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Offlinedr.alkaline
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Morning glory fungal culture
    #17398469 - 12/15/12 09:47 PM (8 years, 10 months ago)

A few words of warning: I am new to posting on this site, so please bear with me.  Also, I have training in chemistry and biology, but have little experience with microbiology, so I do not have expert knowledge on the following topic.

I used the shroomery search engine, but did not find much information on to isolation of endophytes/epiphytes from seeds or plant matter. I have been thinking about trying this for several years, but have not moved forward on it and would appreciate any input from the community.
Epiphytes of Convolvulaceae have been observed, and in some cases characterized (1,2,3,4). These epiphytes may be responsible for the production of ergoline alkaloids in morning glory flowers. It is also possible that the presence of ergoline alkaloids in Hawaiian baby woodrose are produced from similar fungi, although I cannot find references that state so. 

I have some interesting references that outline the isolation and culture of symbiotic fungi of tall fescue grass, which also produce some ergoline alkaloids. These procedures are very simple, and might be applied to culture of an ergoline producing fungi of morning glory and Hawaiian baby woodrose.
The outline of what I hope would be a simple method for producing larger quantities of these alkaloids at home is presented as follows:


- Observation of fungal growth on/in seeds of the aforementioned plants by crushing the seed or staining plant matterand observing fungal growth under microscope
- Sterilization of fungi  containing tissue with ethanol/bleach wash
- Inoculation of potato dextrose agar(PDA) media with fungi
- Inoculation of liquid media for culture on a larger scale


The ergoline producing  entophytic fungus Neotyphodium coenophialum  of tall fescue and other grasses can be isolated from seeds and grown on PDA by simple methods (5,6)



“Initial screening for the detection of fungal endophytes was done by squashing 15 to 20 seeds of each
acces s ion and examining the aleurone layer and adjoining seed coat for fungal hyphae. At first, the seeds were
soaked in 5% NaOH for 16 h at room temperature, washed thoroughly in sterile deionized water, and stained
for 36 to 48 h in 5% aqueous ethanol, rose Bengal (Sigma) (Saha et al., 1988). Then, individual seeds were
placed on a microscope slide and squashed under a cover slip, and observed microscopically for fungal hyphae.
In this way, the percentage of infected seeds was determined.

Seeds were sterilized in 50-ml Cos tar tubes with 70% ethanol for 1 min, followed by rinsing with sterile
water, then soaked for 5 min in 5 % sodium hypochlorite and plated on potato dextrose agar (39 g per liter
of PDA, Merck KGaA) with penicillin (167 units ml ) and streptomy c in (76.3 u nits -1 ml-1). The plates were held
in dark at 25û C. Speed of fungi growth measured. After 30-60 days, fungal mycelium growth o ccurred. Then,
5 pieces of fungal mycelium grown out (approximately 0.5 cm) of the plant material was removed from the
agar and sub cultured in new PDA media to growth (Bacon et al. 1994).”

With careful technique and observation I do not think the antibiotics will be completely necessary. After the cultures are produced on agar, hopefully this can be taken further by liquid culture.  It would be interesting to try the above method on both morning glory seeds and Hawaiian baby woodrose seeds.

Procedures also exist for isolating endophytes from the leaves of tall fescue(7,8), which might be applied to morning glory plants, which has symbiotic fungi growing on the surface of its leaves(3).

These are just a few examples of endophyte/epiphyte culture that I have found recently, and there exist several more on the internet.
One obvious next step to experimentation with the fungi would be isolation of active lysergamides. As a final note, finding the culture of an ergoline producing fungus from Hawaiian baby woodrose seeds successful is especially interesting. One study finds ergine is in such high concentration in these seeds that it can be fractionally crystalized out of extracts without the need resolve with chromatography(9). One can only hope liquid culture of this symbiont would still produce high levels of ergine.


1. http://www.mycologia.org/content/103/5/1133.short
2. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2634565/
3. http://pubs.acs.org/doi/abs/10.1021/np070315t
4. http://www.plantphysiol.org/content/147/1/296.full
5. http://www.ajbasweb.com/ajbas/2009/2544-2548.pdf
6. http://www.ncbi.nlm.nih.gov/pubmed/18333513
7. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC204344/
8. http://www.google.com/patents/US20080207451
9.    http://onlinelibrary.wiley.com/doi/10.1002/jps.2600620409/metrics


Edited by dr.alkaline (12/15/12 09:52 PM)


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InvisibleEntersandman
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Re: Morning glory fungal culture [Re: dr.alkaline]
    #17401577 - 12/16/12 03:09 PM (8 years, 10 months ago)

hy and welcome here :wink:

i am very impressed by your text since i´ve never heard of neophytic fungi in woodrose and MGs :thumbup:

none the less i´ve been planning to isolate Acremonium sp. from sleepy grass (plant material and seeds) for quite a while and coincidencly i had thought about it today as i had my after noon walk so your thread showed up at the right day :eek:


have u make any isolates yet??
keep us updated plz :super:


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Re: Morning glory fungal culture [Re: dr.alkaline]
    #17401814 - 12/16/12 04:01 PM (8 years, 10 months ago)

I've read in some papers that the ergoline producing fungus is the only endophyte in MG that won't grow on agar.

The fungus is contained inside the seeds, so do the seeds have to germinate on the plate to get the fungus? The protocol seems to suggest that first the outside of the seeds is sterilized of all microbials so only what's inside is cultured.

Do post your results, would be interesting to see. The psychoactives inside MG are unlike any other.


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Offlinedr.alkaline
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Re: Morning glory fungal culture [Re: Entersandman]
    #17401930 - 12/16/12 04:21 PM (8 years, 10 months ago)

I have come across many references, some of which are in my bibliography, that describe a similar procedure for isolation of Acremonium sp. from tall fescue or sleepy grass. This is actually more or less where I got started on this whole venture. The advantage to working with those fungi is that they are identified and described in morphology, so with a microscope you can confirm the isolate's identification. 

This brings me to my next point, and one of the top reasons for posting this idea on Shroomery. One of the main problems I see with my methods is confirming the isolation is in fact the ergoline producing symbiont. Since, to my knowledge, the possible fungal symbiont of Hawaiian baby woodrose seeds has not been characterized I cannot think of a good way other than PCR to identify the presence of genes that are responsible for synthesis of ergoline alkaloids in an isolated culture. The next best thing may be to get several seeds from independent sources and see if the morphology of the fungi match on a consistent basis. This method seems unreliable and I am hesitant to trust it.

The advantage to morning glory seeds is that two fungal symbionts have been identified(1). Genetic analysis has been performed to identify the fungi as belonging to clade Clavicipitaceae, and further sequence analysis has found genes the encode enzymes in the ergot alkaloid biosynthetic pathway. Reference 1 is the only reference I do not have from the bibliography, and if anyone can get it please let me know! It may have good information describing the fungi morphology, so that I can accurately identify it without PCR methods, which I don't have lol.

I have not even started this yet, but I have been thinking about it for several years. I think I may first try to isolate Acremonium sp. or  Neotyphodium sp. from tall fescue seeds. However, that would just be proof of concept, as the loline alkaloid concentration of tall fescue is several thousand times larger than the ergot alkaloid concentration, making the grass not the best source for these alkaloids.

Quote:

teal said:
I've read in some papers that the ergoline producing fungus is the only endophyte in MG that won't grow on agar.




Please, please cite these sources, otherwise I  have no way of knowing what you are talking about(plus I would love to see them). I was concerned that the fungus would not be able to live outside the seed as well, but with fungi being one of the most forgiving organisms in microbiology and the success of epiphyte culture in tall fescue, my hopes are high. I have a suspicion that reference 1 outlines a culture method, which is why I am so anxious to see if anyone can get it.

Quote:

teal said:
The fungus is contained inside the seeds, so do the seeds have to germinate on the plate to get the fungus? The protocol seems to suggest that first the outside of the seeds is sterilized of all microbials so only what's inside is cultured.





In addition to using the method from my last post, I also plan on surface sterilizing and breaking the seed open; a similar technique is explained for leaf tissue in reference 7.

"Infected material was freshly collected by cutting leaves 0.5 cm from the crown of the grass. Leaf blades were removed, and the resulting sheaths were washed free of all debris. Sheaths
were again washed and cut into approximately 3-cm sections.
Sheaths were placed in sterile disposable cups, 30 ml
of full-strength bleach (5.25% hypochlorite) was added, and
the sheaths were agitated for 5 min. The bleach was poured
off, and sheaths were rinsed once in sterile distilled water.
Grass tissues (sheath or inflorescence stem) were aseptically
placed in sterile petri dishes, and 0.5-cm segments were cut
from the ends of each sheath and discarded. Sheaths were
halved, and one half of each sample was teased (macerated)
throughout its length with a fine needle to expose internal
plant tissue. The remaining half of each sheath was not
teased but was used directly for comparison of tissue preparation.
Inflorescence stems were treated similarly, except
that they were cut longitudinally into halves and that the
central pith tissue was removed for inoculations.
One to several pieces of either teased or unteased segments"


Edited by dr.alkaline (12/16/12 04:50 PM)


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InvisibleAmphibolos
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Re: Morning glory fungal culture [Re: dr.alkaline]
    #17402874 - 12/16/12 07:25 PM (8 years, 10 months ago)

You should tell us what do you have at hand for the analysis of the strain.  Is the gene sequence in the databases?


Also, you could a FISH probe? Simpler than PCR though you have to make it (or buy it)


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Edited by Amphibolos (12/16/12 07:34 PM)


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Offlinedr.alkaline
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Re: Morning glory fungal culture [Re: Amphibolos]
    #17403045 - 12/16/12 07:56 PM (8 years, 10 months ago)

Quote:

Amphibolos said:
You should tell us what do you have at hand for the analysis of the strain.




Nothing, I don't have any culture equipment, stains, or even a microscope. Molecular techniques are not even an option at this point, although with the DIY bio community they may be in the future.

What would be the best way to identify and unknown?

I am just thinking through the problem at this point.


Edited by dr.alkaline (12/16/12 07:59 PM)


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Offlinedr.alkaline
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Re: Morning glory fungal culture [Re: dr.alkaline]
    #17403621 - 12/16/12 09:21 PM (8 years, 10 months ago)

http://www.sciencedirect.com/science/article/pii/S1754504811000444

I found the above reference today, which has some images of the mycelia of Periglandula ipomoeae the previously characterized epibiotic ergoline producing fungi associated with Convolvulaceae(1). If anyone has or can get this reference there may be some descriptions to go with the great looking pictures to help identify an isolation. I feel that with this paper and reference 1, both of which describe Periglandula ipomoeae, we could really be on our way with this.

I also found that this year Indiana University reported on the molecular, chemical, morphology descriptions of Convolvulaceae fungal symbionts in six species of morning glories. I think this was presented in a poster at the 80th meeting of the mycological society of america in the form of a poster, and a description is in the following link

http://msafungi.org/wp-content/uploads/Inoculum/63(3).pdf

They claim that a fungi with morphology consistent with Periglandula sp. was found on all six host species.


Edited by dr.alkaline (12/16/12 09:59 PM)


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Offlineteal
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Re: Morning glory fungal culture [Re: dr.alkaline]
    #17403795 - 12/16/12 09:46 PM (8 years, 10 months ago)

Quote:

dr.alkaline said:
Nothing, I don't have any culture equipment, stains, or even a microscope.

I am just thinking through the problem at this point.




Nah man, that's the wrong approach. Buy some agar and experiment. Do the literature readings after a few failed attempts.

There's no one here that can help you with that, you're going into new territory. You've already got the right idea, just do it. It's money well wasted (don't forget to take pictures!). :thumbup:


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Offlinedr.alkaline
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Re: Morning glory fungal culture [Re: teal]
    #17404004 - 12/16/12 10:17 PM (8 years, 10 months ago)

Quote:

teal said:
I've read in some papers that the ergoline producing fungus is the only endophyte in MG that won't grow on agar.





After some searching I found reference 1, and as I suspected they did have successful culture of the fungi Periglandula on PDA. They used a simple method similar to the methods I described above for tall fescue.

"The fungi were
isolated from young, unexpanded leaves. Closed leaves were surface
disinfected with 1.3% sodium hypochlorite for 1 min,
rinsed in sterile water and opened under sterile conditions.
Mycelium samples from the adaxial surfaces of the leaves
were transferred onto potato dextrose agar"

" Cultures were incubated at 23 C in the dark.
Fungal morphological characters were observed and radial
growth rates were measured after 4 wk incubation."

The paper continues to provide very detailed descriptions of morphology and contains great pictures.


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Re: Morning glory fungal culture [Re: dr.alkaline]
    #17420562 - 12/20/12 06:13 AM (8 years, 10 months ago)

Interesting experiment, I hope you make some progress and tell us all about it!

Here is a couple of papers you might want to read:

Molecular characterization of a seed transmitted clavicipitaceous fungus occurring on dicotyledoneous plants (Convolvulaceae)  PDF is free
http://link.springer.com/article/10.1007%2Fs00425-006-0241-0

Elimination of ergoline alkaloids following treatment of Ipomoea asarifolia (Convolvulaceae) with fungicides
http://www.erowid.org/references/refs_view.php?A=ShowDocPartFrame&ID=7684&DocPartID=6799


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Re: Morning glory fungal culture [Re: knarkkorven]
    #17460285 - 12/28/12 12:19 PM (8 years, 9 months ago)

A fungus that wont grow on agar?? Strange. Perhaps make an agar based on MG's or WRS's.... Just my 2 cents.


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Re: Morning glory fungal culture [Re: Sillyputty67]
    #17513032 - 01/07/13 06:47 PM (8 years, 9 months ago)

I'm very interested in your experiments. Do you have any new updates? Also it is interesting that the first article posted by knarkkorven indicates the claviceps fungus is epibiotic which would refute teal's claim that the fungus is found on the inside.


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Re: Morning glory fungal culture [Re: crowfoot]
    #17523819 - 01/09/13 07:54 PM (8 years, 9 months ago)

Cool


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Re: Morning glory fungal culture [Re: Yesum]
    #17524232 - 01/09/13 09:04 PM (8 years, 9 months ago)

Morning glory seeds and hawain baby woodrose seeds have lsa in the seeds and ergot mold grows on grain like wheat and and some grasses which is where lsd is derived from  dont know of any ergot growing on morning glory or hawain baby woodrose seeds


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Re: Morning glory fungal culture [Re: stonepony]
    #17548124 - 01/14/13 07:17 PM (8 years, 9 months ago)

No new developments. I have everything in place to pull the trigger on this experiment, but I have become extremely busy over the past few weeks. I intend on doing this in the next few weeks though, and I will keep everyone updated.

You guys may be able to help me with this though. I bought some seeds, but would like to test many kinds from several sources. PM me if you want to donate fresh seeds for science! I only would need a few, like around five or so. 

I am no microbiologist, so if anyone would like to get a copy of ref 1 and give me their 2 cents I will send a copy to your email.


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OfflineGoFindLucy
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Re: Morning glory fungal culture [Re: dr.alkaline]
    #17548725 - 01/14/13 09:24 PM (8 years, 9 months ago)

Try a cold water extraction on the HBWR. Keep it sterilized and used distilled water, maybe you could get some mycelium through a liquid culture like that or of another. Then you could transfer it to a variety of different agar solutions. I've had a similar idea but reintroducing a different fungi on agar and see if the lsa production could be shared through plasmogamy over a long period of time and with a stroke of luck. all you need is one right solution, from there clone it. Force asexual reproduction for a while, reintroduce the other fungi and and see if they will fuse again. Fungi are very adapted to exchange DNA with one another. Its well worth the materials I think, if you got it, go for it.


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Re: Morning glory fungal culture [Re: GoFindLucy]
    #17554139 - 01/15/13 10:34 PM (8 years, 9 months ago)

:popcorn:


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Re: Morning glory fungal culture [Re: ken1993]
    #17555788 - 01/16/13 09:01 AM (8 years, 9 months ago)

I'd be willing to toy around with this.  Having read the posted sources so far I can't think I'd have a shortage of options to play with...  I can take samples back home and use my dad's scopes from work if I can get a viable, clean isolate (he's a microbiologist at a hospital and has always been excited to let me toy around with the scopes if I do my own work up in advance - for scientific purposes only of course :P ).  If I have any luck I'll take some measurements and pictures (hopefully) and report back... :peace:


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Offlinedr.alkaline
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Re: Morning glory fungal culture [Re: GoFindLucy]
    #17557940 - 01/16/13 06:43 PM (8 years, 9 months ago)

Quote:

GoFindLucy said:
Try a cold water extraction on the HBWR. Keep it sterilized and used distilled water





Surface sterilization of leaf/seed and plating is a pretty standard procedure for epiphyte isolation, so I am going to stick with that. It is documented well in the primary literature, why reinvent the wheel? I believe the reason for the sterilization is to kill all of the "non-target" microbes that may be living on, or have settled to, the surface of the seed/leaf. Although much of the fungi is likely killed in this step, there is so much of it relative to the other microbes, that there is still plenty left over to inoculate a plate.

I think what the previous post suggested with a "cold water extraction" would include far too many contaminates to be a viable approach. Although by all means give it a try!

Quote:

420Experience said:
I'd be willing to toy around with this.  Having read the posted sources so far I can't think I'd have a shortage of options to play with... 




Check your inbox I PM'd you with some information...

We can do this guys, pioneers of science! We may be able to grow Periglandula like any other fungi. First isolation and identification, and then we can move onto the nutrient agar/conditions to promote the growth we want.



Edited by dr.alkaline (01/16/13 06:52 PM)


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OfflineDa2ra
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Re: Morning glory fungal culture [Re: dr.alkaline]
    #17562071 - 01/17/13 01:58 PM (8 years, 9 months ago)

Here's another god source of info on this: http://www.shroomery.org/forums/download.php?Number=17534787
I apologize for not making this a PDF. I tried, but the order of the pages kept getting messed up.

Also, I recently ordered some Ipomoea tricolor seeds from World Seed Supply; and I'd be happy to send you some.


Edited by Da2ra (01/25/13 01:47 PM)


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