Claviceps paspali Season!

[Bubbleplate]

For those of you considering growing ergot fungi, in particular Claviceps paspali, I recommend that you take a walk outdoors.
Recently, I decided to take a stroll along a country road, where the forest gives way to open meadows, and a small stream passes through. Examining the wild grasses growing there, I soon discovered that a number of the grass seed heads had wild Claviceps pasapli ergot sclerotia growing on them!
Close examination found purple Banana-shaped sclerotia growing where a seed would normally be. They are anywhere from .25 to 1 cm in length, the average being .5 cm. They grow on about 5 different species of grass in this area.
In about an hour of collecting I had a few hundred sclerotia.
Since each sclerotia can be a different "chemo race", one can expect different types of ergot-Lyseric acid and Clavine type alkaloids to be produced by each.
To isolate the Claviceps fungi, it is important to obtain a sterile portion of the sclerotia. Following the technique of Groger, the sclerotia were shaken for 2 minutes in a 50% solution of Propanol, then a 4% Formaldehyde solution, then rinsed 3 times with sterile distilled H2O. Under a microscope, the outer "skin" was cut away with a razor or scapel, and small slices of the inside of the sclerotia taken. These were placed on petrie dishes of Potato-Dextrose Agar, and incubated for 2 weeks at 26 C. Various small round  colonies develop, and these can then be transfered to other dishes or test tubes of agar for testing and mutation to high yield alkaloid strains.
(A UV light such as an EPROM eraser or an old Sunlamp is an excellent source of high energy UV to mutate the fungi)

[POPTART]

Is there a way of knowing if it is C. paspali or C. purpurea?

Never mind, got. SWIM will be going again today to get a few samples  

[Bubbleplate]

C. pasapli almost always infects Grasses like Paspalum distichum and other "wild grass" types that grow in open fields near edge of woods, or side of road or drainage ditches etc.
Claviceps purpurea almost always infects "Cultivated Grain" type plants like Rye, Wheat, Sorghum, etc. These are more commonly found in farmers fields under cultivation.

The size of the Sclerotia is also an indication:
Claviceps paspali are usually 1 - 2 mm in diameter, and 4 - 15 mm long, while C. purpurea are 4-10 mm diameter,(fatter) and 5-10 mm long. Do a Internet search for pictures of each!

[Aurelius]

Spread of sorghum ergot (Claviceps africana) across the U.S. in 1997. The month of first observation is shown in the states (red) and the hybrid seed production area (blue)..



Remind you of anything?



C. Purpurea growing on Spec. Phalaris

[POPTART]

Good, according to your description it fits the C. paspali stain. Swim is v happy.  

[midway]

I've read a few pages on claviceps africana on american sorghum crops which say that the toxicity potential for animals for 'sorghum ergot' seems to be negligable....perhaps indicating low alkaloid contents in the claviceps africana strain that has landed here in the US? The OZ papers report high toxicity.
Ive also read others that contradict the low toxicity assertion for the US.

http://www.ksgrains.com/sorghum/ergot.html

http://www.dpi.qld.gov.au/news/newsreleases/13547.html

http://www.ianr.unl.edu/pubs/plantdisease/EC1879.pdf

http://www.dpi.qld.gov.au/health/3568.html

The discrepancy has me curious as to alkaloid percentage are in the american version, since infected sorghum would be easier to obtain than other crops, and is available in a wider range of localites. Anyone positively ID Africana in the us and bother with an analysis?

[midway]

This is an interesting article for, identifying 3 distinct strains of claviceps purpurea, including thier different propensities to produce individual types of ergot alkaloid. Unfortunately, there is no mention of if or which strains produce more total alkaloid, reason cited: the samples were too small. Samples were mostly european, but included some eastern US samples.

http://www2.biomed.cas.cz/~pazouto/purpurea.html

the pdf

http://www2.biomed.cas.cz/~pazouto/inoculation.pdf

Strains are distinguished by wether or not the sclerotia floats on water, (an adaptation for g3 strain) and the size and shape of conidia.

Below is a PDF with a table vaugely showing geographical distribution. mildly interesting. Also mentions cultivation with t2 or pda agar.

http://www2.biomed.cas.cz/~pazouto/inoculation.pdf

Anyone have this

http://reo.nii.ac.jp/journal/HtmlIndicate/Contents/SUP0000001000/JOU0001000028/ISS0000011679/ART0000124447/ART0000124447_abstract.html

[sYnThOmAtIc]

Well this answers my question on how you got yout fungi!!

But what wavelength of UV is used specifically and how long?? On how old of what type of cultures... Like how large should the colonies be before attempted mutation? Also do you have any references or readily uploadable text detailing a method/s for mutation??

[Bubbleplate]

Almost all the papers in my collection on Claviceps, where the authors mutated it, say "exposed to UV light according to known art". You can probably find examples in college type text books on microbiology. I've used an old (circa 1960's) "Sun Lamp" bulb that produces 100 watts (!!) of UV light with varying success. At that level 20-30 seconds is enough to kill off most of the culture and leave some alive. The problem with this method is that the dead fungi then plays host to bacteria and molds...
A better method is the "Dilution" method: chop young culture in sterile blender with DH2O. Take a small amount and dilute with large amount of DH2O, say 10 ml into 500 ml. Take a very small amount from the 500 ml, .1 ml and plate out onto agar. Individual colonies will form and these can then be re-plated out by themselves and tested for alkaloid production, purple color, etc. Many agar plates, sterile conditions, and patience are a must!

[sYnThOmAtIc]

Yea I know, that is why I wanted to see specific instructions on how it is carried out. I tried mutating some cells once with the germicidal uv lamp in my tissue culture enclosure and fried the whole thing in ten seconds. I guess just flashes of light are needed.

I hate reading all these papers and patents to only see "mutated by known methods" without any references for their mehtod used.

Serial dilution is not an effective method for isolating high alkaloid producing strains, someone told me. This is a process that should be carried out after the attempted mutation to isolate colonies of the living and hopefully mutated cells. Serial dilution to obtain isolated colonies takes more dilution than that, but I see that ou are only refering to the general idea of things. After all, it's hard to estimate the exact dilution and requires plating of each dilution to figure it out. Why I just make several with the last expected to have nothing and plate two dishes of each dilution. Having a microscope and a good microliter pipet helps.

I was just hoping you had some papers on the mutation methods. I guess I'll have to find some references or somethign and post it.

[Bubbleplate]

Well, I actually believe serial dilution and subsequent isolation of unique colonies is a good method of obtaining high yielding Claviceps paspali strains. The reason can be found in Arcamone's original work and article. Arcamone was the first person to grow Claviceps AND actually get alkaloids in more than trace amounts. His technique was to isolate individual colonies on agar, grow each out in shake flasks and test for alkaloid yield/look for purple coloration. Then start the whole process over. In 3 times through that process he was able to improve a strain that yielded significant amount of alkaloids:

https://www.thevespiary.org/rhodium/Rhodium/pdf/arcamone.submerged.claviceps.paspali.pdf

The reason one can do this is that Claviceps paspali strains that produce large amounts of alkaloids are purple colored, while lower yielding strains are brown or white. So there’s a “built-in” indicator which to look for.
I myself have had similar success using that technique, although it would be nice to try and mutate some strains and look for a better yielding one. Never-the-less, even if one is able to induce mutations, you still need to go through the procedure of growing them out, testing for alkaloids, repeating the cycle, etc. It goes without saying that a microscope, rotary shaker, autoclave/sterilizer, and other equipment is a necessity.

[sYnThOmAtIc]

I'm not saying it can't be done!

Never-the-less, even if one is able to induce mutations, you still need to go through the procedure of growing them out, testing for alkaloids, repeating the cycle, etc. It goes without saying that a microscope, rotary shaker, autoclave/sterilizer, and other equipment is a necessity.

Of course! This has to be done in order to ensure satisfactory results. It is about the only way one can produce measurable quantities of alkaloids from a culture obtained from a grass field. I'm just saying better results come from this procedure after a mutation. I've obtained wild strains before and isolated many different substrains, none of which produced anything desirable. And after repeated plating it was obvious that the cells were dying and soon quit growing all together.

It goes without saying that a microscope, rotary shaker, autoclave/sterilizer, and other equipment is a necessity.

Again, of course! Anybody attempting such an endeavor without would be a fool! Well, not exactly, but autoclave and shaker are a must. Microscope is worth a million bucks in this field. Especially when analyzing solutions of diluted cells for plating. Very useful for ensuring a solution is dilute enough for growing single colonies. Although it can be done without, guess work leads to more work. Your definatly right about needing lots of equipment a Glovebox, inline hepa-caps, silicone micron filter flask plugs, air pump, transfer peristaltic pump, small cleanroom, one could go on for a day here. Some being nessecary and others being extremely helpful. Although some people can get away without contaimination with the most ghetto and primative setups. Some people I know who have had luck in producing this stuff just use aquarium pumps into five gallon wine jars in an open enviroment room. Never isolate strains ar anything! Although, they get there strains from cellular banks and are damned lucky throughout the entire process. I guess their sub-zero climate where they live isn't a major producer of spores and other contams, but still WTF!

It's a shame lysergic or paspalic acid can't be synthesized, or at least not in a pratical way