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Growth Inhibitory Compound Discussion (BLIS-Chitinase) * 8
    #27525216 - 10/31/21 10:37 AM (2 years, 2 months ago)

Growth Inhibitory Compound Discussion (BLIS-Chitinase)





Chitinases

Quote:

As chitin is a component of the cell walls of fungi and exoskeletal elements of some animals (including mollusks and arthropods), chitinases are generally found in organisms that either need to reshape their own chitin or dissolve and digest the chitin of fungi or animals. Chitinivorous organisms include many bacteria(Aeromonads, Bacillus, Vibrio, among others), which may be pathogenic or detritivorous. They attack living arthropods, zooplankton or fungi or they may degrade the remains of these organisms




Quote:

Chitinases have the ability of chitin digestion that constitutes a main compound of the cell wall in many of the phytopathogens such as fungi.




Characterization of a chitinase with antifungal activity


Bacteriocins

Quote:

Bacteria produce and excrete a versatile and dynamic suit of compounds to defend against microbial competitors and mediate local population dynamics. These include a wide range of broad-spectrum non-ribosomally synthesized antibiotics, lytic enzymes, metabolic by-products, proteinaceous exotoxins, and ribosomally produced antimicrobial peptides (bacteriocins). Most bacteria produce at least one bacteriocin




Bacteriocins from the rhizosphere microbiome



https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6963780/

It's notable that many of the infected cultures  did not exhibit any morphological traits and where white, some possesed rings, others fluffy mycelium with exudates. With all white-colored colonies having villous texture, these white cultures were less virulent and lacked the presence of conidia. So even under the scope we wouldn't likely be able to identify.

This mentions one mentions inhibitory zones:

"There was no zone of inhibition, found in this interaction."

https://www.semanticscholar.org/paper/Isolation-and-identification-of-Mycogone-causing-in-Kouser-Shah/2c5b2af21be4a7c1f98ea282998646638af280db#paper-header

This seems like an odd mention. I'd like to find out more about culture morphology that includes zones of inhibition.

"Chitinases of pathogenic fungi not only play vital roles in spore germination, septum formation, cell division, and morphogenesis, but the enzymes are also important in the host interaction . In addition to degradation of the host fungal cell wall, chitinases also inhibit hyphae growth and bud tube elongation.

https://www.frontiersin.org/articles/10.3389/fmicb.2020.596719/full

I am convinced that scalloped edges are morphological identifiers for some inhibitory substance like chitinases or BLIS (bacteriocin-like inhibitory substances), I refuse to believe that inhibitory zones are healthy growth patterns.




The left hand plate was used to produce the following:




Front and back of the same plate . These plates are heavily scalloped, floccose and thicken toward the margin which prevents light from passing through the culture.



Inhibitory zones are used to test the strength of both antibiotics and fungicides:

Antifungal properties of Chitinases:



https://www.researchgate.net/figure/Antifungal-Activity-of-Chitinases-against-Various-Fungi-The-fungi-used-are-as-follows_fig4_7145317

Considering that Mycogone, Aspergillus,  Trichoderma, Bacteria, etc, can produce Chitinases; all bacteria can produce at least one form of bacteriocin. It seems reasonable to suspect that these compounds will be found on our plates and certainly found in grain processed for spawn production, furthermore it seems reasonable to suspect that these compounds if present would effect morphology by preventing growth of the host organism where these products are found.

I believe that many of the positive visual identifiers for generally stressed grain masters may be the result of these compounds, or similar compounds; live vegetative bacteria may not be present but their byproducts may very well be. Chitinases, BLIS, or some similar mechanism seems reasonable, it also appears to be reasonable to state that  symptom severity will reflect stage of infection, virulence etc.


Edited by Stipe-n Cap (12/24/22 12:46 PM)


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Re: Myco Parasites [Re: Stipe-n Cap] * 1
    #27525229 - 10/31/21 10:47 AM (2 years, 2 months ago)

Endochitinases (E.C 3.2.1.14) and exo-chitinases. The endochitinases randomly split chitin at internal sites, thereby forming the dimer di- cetylchitobiose and soluble low molecular mass multimers of GlcNAc such as chitotriose, and chitotetraose.[19] The exo- chitinases have been further divided into 2 subcategories: Chitobiosidases (E.C. 3.2.1.29),[20] which are involved in catalyzing the progressive release of di-acetylchitobiose starting at the non-reducing end of the chitin microfibril, and 1-4-β-glucosaminidases (E.C. 3.2.1.30), cleaving the oligomeric products of endochitinases and chitobiosidases, thereby generating monomers of GlcNAc.[19]

Our meds tend to target steroids, cell walls, or protein production. Went down a rabbithole of trying to find what bacteria we used to develop anti-fungals and what their mechanism of action is.

https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1007184

Best I could find. I like cell biology and microbiology...


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Re: Myco Parasites [Re: rockyfungus]
    #27525251 - 10/31/21 11:26 AM (2 years, 2 months ago)



Mycogone Wet Bubble Disease:







Cobweb: Please note I have confused cobweb with pin molds in the past. Real cobweb can look nearly the same as cubensis mycelium.















Some pic dumps


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Re: Myco-Parasite Discussion [Re: sandman420]
    #27525304 - 10/31/21 12:29 PM (2 years, 2 months ago)

Off to a good start, guys. I reposted my earlier comments from general discussion for ease of reference.

The pic set directly under wet bubble heading, photo D exhibits the very typical flocculant mycelium I have experienced with scalloped plates.


Edited by Stipe-n Cap (10/31/21 12:48 PM)


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Re: Myco Parasites [Re: Stipe-n Cap] * 1
    #27525325 - 10/31/21 12:48 PM (2 years, 2 months ago)

Just for the record, the picture of the culture of mine you posted here cleaned up to normal looking growths and fruited very well on bulk substrate. As I've said elsewhere, I believe this morphology to sometimes occur due to somewhat genetically distinct mycelial colonies not blending well with each other. Those areas you call "inhibitory zones" actually had mycelium growing on them but just very sparse and thin mycelium. The plates pinned nicely as well over time. So I'm not sure if you should use that photo as an example to this argument. It's very possible that similar growth patterns could at time be indicative of some sort of contamination but I'm quite sure that was not the case here based on how the culture behaved like a totally clean culture while fruiting.

This plate is a child of those:


I see the pattern not infrequently on germination plates. You can see it here on the right:



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Re: Myco Parasites [Re: multifractal]
    #27525327 - 10/31/21 12:49 PM (2 years, 2 months ago)

Yes, but I clearly dont think thats the end of the conversation.

And yes, there are times when visually similar traits will manifest on plates that do not represent an infection, however there is clearly something else going on, I have enough experience to know when my plates are infected.


Edited by Stipe-n Cap (10/31/21 12:59 PM)


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Re: Myco Parasites [Re: Stipe-n Cap]
    #27525340 - 10/31/21 01:03 PM (2 years, 2 months ago)

Never said it was the end of a conversation. I just don't think it's so black and white that if a plate has these features then it is necessarily being parasitized and thought that those plates might not be good examples of the topic that you are discussing due to their apparent cleanliness in producing healthy and copious mushrooms. I understand using those plates to add nuance to the discussion, offering a counter example, but not using them to exemplify contamination. As you mentioned, I looked at some plates exhibiting similar patterns under scope and saw nothing to indicate the presence of any organism besides cubensis.


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Re: Myco Parasites [Re: multifractal]
    #27525366 - 10/31/21 01:27 PM (2 years, 2 months ago)

Are you trained in microscopy to identify such things? I only ask because it seems germane.

To be clear I'm not attempting or suggesting a black or white approach to something as complicated as microbiology.

the first link outlined the lack of observable conidia:

"On PDA chlamydospores were not observed on Hp2 and Hp9, similarly, conidia were not observed for H2, Hp8, and Hp9 . The isolates with brown colony morphology produced more conidia compared to the isolates with white colony morphology."

I'm willing to wager that a laymen may not be able to positively ID mycoparasites under some condiotions, at least.

I cannot comment on your plates, I don't have any experience with your cultures, I borrowed you pic as an example of what to expect.

I should probably remove the picture. (Removed)

I think that its reasonable to assume that there are:

1. Mycoparasites growing alongside our plate cultures;

2. These mycoparasites produce metabolites;

3. Pathogenic parasitic fungi produce Chitinases ( both Mycogone perniciosa and Trichoderma);

4. Chitinases effect culture morphology, including visible inhibatory zones:

"Chitinases of pathogenic fungi not only play vital roles in spore germination, septum formation, cell division, and morphogenesis, but the enzymes are also important in the host interaction . In addition to degradation of the host fungal cell wall, chitinases also inhibit hyphae growth and bud tube elongation."

https://www.frontiersin.org/articles/10.3389/fmicb.2020.596719/full

I beleive these statements to be reasonable and supported by evidence, cleary there will be some overlap in morphological features that apply to cultures that may be healthy.


Edited by Stipe-n Cap (10/31/21 01:46 PM)


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Re: Myco Parasites [Re: Stipe-n Cap]
    #27525413 - 10/31/21 02:35 PM (2 years, 2 months ago)

Speaking from a human physiology point of view. Scalloped edges, uneven borders, thicker borders, raised areas, change in colors, etc are not NORMAL. Basically things that human pattern recognition software picks up. Are these abnormalities idiopathic (genetics for simple sake) or true pathology?

Something is either impeding growth, taking up mass, stealing nutrients or blood (let's call it water?), or hacking cellular mechanisms. Obviously these mycoparasites like most human pathogens don't want to kill the host outright. They need to parasitize a host but not disrupt it's lifeblood.

We are only talking about "biotic" life here aren't we. What about the billions of viruses that most likely causing issues as well.

Wouldn't a germination plate have the highest concentration of microbes if we are working in true aseptic fashion?

We try and grab way from those notches as best chances are far away from the "odd" part. Did those clean plates come from anywhere near the notch?


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Re: Myco Parasites [Re: rockyfungus]
    #27526089 - 11/01/21 07:38 AM (2 years, 2 months ago)

I'm pretty sure mycogone is strictly a fruit body pathogen. It's not something that could hide away in mycelium like Trichoderma can. I seem to recall they did some experiments with adding it to the compost and it had no effect. The spores had to be in the casing layer where they would come in contact with the fruit bodies, same with verticillium. Presumably the mycelium can produce metabolites that inhibits in growth and fruit bodies can't.


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Re: Myco Parasites [Re: Kizzle]
    #27526109 - 11/01/21 08:15 AM (2 years, 2 months ago)

Quote:

Kizzle said:
I'm pretty sure mycogone is strictly a fruit body pathogen. It's not something that could hide away in mycelium like Trichoderma can. I seem to recall they did some experiments with adding it to the compost and it had no effect. The spores had to be in the casing layer where they would come in contact with the fruit bodies, same with verticillium. Presumably the mycelium can produce metabolites that inhibits in growth and fruit bodies can't.




I know what you are talking about, here is my explanation of these studies:

Big commercial growers do not grow their own spawn, and if they do it is in a totally separate facility with that goal in mind. They use elite worldwide banked monocultures and they know what they are doing. They are not running foil prints to agar made in some hippies closet.

Clearly they can grow while not on a mushroom fruit body, see the pics of it growing on agar as proof, it goes without saying that is not a fruit body.

There are studies I have seen that show it can germinate in sterile water.

So it would make sense that their studies are mainly in line with after spawning because that's all they do is spawn the bulk and case and assume that they spawn is clean because that is a separate professional thing with it's own concerns. Does that make any sense? Mushroom growing isn't the same thing as spawn making or culture making, commercially/professionally that is.

Verticillium in particular is interesting because it sporulates on the caps of mushrooms only, which would be a big ass problem for a print...

PLEASE NOTE I DO NOT HAVE A FULL UNDERSTANDING OF THIS JUST AS ANYONE ELSE COMMENTING JUST CONTRIBUTING THE BEST I CAN, I'M NO AUTHORITY

edit: I think that was a bad explanation let me try again.

Commercial growers of the type in these studies don't use spore prints, spores at all, agar, or probably even have anything to do with their grain spawn. The just spawn bulk substrate from grain spawn that is provided to them professionally. And when grain spawn and cultures ARE made they aren't made from spore prints, only elite cultures. So therefore this is not a concern of these studies, they all seem to only ever discuss spawning and casing not the in between print life cycle that we do.

Those studies mainly are saying that for them, they get it at casing and the stuff wont really grow in the casing until it touches primordia. But it germinates and waits. I think.


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Edited by sandman420 (11/01/21 08:44 AM)


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Re: Myco Parasites [Re: Kizzle]
    #27526120 - 11/01/21 08:39 AM (2 years, 2 months ago)

The information linked above does show mycogone growing on PDA as a pure culture, however when it came time to infect the host mushroom they inoculated the casing layer, which is definitely an important bit of information
that they didn't culture them side by side. Having said that mycogone is clearly capable of growing on agar and the sources don't specifically mention that the parasite is incabale of growing with the host mycelium.

More info is needed to confirm that it doesn't parasitize growing mycelium, do you have a link?

Same here, I'm just a glorified monkey trying to stumble around in the dark to find out what's going on with mycelium at a microscopic level, without a microscope:lol:

I know next to nothing about mycoparasites, so the best way to educate myself is to get this conversation going so that we can all engage in a group exploration of the subject through discussion.


Edited by Stipe-n Cap (11/01/21 08:49 AM)


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Re: Myco Parasites [Re: Stipe-n Cap]
    #27526132 - 11/01/21 08:55 AM (2 years, 2 months ago)

Also it just Must be contagious to the spore print. Surely you've all got a swab or print that will grow nothing but fucked up "white mold" off looking myclium or wont fruit or bubbles or wahtnot. It just must be from the print.

if my above post could be distilled this is it:

The spore print > agar > grain > fruit > spore print > on and on life-cycle does NOT EXIST in commercial cultivation and therefore is not part of any studies on this I have seen.


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Re: Myco Parasites [Re: sandman420]
    #27526174 - 11/01/21 09:42 AM (2 years, 2 months ago)

Quote:

sandman420 said:
Also it just Must be contagious to the spore print. Surely you've all got a swab or print that will grow nothing but fucked up "white mold" off looking myclium or wont fruit or bubbles or wahtnot. It just must be from the print.





This has been my only experience with what I presume to be mycoparasites, always from swab or print. If what we're experiencing is a mycogone, this mechanism of infection seem most likely due to the organisms preference for infecting basidiocarp and basidium. This makes sense to me.

Quote:

The spore print > agar > grain > fruit > spore print > on and on life-cycle does NOT EXIST in commercial cultivation and therefore is not part of any studies on this I have seen.




Good point.


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Re: Myco Parasites [Re: Stipe-n Cap]
    #27526242 - 11/01/21 10:52 AM (2 years, 2 months ago)

I could be completely off base. For commercial growers they just transfer these known commercial strains? Are they reaching senescence and weakening the "immune system" of the myc.

Like my 3015 is god knows how far out it is? It came from agar, I've received LCs for gourmet. I've tried to go back to spores from own grows but at that point it's not 3015 I'd imagine. 


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Re: Myco Parasites [Re: Stipe-n Cap] * 1
    #27526268 - 11/01/21 11:19 AM (2 years, 2 months ago)

It could be determined through experimentation. Isolate the parasite and intentionally introduce it to a culture. I have yet to isolate mycogone from any mushrooms while cloning. It may occur but I haven't seen any evidence of it occurring with cubensis.

Also in all my encounters with mycogone-like fruit body deformations I've never seen any indication of a disease spreading. They usually appear in late colonization or early pinning and then normal fruit bodies follow.


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Re: Myco Parasites [Re: Kizzle]
    #27526299 - 11/01/21 11:40 AM (2 years, 2 months ago)

It's also been my experience with "wet bubble looking" infections appearing early and leading to a normal second flush. But probably related to severity/time of infection just like aspects with button mush. Maybe that's a sign of infection after spawning for example (out of my ass)


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Re: Myco Parasites [Re: sandman420] * 1
    #27526326 - 11/01/21 12:06 PM (2 years, 2 months ago)

We shouldn't be married to the idea that it's even mycogone, there's clearly something going on but it seems like mycogone isn't a perfect fit for a lot of the issues that we're seeing, not to say that it isn't an issue at times.

I'm attempting to move to Vancouver Island, atm. When I get out there I know people with connections at UVic, I'm hoping going to make some friends with actual mycologists over there.



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Re: Myco Parasites [Re: Stipe-n Cap] * 1
    #27526330 - 11/01/21 12:10 PM (2 years, 2 months ago)

no doubt, it's just that it fits the wet bubble disease expressions. The pathogens may be even a complex of some things. Mycogone p. being the pathogen in the known case. But the disease appears the same at least.

Also we should include mycoviruses in this thread. I suspect they are much more common. Very little known but there are some stuff. Mushroom Virus X and La France (? probably remember that name wrong ill double check that one lol) among an endless amount of others.

I read one report that infected fruits make 25% spores that carry the mycovirus from one type, it hink it was MVX. (actually I might not have read the report just the abstract i am waiting to get free report from authors)

edit here it is. Do I read that as 25% of of all spores contain it or 25% of infected fruits spores are infected?

Quote:

The identification of a novel Pleurotus ostreatus dsRNA virus and determination of the distribution of viruses in mushroom spores
Yeo Jin Kim, Ji Yeon Kim, Ji Hye Kim, Seon Mee Yoon, Young-Bok Yoo & Se Won Yie
The Journal of Microbiology volume 46, pages95–99 (2008)Cite this article

307 Accesses

10 Citations

Metricsdetails

Abstract
Double-stranded RNAs and virus particles were identified in Pleurotus ostreatus strain Shin-Nong in Korea. Isometric virus particles with a diameter of 33 nm were purified, which are similar to other Pleurotus viruses reported previously. This strain contains 5 dsRNAs, 8.0, 2.5, 2.4, 2.0, and 1.8 kb in size. The virus particles contain 2 dsRNAs, designated RNA-1 (2.5 kb), and RNA-2 (2.4 kb) which is a typical pattern of Partitiviridae. A non-encapsidated dsRNA of about 8.0 kb also was identified. Partial cDNA from RNA-1 was cloned, and sequence analysis revealed that this gene codes for RdRp. The comparison of the sequence from partial cDNA clone showed 35% amino acid homology with the C-terminal end of the RdRp gene of Helicobasidum mompa virus and Rosalinia necatrix virus. Specific primers designed from the partial sequences successfully amplified RT-PCR product from the infected mycelium and a single spore culture. We used these primers to determine the pattern of distribution of viruses in spores. Of the 96 different single spore cultures generated from Shin-Nong strain, a specific RT-PCR product was identified in 25 cultures, indicating that about 26% of basidiospores contain viruses.




if anyone has access to this report please msg me. $40? You think I'm made of money?

more

Quote:

Minor Pest Title:
Die-back disease (Virus)

Minor Pest Description:
It causes spots in the casing soil where no mycelial growth occurs. Around these spots, mushrooms of low quality appear with long stems and dirty caps. Sometimes the only indication of a virus infection is low yield. In severe cases, a few deformed mushrooms are produced. The disease can be introduced to the farm by infected spawn. It is spread by spores and mycelium from infected mushrooms. Mushrooms affected with the virus open fast, releasing infected spores. Sometimes, mushrooms that were formed inside the casing layer come out already open. Spores from infected mushrooms are easily carried by wind, insets, on implement, clothes and hands of personnel.

Minor Pest What to do.:
Observe sanitation and hygiene during growing cycle
Cover the beds after spawning with a paper, which must be sprinkled with 2% formalin solution every 3-4 days in order to kill all settling spores
Harvest mushrooms in proper time, not allowing them to open
Clean and disinfect growing rooms after growth cycle
Grow tropical mushroom (Agaricus bitorquis). It is resistant to the virus




would like access to these papers also, I feel like we could get somewhere together with this for the OMC.

Quote:

Viruses Associated with A Die-Back Disease of Cultivated Mushroom
M. HOLLINGS




Quote:

Kim, Y.-J., S. Park, S.W. Yie, and K.H. Kim. 2005. RT-PCR detection of dsRNA mycoviruses infecting Pleurotus ostreatus and Agaricus blazei Murrill. Plant Pathol. J. 21, 343–348.





Quote:

Ihrmark, K., H. Johannesson, E. Stenstron, and J. Stenlid. 2002. Transmission of double-stranded RNA in Heterobasidium annosum. Fungal Genet. Biol. 36, 147–154.




Quote:

Morris, T.J. and J.A. Dodds. 1979. Isolation and analysis of double-stranded RNA from virus-infected plant and fungal tissue. Phytophalology 69, 854–858.




Quote:

Preisig, O., B.D. Wingfield, and M.J. Wingfield. 1998. Coinfection of fungal pathogen by two distinct double-stranded RNA viruses. Virology 252, 399–406.




Quote:

Rao, J.R., D.W.A. Nelson, and S. McClean. 2007. The enigma of double-stranded RNA (dsRNA) associated with mushroom virus X(MVX). Curr. Issues Mol. Biol. 9, 103–122.




Quote:

Raper, J.R. and C.A. Raper. 1972. Genetic analysis of the life cycle of Agaricus bisporus. Mycologia 64, 1088–1117.




Quote:

Revill, P.A. and P.J. Wright. 1997. RT-PCR detection of dsRNAs associated with La France disease of the cultivated mushroom Agaricus bisporus (Lange) Imbach. J. Virol. Methods 63,




Quote:

Romaine, C.P. and M.M. Goodin. 2002. Unravelling the viral complex associated with La France disease of the cultivated mushroom Agaricus bisporus, 237–257. In S.M. Tavantzis (ed.), dsRNA Genetic elements: Concepts and applications in agriculture, forestry and medicine. CRC Press, USA.




Quote:

Rong, R., S. Rao, S.W. Scott, and F.H. Tainer. 2001. Common multiple dsRNAs are present in populations of fungus Discula destructiva originating from widely separated geographic locations. Curr. Microbiol. 42, 144–148.




Quote:

Seo, J.J., W.-S. Lim, J.H. Jeong, Y.B. Yoo, S.W. Yie, and K.-H. Kim. 2004. Characterization and RT-PCR detection of dsRNA mycovirus from Oyster Mushroom, Pleurotus ostriatus.




Quote:

Van Diepeningen, A.D., A.J.M. Debets, and R.F. Hoekstra. 2006. Dynamics of dsRNA mycovirus in black Aspergillus populations. Fungal Gen. Biol. 43, 446–452.




Quote:

Yu, H.J., D. Lim, and H.S. Lee. 2003. Characterization of a novel single-stranded RNA mycovirus in Pleurotus ostreatus. Virology 314, 9–15.




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Edited by sandman420 (11/01/21 01:45 PM)


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Re: Myco Parasites [Re: sandman420] * 1
    #27526398 - 11/01/21 01:40 PM (2 years, 2 months ago)

Didn't read the whole thing just what you got.
Viruses are in you, part of your DNA. Probably along those lines.

All this genetic material is prayed on by viruses. So spores are a honeypot I guess.


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