Sciencemadness Discussion Board - Some fungus growing on grass

Some fungus growing on grass

JJay - 24-7-2018 at 07:08

I have been studying various species of wild grass for the past few weeks after I noticed some odd growths on their seed heads. At first I wasn't sure if they were some sort of pollinator or a fungus, but over time I saw more and more of them. Some were red, some black, purple, white, green, yellow.

It has been raining the past few days, and the growths on the grass have grown much larger and more plentiful. I see them on many species of grass, and though the colors vary widely, from white to green to black to purple, and the level of infestation varies from just a few growths to the colonization of almost the entire seedhead, the shape of the growths is fairly uniform no matter what kind of grass they are growing on. The growths are long and skinny and do not resemble the ergot that I have seen in photos.

So far I have left all of the suspected fungus in its natural habitat, but I am wondering: Are these growths actually ergot? What other kinds of fungus grow on grass seedheads? If they are ergot, is it safe/legal to do anything with them except take pictures?

MJ101 - 24-7-2018 at 07:34

@JJay: Please post pictures. Maybe someone can identify them for you.

Here's something I found on ergot fungus. They have a picture of ergot on a wheat stalk.

https://www.britannica.com/science/ergot-fungus

I hope this helps.

JJay - 24-7-2018 at 13:14

The growths do not look like those at all... I think maybe the growths are just anthers. I'll try to get some pictures.

S.C. Wack - 24-7-2018 at 14:57

If it looks like mini-ergot it probably is. Technically if your intent is extraction and lysergic acid that's OK unless it's to be converted to something interesting. Don't eat bread made from the grass seed.

JJay - 25-7-2018 at 08:59

Well, I am pretty sure I found a very tiny ergot today. It was on a very short common lawn grass (a Festuca I believe) that has black seeds. Its seed heads often look like they have mildew or something growing on them, and the seed stalks are not very tall, so they are easy to overlook.

I believe that this particular Festuca colonizes entire lawns, but the seed heads are typically visible only on the edges of lawns and other places that are hard to mow. I saw a few that looked like they might have ergot growing on them, but on one specimen about a block from my home on the sidewalk's edge, I saw a very tiny ergot-looking growth protruding out of one of the seed pods. I removed the seed from its pod and looked at it to make sure that I wasn't just seeing a seed protruding from its pod, and there was definitely a black growth with its base in a seed. I didn't keep it, mainly because I had nothing to put it in. If it was really ergot, I think I will find some more.

I have checked several varieties of wild rye but haven't seen any ergots.

It might be fun to try to grow some ergot and see if it is possible to get any ergot alkaloids out of it. I think probably the best way would be to start it on agar with suitable nutrients and use a healthy culture to inoculate some kind of fermenter.

I thought I had read somewhere that isolating lysergic acid is illegal because it is a controlled substance as well as a listed substance, but I will have to look into it to have peace of mind. I think ergot alkaloids are uncontrolled, though... I will definitely research the laws on them thoroughly before gathering any ergot.

Hopefully, I can get some decent pictures the next few days....

[Edited on 25-7-2018 by JJay]

clearly_not_atara - 25-7-2018 at 12:16

Lysergic acid is a Schedule III compound:

https://en.wikipedia.org/wiki/List_of_Schedule_III_drugs_(US)#Hallucinogens

However, ergotamine itself is only listed, IIRC.

CouchHatter - 27-7-2018 at 10:26

Ergonovine and ergotamine are List I, at 10g and 20g respectively. Page 34.
The thing to research would be threshold possession of controlled substances - your state might have different laws.

[Edited on 27-7-2018 by CouchHatter]

JJay - 27-7-2018 at 22:06

I didn't see any well-formed ergots today, but I did see a lot more diseased grass than I did the other day... fingers crossed for the next rain....

JJay - 29-7-2018 at 23:34

I went looking today and still didn't find any well-formed ergots, but I took some pictures.

This sort of growth is what caught my attention initially. I no longer believe that growths of this sort are ergot; instead, I think they are anthers:

This I believe might be ergot. It's hard to get a decent picture of it, but I am almost 100% positive that it is some sort of fungus. I found a better-formed specimen last week on this same variety of grass, but it was very tiny.

Any ideas how I could conclusively identify the fungus on the second specimen? I was thinking I might use microscopy, but the local libraries have a positively dismal selection of journals, so I'm not sure what references to use....

Tsjerk - 30-7-2018 at 04:20

You probably need more than one infected top, actually quite a lot, but you could test with Marquis reagent. I expect the test to become black with ergot, but I can't find conclusive examples.

JJay - 30-7-2018 at 18:34

Quote: Originally posted by Tsjerk

You probably need more than one infected top, actually quite a lot, but you could test with Marquis reagent. I expect the test to become black with ergot, but I can't find conclusive examples.

That is a good idea, and I think there are some more exotic reagents that are used specifically for ergolines. Given that the alkaloid concentration is probably very low, I think it will be necessary to grow cultures first to perform a positive identification.

I already have agar on my shopping list, but according to everyone's favorite encyclopedia, they usually use agar fortified with dextrose and potatoes for ergot, sterilized at 15 PSIG (which is about what a pressure cooker does). I don't have any petri dishes, but I imagine that baby food jars would work.

A terrarium should be sufficient for developing cultures on agar, but I'd need a clean room or a glove box for inoculating them, and I'm not sure a HEPA filter is going to cut it... I know a neighbor who has a bio lab, but I'm not sure if he'd like this project.

Tsjerk - 30-7-2018 at 23:13

Cool! More people doing microbiology!

When starting I would just use a camping gas stove burner instead of directly working with a glovebox or HEPA filters. If you never did sterile working I would try isolating some species from air or dust or something and see if you can propagate them.

JJay - 31-7-2018 at 19:49

This has some kind of black growth that I've seen a few places. It looks almost like motor oil on grass. On this one, I can also see a couple of tiny growths that look like ergots where there should be florets.

JJay - 1-8-2018 at 18:35

Jackpot.

happyfooddance - 2-8-2018 at 16:20

Quote: Originally posted by JJay

A terrarium should be sufficient for developing cultures on agar, but I'd need a clean room or a glove box for inoculating them, and I'm not sure a HEPA filter is going to cut it... I know a neighbor who has a bio lab, but I'm not sure if he'd like this project.

If you have a room (bathroom maybe) that is not carpeted and has mostly non-porous surfaces, DOESN'T have a draft, which you can easily clear out (clear counters, shelves, etc), it isn't too hard to sterilize with Lysol or Oust. Buy a couple of cans. Also you will need a flame and some 50%ish alcohol. I am sure you have read up on the basics, but it isn't too hard to make it happen in a home setting. I used to grow mushrooms (lentinula edodes "shiitake", pleurotus ostreatus "oyster", agaricus brunescens "crimini/portabella/button" and others) at home, and I would inoculate them at home, with a very low contamination rate (less than 2%, easily). I used to even make my own spore syringes and liquid cultures.

Just to say it can be done. Wipe down the walls (and ceiling if you can), then the floor, then spray everything down with the Lysol, then bring in your equipment and place it on a table or counter, preferably a tall one, because 90% of contaminants hang out within 3 ft of the ground. Then spray all that down, then go fix yourself (shower, fresh clothes). I wear long sleeves and spray myself down with the Lysol.

It might sound like a lot of work, but you could set up in half an hour if you do it right.

JJay - 3-8-2018 at 15:55

There are some photos of fruiting ergot on some of the mushroom forums.

Right now I have the four specimens sitting in plastic bags in my refrigerator. If I can find any dextrose, I'm going to try growing each on agar while I research chemical reagents that can identify ergot alkaloids, but I'm not too sure about going any further.

In a way, it would be kind of cool to isolate some ergotamine, but I'm a bit doubtful that I could get my bathroom clean enough for inoculating a fermenter... I guess something like a temperature-controlled five-gallon bucket with a filtered aquarium bubbler would work, but the conditions would have to be ultra-clean, and I have too many other projects going on.

I wonder how hard it is to get a license to sell ergotamine....

Tsjerk - 4-8-2018 at 10:55

You can always consider the use of antibiotics against contaminations. It makes working "sterile" a whole lot easier.

happyfooddance - 4-8-2018 at 18:40

Quote: Originally posted by Tsjerk

You can always consider the use of antibiotics against contaminations. It makes working "sterile" a whole lot easier.

This is a really great suggestion for preventing some tenacious bacteria like clostridium, whose endospores can survive sterilization (although there are ways to destroy the endospores if you are in the know). However, it won't do any good against fungi like trichoderma, which love a dextrose medium and are ubiquitous.

Quote: Originally posted by JJay

If I can find any dextrose, I'm going to try growing each on agar while I research chemical reagents that can identify ergot alkaloids, but I'm not too sure about going any further.

Splenda sweetener is mostly dextrose (and maltodextrin). The small amount of sucralose shouldn't interfere, but can be easily removed with dry alcohol. I used splenda packets that I got for free to make the aforementioned liquid cultures.

Quote: Originally posted by JJay

I'm a bit doubtful that I could get my bathroom clean enough for inoculating a fermenter... I guess something like a temperature-controlled five-gallon bucket with a filtered aquarium bubbler would work, but the conditions would have to be ultra-clean, and I have too many other projects going on.

Getting a bathroom clean enough for inoculation is easy. I already told you how to do it. Trust me: nothing is going to live coated in Lysol.

As to the five-gallon bucket, how would you go about sterilizing it? Unless you have a 20-gallon pressure cooker I guess...

Think a little smaller, maybe quart canning jars? As a chemist you should know, scaling up before trying out your method is a great way to waste your time, energy, and resources.

An aquarium bubbler with an inline hepa filter sounds like it would work, but probably won't: the filters have a shelf life and the volume of air you would be pushing, plus the airlock with H2O2 (which will need to be replaced often, while maintaining the integrity of the seal - good luck with that) sounds like a major pain in the ass.

Better would be canning jars with holes in the lid, with Tyvek under the lid. That would give you enough air flow, plus you can put the whole shebang (including your medium) in a pressure cooker.

Tyvek can be gotten for free, FedEx, UPS, USPS all have free envelopes made of Tyvek.

[Edited on 8-5-2018 by happyfooddance]

[Edited on 8-5-2018 by happyfooddance]

JJay - 5-8-2018 at 17:15

I went for a walk earlier and saw more well-formed ergots. They seem to be most plentiful along roads with heavy traffic (I'm guessing that has something to do with how the spores spread).

A 5-gallon bucket filled with potato dextrose broth is not exactly a huge investment....

I never even thought of using an airlock filled with H2O2... I figured a tube filled with cotton would be sufficient as long as the cotton stays dry. The volume of air would be tiny... I've never done anything like that before, but I'm pretty sure that the biggest problem would be keeping out contamination when inoculating the medium.

happyfooddance - 5-8-2018 at 17:41

Quote: Originally posted by JJay

I've never done anything like that before, but I'm pretty sure that the biggest problem would be keeping out contamination when inoculating the medium.

Sorry to say, but you're pretty wrong about that. That is literally the easiest part!

For example, transfering your sterile medium (not to mention sterilizing that quantity) into your "sterile" 5 gallon bucket is a vastly more difficult operation, with much, much more opportunity for contamination to be introduced. And keeping that sterile is going to be pretty close to impossible. Innoculating anything is extremely simple, just flame a syringe until its red, lift lid, squirt, shut lid, done. If this is done in a mist of Lysol it is near foolproof. I've done it more times than I can count.

But, you learn from experience, so I guess try what you think will work.

JJay - 5-8-2018 at 18:46

You're missing the point, happyfooddance. The process of transferring the medium into the 5-gallon bucket is part of the whole process of inoculating the medium. The point is, once you have the medium inoculated, it's going to be pretty easy to keep stuff out. While there are lots of mushroom teks on inoculating from agar, there is a serious risk of contamination when doing so, especially in a filthy place like your bathroom, and they suggest using special flow hoods (which is where the HEPA filter comes into play).

happyfooddance - 5-8-2018 at 19:08

Quote: Originally posted by JJay

You're telling me that you can load agar and mycelium into a syringe? You really think you can squeeze agar through a hypodermic needle? I don't care how many times you claim to have grown random species of mushrooms in your bathroom at home; this is nothing like that. You are not an expert.

The process of transferring the medium into the 5 gallon bucket is part of the whole process of inoculating the medium. The point is, once you have the medium inoculated, it's going to be pretty easy to keep stuff out.

Lol. Don't get mad just because you don't know what I'm talking about. Maybe just read more about things such as isolating cultures and streaking petris. It's all pretty simple stuff. I may not be an expert but I am certainly experienced.

No you don't put agar in the syringe, that is ridiculous, I never said anything about that. What I have done is take a small sample of mycelium growing, or a spore print, and place them in sterile syringes with a couple small bearings (sterilize them with the bearings inside). You don't use hypodermic needles, why? Because hypodermic needles are for injecting into human veins. You use luer locks of the proper gauge, I got mine in a few gauges from a livestock supply store. Horse syringes. You fill the syringe with sterile water. You shake it well to diperse the cells a bit, and use it to inoculate your medium. Agar is used to ISOLATE the culture.

I didn't grow "random" mushrooms, I grew very specific ones (I told you some), and very little else in the way of contamination, though I have dealt with frustrating contamination (namely, CLOSTRIDIUM). Like I mentioned, trichoderma is also one that likes to sneak in. You say that it is nothing like this, but that is ignorant to the fact that you will be trying to stave off the same contaminants, because the same contaminants are everywhere on the planet and thrive/die in the same conditions. And I have isolated cultures and inoculated them onto the same media that you propose to use.

I never inoculated in my bathroom, that was just a suggestion I made for YOU, as they usually have non-porous surfaces. I usually work in the kitchen.

I don't "claim" to have done any of this because I give a shit about what you think, I just thought I could help. In the spirit of ScienceMadness.

JJay - 5-8-2018 at 19:30

I am well aware of how big of a problem trichoderma is. Again, your experience growing random mushrooms in various potentially unsanitary conditions is not really applicable, and stating that I'm ignorant to blahblah when I'm talking about filters and flow hoods while you're working in your kitchen - seriously man, you must be on drugs or something.

happyfooddance - 5-8-2018 at 20:35

Quote: Originally posted by JJay

seriously man, you must be on drugs or something.

Bro. You are talking about making ergotamine.

No it's not legal, and it is impossible for you to get a license.

It is okay, I won't post in your thread, good luck

JJay - 5-8-2018 at 21:07

Look, please stop spouting off on things you have no knowledge of as if you are an authority. You mentioned that Splenda is mostly dextrose, cool. That is your only contribution to this thread. There's no legal barrier to me for getting a DEA license with a legitimate reason, and it's perfectly legal to make ergotamine without a license (under the threshold quantity, which IIRC is 20 grams).

Am I going to do that? Probably not, but it's something to think about.

Tsjerk - 6-8-2018 at 01:28

Don't transfer sterile broth to the incubator, sterilize the broth in the incubator. If you can't do 120 degrees (which kills anything including spores), just heat the container including the broth too 100 degrees, wait for 24 hours for any surviving spores to germinate, and heat too 100 again (one hour orso).

This way you only have to sterile transfer the small amount of inoculation culture, not the whole lot of broth, which would be pretty tedious without a clean bench.

Edit: one hour at 100 already kills almost anything, I don't know if the two times boiling for an hour with 24 in between is really different from just boiling once for two hours, but it works. Bacillus subtilis spores are quite hardy and don't really survive more than ten minutes boiling, after twenty they are gone.

[Edited on 6-8-2018 by Tsjerk]

JJay - 6-8-2018 at 11:31

That seems reasonable.

I tend to think that if a lot of culture can be used for inoculation, the ergot would have a big head start and would hopefully manage to defeat minor bacterial infections (it's antibacterial, right?), but that intuition may well be wrong, and of course it would be best to utterly eliminate all bacteria. Since ergot is a parasite, it stands to reason that it won't compete very well with saprophytes like yeast and trichoderma (or even common mushrooms like agaricus).

I've boiled water in a 5-gallon bucket before... the plastic degrades after days of boiling, but it would be fine for an hour or two.

Apparently, ergot is pretty common on Festuca grasses: https://dl.sciencesocieties.org/publications/cs/abstracts/34...

S.C. Wack - 6-8-2018 at 14:59

I didn't read the article but BTW it looks like that's not ergot they're writing about. So, tall fescue may already have alkaloids detectable without any Claviceps. Infection with the common endophyte is sold as a superior lawn product; such infected seed is reported to have only 1% of the alkaloid there would be if it was ergot. Obviously not all fescue ergot is the same and different seed heads could have different strains and have more or less or different alkaloids.

BTW in some places those with some interest in ergot and an abundance of time and laziness can deposit rye on neglected tall weed type areas in late summer into fall before a little rain. Rye will establish itself in fall while everything not a grass dies. Some years the ergot will be just ridiculous. It's hard to imagine that even a thousand years ago rye bread countries didn't bother screening out the ergot, which is bigger than the grain...it says something about us fundamentally...none of the human condition problems involved seem to be going away any time soon.

JJay - 6-8-2018 at 15:24

That's interesting... about half of the ergots I have found so far were growing on fescues. This article suggests that the ergolines in fescues are caused by Claviceps: www.ncbi.nlm.nih.gov/m/pubmed

Wild rye is pretty common here, but the small variety that is maturing seems to be immune to ergot, and I'm not sure about the larger wild varieties. I just might give that a shot with a commercial variety.

S.C. Wack - 14-8-2018 at 14:34

BTW this shows helpful hints like ergot growing on different species.

PS speaking of sacks of grass seed I'd really like to have one of Achnatherum inebrians...if anyone in China is listening...My lawn needs to be replaced and it's supposed to be tough.

[Edited on 14-8-2018 by S.C. Wack]

andy1988 - 14-8-2018 at 15:12

Quote: Originally posted by Tsjerk

, just heat the container including the broth too 100 degrees, wait for 24 hours for any surviving spores to germinate, and heat too 100 again (one hour orso).

This is called the tyndallization process, also called fractional sterilization. A good write up here. I don't have a pressure cooker... this is what I do.

JJay - 2-9-2018 at 18:17

I think I have all the materials I need to grow small quantities of ergot in petri dishes on agar. I've done some reading on the subject, and a few amateurs have tried it before. Apparently, there are some factors that keep the ergot colonies from growing beyond a certain point. In industry, they do something akin to giving the ergot cancer with UV radiation to create a mutant strain that can multiply infinitely.

Tsjerk - 2-9-2018 at 22:17

Ethyl methanesulfonate is also used to induce mutations, the procedure is straight forward and mutants are easy to select as they keep growing. There is a small chance you also mutate the ergoline synthesis pathway though.

[Edited on 3-9-2018 by Tsjerk]

JJay - 3-9-2018 at 20:42

Ethyl methanesulfonate is probably something that could be produced at home... (perhaps reflux sodium sulfite in molten potassium methyl sulfate, purify and esterify). I'm not really equipped to mess around with it, though... at this point, I'll be happy if I get a single ergot culture growing.