlets takes place, then forming mycelium. If you happen to use the same species, and that germination and entanglement happens to differing genetic partners, a simple or many simple hybrid will form. I gather this is the basics of mycology morphology in a jar. Selection is made from the final characteristics of the fruiting body, then cloned to propagate your new selection.

SO to answer your question, mixing like spores can/will create hybrids if done in a nutrient rich solution(or maybe in situ) but mixing species wont do much except cause competion problems. One is most likely to overrun the other, or be grouped/ segregated like UG said.

Hope that helps.

[Chiral]

That is exactly where my mind was heading with the hybrid thing..so if you where using say 2 types of cube in the one syringe the likeliness is that a hybrid will form..this is exactly the basis of my question. The question wasn't asking about mixing say subs with cubes it was simply about mixing the same type of spore with another from the same family.

H.

[Hyphal]

As far as I know, even different cube strains won't mate to form hybrids without specific catalysts to encourage this.

Snake venom is used to encourage mating and is how some hybrids have been created, but I don't know anything about it.

I'm sure you can't create hybrids by simply mixing two different strains but could be wrong...

[Chiral]

Snake venom is used to encourage mating and is how some hybrids have been created

Snake Venom..care to elaborate on that...sounds interesting.

H.

[Hyphal]

I dont have a lot of info, but a few google searches should turn something up.

[Monk]

Actually, to do traditional breeding, it is only necessary to create monokaryotic mycelium through serial dilution of spore solutions. Basically, you dilute the spores so much that you can get a single spore to germinate in each area and culture them away from the other monokaryotic myc. Then, two different monokaryotic strains of the same species (regardless of strain) can be plated together on agar to cross and form a dikaryotic strain that is much more likely to fruit etc. Of course, it's not that simple as not all monokaryons will cross with each other, even from the same species or strain, much less fruit or fruit well. Therefore, the mushroom breeder must do many, many plates like this and grow out the different successful crosses to determine which ones worked and fruit and have the characteristics they are looking for. This is the technique that Roger Rabit used to cross the red-spore with another cubensis to bring the genes back or some such. Workman has also used this technique with his PF Albino/Penis Envy cross that is now available as APE 1.0 (albion penis envy.) Edible and medicinal fungus growers use the same tek the world over to create new strains.

Now, the rattlesnake venom thing is actually in reference to hybridization between different species through protoplast fusion. In most fungi, different species cannot hybridize naturally, so people use different techniques to create interspecific, intergeneric and even inter-order hybrids. Most researchers use lysing enzymes to dissolve the cell walls to allow the protoplasts to fuse to create hybrids, but aloha medicinals used rattlesnake venom to fuse various Cordyceps species and strains. Here's their article on it: http://www.alohamedicinals.com/cordy_IJMM_hybrid_article.pdf

Pretty good read, but there are other ways to do it, too. Either way, you gotta get monokaryotic mycelium first.

The reason that simply mixing spores doesn't create hybrids is that spores clump. That is, in a spore 'solution,' the spores of any given strain clump together and germinate in these little masses. So, they become dikaryons before they ever encounter the other strain. So, you just get a mix of the strains you used, rather than a hybrid. I've not tried that, so I don't know how this would affect the outcome, though.

[Undergrounder]

wow very interesting, who knew there was such a thing as a "mushroom breeder"!

[Hyphal]

Thanks for the detailed info FM!

[Inyan]

Serial dilutions are a definite must, but just as important is your plating practices if not more so. You will need several plates each with the designated dilution marked on them so you know what was your best dilution given your own streaking technique. You will want to take a dropper and take up just a single drop of your dilution starting with the most dilute first and add this drop to your plate on the edge. You will then need to streak this out by making at least 4 quadrants. Now, if you've done this right, you should be able to isolate a few individual spores which will then begin to grow. The better your streaking technique, the less you have to dilute. With extremely dilute samples you can simply pour a good euro sized drop or larger onto your plate and roll this around until your entire plate is covered. Watch for growth and once you have a few isolated strains of each variety you can then subculture onto another plate. Obviously, your going to want to right down the specific variety on each plate before hand and work with one strain at a time. Permanent markers work well for this. After you have allowed your plates to settle, turn them upside down as moisture will collect in the tops otherwise and ruin your culture as it drops down into your mycelium. Contamination also occurs this way. If you don't have the time to make serial dilutions you can sub your plates one after another taking each culture from the last quadrant of the last plate you worked on. This should be done prior to growing them out.

One more note, each single isolated culture can be kept in its pure form via plating to another and labeling which plate each one came from originally. In this way, you can make multiple crosses with a single isolate and test each cross against each other to see which one performs better. You can think of each cross as a single variable seed from a plant for comparison purposes. When you make a cross with plants or cacti you want to grow a number of seeds from that same cross to get a variation that is worthy of introducing. For what its worth, shaking, vibrating, rotating, etc. are all methods of getting your spores to separate out from each other in their dilution. If you have a microscope, it would be advised that you check a drop out first to see approximately how many spores you have in a drop. If you have just 1-10 spores in a single drop you can forget streaking and simply roll that drop around on your plate until all portions of your media have been covered. I like to rotate the pate continuously for a few minutes when utilizing this method. If you see more than 50 spores in a single drop or dilution... streaking is going to be a must.

http://www.studentsguide.in/microbiology/m...ate-method.html

The above is a simple diagram showing different streaking methods. Practice makes perfect.

http://www.fungi.com/tools/media.html

A good agar has antibiotics in it to prevent/control contamination... in my opinion. Plastic plates can also be used and reused with little fear of contamination via cleaning them in h202 hydrogen peroxide bath followed by an ethanol bath and lastly a 10% bleach bath for a one hour soak in each bath. Allow your plates to air dry between each batch.

Edited January 19, 2009 by Inyan

[Monk]

Thanks a million for those details, Inyan! Those'll be coming in handy soon

[Inyan]

FM,

Just trying to fill in a few gaps I saw in an otherwise very beautifully detailed piece of work. The 4 quadrant streak is the best though in my opinion for isolation if your going to choose just one method. One thing of importance is you only want to cut into each quadrant streak with one or two swipes max.

I found a few examples to show you a halfway decent attempt at isolation... notice that each quadrant is only went into twice... resulting in better isolation.

http://www.microbelibrary.org/microbelibra...Sreak_fig10.jpg

Another good attempt...

http://www.microbelibrary.org/microbelibra...Streak_fig6.jpg

Now, for a piss poor technique where each quadrant is cut into repeatedly....

http://www.microbelibrary.org/microbelibra...treak_fig13.jpg

As you can see, it is possible to get a few isolates even with poor technique. While this is generally true, you must keep in mind that spore syringes as well as prints are loaded with many more spores than you need so you may still want to dilute 50% or more before you take a single drop to plate out via this method.

Edited January 21, 2009 by Inyan

[Monk]

Very nice and practical info.

In your experience, is it also helpful to add a bit of wetting agent to help break up spore clumps when attempting to isolate monokaryons? (Assuming of course, the solutions will be used before the wetting agent encourages germination....)

Thank you

[TheDudeAbides]

why couldnt the mycelium thatch together? after all when u do one inoculation of one type of spore print u get all differet types of mycelium growth. hence why people grow mycelium then transfer the better mycelium to other jars and keeping that one gene of mycelium

[Monk]

why couldnt the mycelium thatch together? after all when u do one inoculation of one type of spore print u get all differet types of mycelium growth. hence why people grow mycelium then transfer the better mycelium to other jars and keeping that one gene of mycelium

Not exactly sure why this is, mike. If you plate two different varieties of the same species on agar, for instance, they won't thatch together.

Also, I have now actually tried this experiment and shot two different varieties of spores into the same grain jars. It became a weird, clumpy, knotted mess of non-blended mycelium just as others had told me it would.

I can't explain why they wouldn't share, but that's what happened. I'm not inclined to try it again, but maybe you could with some other varieties and see what happens. I'm all for testing theories

[Chiral]

Perhaps an LC of 2 different spore prints maybe make a difference...like you say FM plenting theory testing to be done here.

[poo]

This is a really interesting thread - always wondered about this, thanks for the explanation guys!