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OfflineMr.Caterpillar
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Starting Claviceps Paspali cultures from wild specimens * 1
    #18937982 - 10/05/13 09:16 PM (10 years, 4 months ago)

I've recently found some Claviceps Paspali growing on heads of Dallis Grass (Paspalum Dilatatum).

I'm interested in culturing this.  I'd like to hear from anyone who has experience culturing Paspali.

I am endeavoring to germinate sclerotia of the ergot by incubating them in sterile moist sand at 10 c for 30 days, and then germinating at 20 c.  This is according to the procedure outlined in:

Quote:

Germination Requirements of Claviceps paspali Sclerotia
Barry M. Cunfer and David Marshall
Mycologia
Vol. 69, No. 6 (Nov. - Dec., 1977), pp. 1137-1141





Successful germination of the sclerotia will result in the tiny eponymous fruitbodies; "Clava", which produce the ergot "honeydew" which will grow into mycelial cultures on agar.

Hmm . . . I see that the Dallis Grass heads are covered with drops of the sticky honeydew.  Why not try culturing directly from there?  Perhaps with antibiotic agar, luck, and many transfers a clean culture can be obtained directly from field specimen rather than from the germinated sclerotia?

Does anyone have any experience with  germinating Paspali sclerotia or culturing the honeydew?

Please advise.

Thanks,
Mr. Caterpillar


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Re: Starting Claviceps Paspali cultures from wild specimens [Re: Mr.Caterpillar]
    #18938133 - 10/05/13 09:57 PM (10 years, 4 months ago)

:pm:


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OfflineMr.Caterpillar
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Re: Starting Claviceps Paspali cultures from wild specimens [Re: Eminence]
    #18938600 - 10/06/13 12:45 AM (10 years, 4 months ago)

Thank you, Frylock, for the info.  I pm'd you back already.

Mr.Caterpillar


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OfflineShroomDoom
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Re: Starting Claviceps Paspali cultures from wild specimens [Re: Mr.Caterpillar]
    #18939869 - 10/06/13 11:43 AM (10 years, 4 months ago)

you found honeydew! congrats :yesnod:.


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OfflineMr.Caterpillar
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Re: Starting Claviceps Paspali cultures from wild specimens [Re: ShroomDoom]
    #18943068 - 10/07/13 12:58 AM (10 years, 4 months ago)

Quote:

you found honeydew! congrats :yesnod:.




Thanks!  It's exciting!!



Above are photos, from left to right (as they appear in my browser . . . ), that show the Claviceps Paspali I've found:

1. Heavily infected Dallisgrass head.  Note the large sclerotia, the blackish fungus, and on the rightmost sclerotium; a glistening bead of "honeydew".

2.  C. Paspali sclerotia separated from the head of grass after having been cleaned.  I cleaned them using a solution of ~.0015% H2O2 in tap water.  Under a laminar flow hood I soaked them for one minute in the H2O2, and then rinsed them in regular tap water, then placed them in the dry petri under the sterile air flow to dry off.

3.  This photo shows one large sclerotia on an otherwise uninfected head of Dallis.  Paspali sclerotia are generally about the size of pot seeds, and, in fact, resemble them a little in their shape, color, hardness, and even their markings.  However, the largest of Paspali sclerotia (that I have found) have a weirdly popcorn-ish texture.  I am not entirely sure if the smaller ones are immature or merely smaller by nature, but I think it is the latter.

4.  This photo shows a sample tube that I have filled with sand and sterilized.  I planted approximately 10 sclerotia in each tube.  Some of the tubes are being stored at 20+ C in hopes of germinating without a cold incubation, but the majority are incubating in the fridge at ~ 10 C.  I speculate that because where I live does not have a very cold winter that this ergot is adjusted to the mild climate and may germinate without the cold incubation that is intended to mimic winter, and so perhaps the sclerotia that are being stored at the warm temp will just germinate directly.  According to the article I cited earlier optimum results are a 40% germination rate.  Hmm . . . it seems to me, however, that the authors basically duplicated the technique of Stevens and Hall who wrote the original paper on Paspali ("Three Interesting Species of Claviceps", Botanical Gazette
Vol. 48, No. 1 (Jul., 1909) pp. i-vi ) and that the technique could be improved upon.  I mean, I don't know why the sclerotia should be in sand instead of some other moist material - I think it is just a very old technique.  Personally I would prefer to put them into something where I can clearly see the sclerotia, so I am thinking of some other methods.

Also, I inoculated a number of PDA plates with the honeydew direct from the Dallisgrass.  I didn't have any antibiotics on hand for this, so I expect only to grow a bunch of contams unless I'm lucky (and I do tend to be lucky!!:cool: . . . well, sometimes . . .:eek: ).  I was impressed by how pungent the caramel smell of burning sugar was that came off the scalpel when I flamed it.  That Paspali exudate is aptly named "honeydew" because it is really sweet, although I don't dare taste it.  The collection of grassheads gives off a distinctly fermented odor that reminds me of a crude malt liquor or palm wine.

I've acquired some antibiotic ("Furan-2" - for use in fish tanks) that I'll use to make some antibiotic agar, and try to get some really fresh honeydew to culture with.  I'll order some gentamycin and try with that too, provided the Ergot is still growing here when it arrives.

Unless anyone has any questions/comments then that's all for now, folks!  I'll update this thread if and when my sclerotia germinate and/or my efforts at honeydew culture succeed.

Mr.Caterpillar


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OfflineRogerRabbitM
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Re: Starting Claviceps Paspali cultures from wild specimens [Re: Mr.Caterpillar]
    #18943884 - 10/07/13 09:30 AM (10 years, 4 months ago)

Gentamicin sulfate (I probably misspelled it) is autoclavable so it's a preferred antibiotic.
RR


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Re: Starting Claviceps Paspali cultures from wild specimens [Re: RogerRabbit]
    #18972613 - 10/13/13 03:33 PM (10 years, 4 months ago)

So reasonably if one found some dew you could just dab some of it on antibacterial agar and clean up the culture as needed? sounds GROOVY :flowerchild:.


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Re: Starting Claviceps Paspali cultures from wild specimens [Re: ShroomDoom]
    #19040255 - 10/27/13 11:04 AM (10 years, 3 months ago)

Why don't you make the culture on PDA straight from the sclerotias?
Anyway, good luck, and keep us updated!


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Re: Starting Claviceps Paspali cultures from wild specimens [Re: Tana]
    #19069032 - 11/01/13 09:53 AM (10 years, 3 months ago)

i sent you a PM as well


great find! im hoping your project is fruitful!


Edited by shopdropper (11/01/13 10:02 AM)


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OfflineShroomDoom
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Re: Starting Claviceps Paspali cultures from wild specimens [Re: Tana]
    #19069061 - 11/01/13 10:05 AM (10 years, 3 months ago)

Quote:

Tana said:
Why don't you make the culture on PDA straight from the sclerotias?
Anyway, good luck, and keep us updated!



Placing the sclerotia on agar would just be cloning the genetic material from that sclerotia , right? Using the honeydew would be like a multispore inoculation that one could have a variety of genetics to isolate and mutate into a more productive strain. Am I correct in this line of thinking? I'm new to ergot culture and know very little about it.


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Edited by ShroomDoom (11/01/13 10:08 AM)


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Re: Starting Claviceps Paspali cultures from wild specimens [Re: ShroomDoom]
    #19069072 - 11/01/13 10:09 AM (10 years, 3 months ago)

Quote:

ShroomDoom said:
Quote:

Tana said:
Why don't you make the culture on PDA straight from the sclerotias?
Anyway, good luck, and keep us updated!



Because the sclerotia to agar would just be cloning the genetic material from that sclerotia no? Honeydew would be like a multispore inoculation that one could have a variety of genetics to isolate and mutate into a more productive strain?





this dude is right, and with Fluorescein and a black light youll be able to find the higher producing sectors for seperation.


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Re: Starting Claviceps Paspali cultures from wild specimens [Re: shopdropper]
    #19076375 - 11/02/13 06:05 PM (10 years, 3 months ago)

One easy way to select a sclerotia for culture would be to take each sclerotia, cut it in half, grind one half with a pinch of lime (or about a ml of conc ammonia) and shake in a test tube with a little DCM and water. Allow it to settle out and shine with black light, the ones that the DCM layer glows the brightest are the ones you want to keep. Germinate the other half as per usual. Good luck on your adventures! :thumbup:


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Re: Starting Claviceps Paspali cultures from wild specimens [Re: Eywa_devotee]
    #19077059 - 11/02/13 08:53 PM (10 years, 3 months ago)

Quote:

Eywa_devotee said:
One easy way to select a sclerotia for culture would be to take each sclerotia, cut it in half, grind one half with a pinch of lime (or about a ml of conc ammonia) and shake in a test tube with a little DCM and water. Allow it to settle out and shine with black light, the ones that the DCM layer glows the brightest are the ones you want to keep. Germinate the other half as per usual. Good luck on your adventures! :thumbup:




Very interesting! Are you sure that a black light is sufficient? I was under the impression that a UV-C lamp (254nm) was generally used for visualisation.

The book Ergot: The Genus Claviceps  confirms your technique and proposes TLC as a simple, fast and cheap method of determining alkaloid content. "Normal phase ergot alkaloid TLC is spotted on silica plates and developed in chloroform:methanol solutions. Plates can be viewed in UV light to visualize the fluorescent ergot alkaloids, or then can be sprayed with Ehrlich's reagent which is a positive test for the indole ring, common to most ergot alkaloids."


Edited by hjalmar (11/02/13 08:58 PM)


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Re: Starting Claviceps Paspali cultures from wild specimens [Re: hjalmar]
    #19077118 - 11/02/13 09:06 PM (10 years, 3 months ago)

UVC is used for mutation 200-280nm  I believe A regular blacklight (UV) will make ergolines fluoresce is 315-400nm  and like I said agar plates made with flourecien will make the more concentrated spots glow darker.


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DON'T TRY THIS AT HOME: no guarantees can be made about the accuracy of the information herein. The information dicussed in these posts is purely hypothetical, and for intelectual purposes only. Any similarity between internet chat and real life is pure coincidence.

:google:


Edited by shopdropper (11/02/13 09:16 PM)


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Re: Starting Claviceps Paspali cultures from wild specimens [Re: shopdropper]
    #19077196 - 11/02/13 09:20 PM (10 years, 3 months ago)

Following this. I tried culturing claviceps  purpurea from sclerotia i found in rye grain last year. It grew pretty well  on wheat straw dextrose  agar. 20 grams powered wheat straw plus another 1gram very finely chopped and just 2 grams of dextrose, 20 grams agar per 1000 ml h2o. I had a very hard time isolating it away from the loads of bacteria and yeast. I finally gave up. I still have a contamimated plate or two floating around somewhere. Keep us posted.


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Re: Starting Claviceps Paspali cultures from wild specimens [Re: Mr.Caterpillar]
    #19099750 - 11/07/13 10:38 AM (10 years, 3 months ago)

Thanks to everyone who has taken an interest in this and posted there comments and questions.  I will respond to all, but first I'm posting some photos of some of the Paspali cultures I have grown by inoculating plates with honeydew:



Left to right:

1. One month old culture grown directly from fragment of what appears to me to be dried honeydew, a blackish crusty substance that covers the heads of grass and often surrounds the sclerotia.  This culture grew out with almost no contamination, and whatever there was seems to have been entrapped in the very center and finally subsumed by the Paspali.  Note the little yellow spots around perimeter - these appear to me to be hyphal aggregates, but I am not sure what they are aggregating for. 

Also note the very dark color of the agar.  Paspali excretes pigmented metabolites into the medium.  These vary in color though typically they start as a purple color and with age turn deep yellow and orange or even dark brown. Actually the colors very considerably and some paspali hosting media are a beet red color.

If you magnify the image you will see that there are many tiny drops of some excretion all throughout the mycelial mat.  Some of the larger drops are purple in color, while most of the tiny ones are clear.  I am wondering if these drops are honeydew.

2.  First transfer from honeydew culture.  Notice the concentric growth rings.  These cultures have not been kept in a controlled environment.  The growth rings reflect changes in temperature.  One night I took the culture with me to show a friend and it spent the night in my (warm) jacket pocket.  I noted that the color of the medium had grown considerably darker for just that one warm night.  This culture was originally purple in the center with a white ring of outward growth, but with age has become rust red/brown, orange, and yellow.

3. Sorry, but I guess this is not the greatest photo!  It's a different culture that grew with a more exaggerated mat, and showed colors that were brown and olive green.  Transfers taken from this culture have new growth that glows violet under black light.  I have noticed that the older pigmented cultures do not glow, however the fresh mycelial growth does.  I am a bit confused on the fluorescence so far since I am not sure what color it should glow, but the color I see is violet.

4.  Once again, a bad photo!  However, it shows an earlier stage of growth.  This culture is the direct inoculation from honeydew.  There was a little green mold of undetermined type growing in the center, however the Paspali completely surrounded  and overtook it.  At this stage you can see the purple center, and the white growth ring moving outward across the plate.  Somewhere in the literature I have read that ergots do not produce antibiotics adequately to defend themselves against contamination, however that does not fit with what I am seeing on my plates.  I am seeing a virulent fungus that not only stands up to competitors but often eats them alive.  Perhaps the academic consensus derives from experiences using highly bred monocultures that may have lost their natural capabilities to defend themselves from competitor fungi?  I think these honeydew cultures are multispore germinations and have the advantage of natural selection that is inherent in a multispore germination.

The medium was plain PDA with no added antibiotics.  I decided against using antibiotics since I thought that it would favor development of strains who are dependent on them rather than developing strains with strong natural resistance.

Another thing to note is that I have kept my cultures under filtered sunlight.  I reasoned that if UV light mutates them favorably towards production of LAH, while blocking ergometrine then why not let them get a little sunshine?  I think the hypothesis that Paspali honeydew was the secret of the Kykeon potion at Eleusis is dead on.  Essentially, Paspali honeydew was grafted onto barley and on the sweeter grain it produced stronger, more palatable alkaloids.  Under cultivation for generations the cultivators certainly must have developed strains of paspali that performed as they wished.  The honeydew's entire natural life is under the sun.  I would suspect that it is photosensitive, and that it produces the LADs in response to sun and sugar.  Laboratory techniques of mutating it with UV take it's natural responses to light to an extreme, as do the sweet growth media.

I did look at mycelium from these cultures under a microscope at 40x and 100x.  I could not detect contaminants.  Mycelium is composed of long segmented filaments that are highly pigmented though translucent.  I would love to post a micrograph, but, alas, my microscope does not have a camera mount.

I also tested the cultures with Ehrlich Reagent.  Mycelium itself stained deep purple immediately.  The reagent itself turned yellow.  Agar tested turned deep purple, but did not turn the reagent purple, only yellow.

Also, the sclerotia that have been in cold incubation were set to germinate at 23 c.  I'll post photos after they have germinated - probably in a month.

- Mr.Caterpillar


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OfflineMr.Caterpillar
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Re: Starting Claviceps Paspali cultures from wild specimens [Re: ShroomDoom]
    #19099770 - 11/07/13 10:40 AM (10 years, 3 months ago)

Quote:

ShroomDoom said:
So reasonably if one found some dew you could just dab some of it on antibacterial agar and clean up the culture as needed? sounds GROOVY :flowerchild:.




I have found that there is no need for antibiotic agar - at least not so far as I can tell at present.


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OfflineMr.Caterpillar
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Re: Starting Claviceps Paspali cultures from wild specimens [Re: Tana]
    #19099787 - 11/07/13 10:43 AM (10 years, 3 months ago)

Quote:

Tana said:
Why don't you make the culture on PDA straight from the sclerotias?
Anyway, good luck, and keep us updated!




I am trying that currently.  The reason I did not initially is because my reading did not lead me to that technique, rather I learned of germinating the sclerotia, and followed that.  Taking culture from the honeydew was my own idea, although I have subsequently learned that it is also an established technique and papers have been written about it.


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Re: Starting Claviceps Paspali cultures from wild specimens [Re: Eywa_devotee]
    #19099800 - 11/07/13 10:46 AM (10 years, 3 months ago)

Quote:

Eywa_devotee said:
One easy way to select a sclerotia for culture would be to take each sclerotia, cut it in half, grind one half with a pinch of lime (or about a ml of conc ammonia) and shake in a test tube with a little DCM and water. Allow it to settle out and shine with black light, the ones that the DCM layer glows the brightest are the ones you want to keep. Germinate the other half as per usual. Good luck on your adventures! :thumbup:




Very clever!  Maybe I'll try this out also.  Thanks for the tip!


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Re: Starting Claviceps Paspali cultures from wild specimens [Re: Mr.Caterpillar]
    #19099839 - 11/07/13 10:58 AM (10 years, 3 months ago)

Quote:

Mr.Caterpillar said:
Thanks to everyone who has taken an interest in this and posted there comments and questions.  I will respond to all, but first I'm posting some photos of some of the Paspali cultures I have grown by inoculating plates with honeydew:



Left to right:

1. One month old culture grown directly from fragment of what appears to me to be dried honeydew, a blackish crusty substance that covers the heads of grass and often surrounds the sclerotia.  This culture grew out with almost no contamination, and whatever there was seems to have been entrapped in the very center and finally subsumed by the Paspali.  Note the little yellow spots around perimeter - these appear to me to be hyphal aggregates, but I am not sure what they are aggregating for. 

Also note the very dark color of the agar.  Paspali excretes pigmented metabolites into the medium.  These vary in color though typically they start as a purple color and with age turn deep yellow and orange or even dark brown. Actually the colors very considerably and some paspali hosting media are a beet red color.

If you magnify the image you will see that there are many tiny drops of some excretion all throughout the mycelial mat.  Some of the larger drops are purple in color, while most of the tiny ones are clear.  I am wondering if these drops are honeydew.

2.  First transfer from honeydew culture.  Notice the concentric growth rings.  These cultures have not been kept in a controlled environment.  The growth rings reflect changes in temperature.  One night I took the culture with me to show a friend and it spent the night in my (warm) jacket pocket.  I noted that the color of the medium had grown considerably darker for just that one warm night.  This culture was originally purple in the center with a white ring of outward growth, but with age has become rust red/brown, orange, and yellow.

3. Sorry, but I guess this is not the greatest photo!  It's a different culture that grew with a more exaggerated mat, and showed colors that were brown and olive green.  Transfers taken from this culture have new growth that glows violet under black light.  I have noticed that the older pigmented cultures do not glow, however the fresh mycelial growth does.  I am a bit confused on the fluorescence so far since I am not sure what color it should glow, but the color I see is violet.

4.  Once again, a bad photo!  However, it shows an earlier stage of growth.  This culture is the direct inoculation from honeydew.  There was a little green mold of undetermined type growing in the center, however the Paspali completely surrounded  and overtook it.  At this stage you can see the purple center, and the white growth ring moving outward across the plate.  Somewhere in the literature I have read that ergots do not produce antibiotics adequately to defend themselves against contamination, however that does not fit with what I am seeing on my plates.  I am seeing a virulent fungus that not only stands up to competitors but often eats them alive.  Perhaps the academic consensus derives from experiences using highly bred monocultures that may have lost their natural capabilities to defend themselves from competitor fungi?  I think these honeydew cultures are multispore germinations and have the advantage of natural selection that is inherent in a multispore germination.

The medium was plain PDA with no added antibiotics.  I decided against using antibiotics since I thought that it would favor development of strains who are dependent on them rather than developing strains with strong natural resistance.

Another thing to note is that I have kept my cultures under filtered sunlight.  I reasoned that if UV light mutates them favorably towards production of LAH, while blocking ergometrine then why not let them get a little sunshine?  I think the hypothesis that Paspali honeydew was the secret of the Kykeon potion at Eleusis is dead on.  Essentially, Paspali honeydew was grafted onto barley and on the sweeter grain it produced stronger, more palatable alkaloids.  Under cultivation for generations the cultivators certainly must have developed strains of paspali that performed as they wished.  The honeydew's entire natural life is under the sun.  I would suspect that it is photosensitive, and that it produces the LADs in response to sun and sugar.  Laboratory techniques of mutating it with UV take it's natural responses to light to an extreme, as do the sweet growth media.

I did look at mycelium from these cultures under a microscope at 40x and 100x.  I could not detect contaminants.  Mycelium is composed of long segmented filaments that are highly pigmented though translucent.  I would love to post a micrograph, but, alas, my microscope does not have a camera mount.

I also tested the cultures with Ehrlich Reagent.  Mycelium itself stained deep purple immediately.  The reagent itself turned yellow.  Agar tested turned deep purple, but did not turn the reagent purple, only yellow.

Also, the sclerotia that have been in cold incubation were set to germinate at 23 c.  I'll post photos after they have germinated - probably in a month.

- Mr.Caterpillar



wow!  looks a lot like morchella myc, which also browns the agar in a similiar fashion.  Does it grow quickly?  If it does, it is often difficult  to detect contams even microscopically, as the myc over-runs them.  They then show up after transfer to grain.  But hopefully not, in your case.


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* long-term storage and maintainance of c. paspali
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piracetam 6,120 46 06/03/11 11:13 AM
by LightShedder

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