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Stipe-n Cap


Registered: 08/04/12
Posts: 7,623
Loc: Canada
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Re: Mycoparasite/Mycovirus Discussion [Re: sandman420]
#27526709 - 11/01/21 06:11 PM (2 years, 2 months ago) |
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I agree, viruses are definitely a great topic of discussion as well. Too bad about the pay wall.
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sandman420
Saint PP



Registered: 06/17/04
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Re: Mycoparasite/Mycovirus Discussion [Re: Stipe-n Cap]
#27526727 - 11/01/21 06:28 PM (2 years, 2 months ago) |
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Some young lad in a mycology PhD trajectory totally should write your thesis on The Viral Infections of Clandestine Psiolcybin Mushroom Cultivation.
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Stipe-n Cap


Registered: 08/04/12
Posts: 7,623
Loc: Canada
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Re: Mycoparasite/Mycovirus Discussion [Re: sandman420]
#27526736 - 11/01/21 06:33 PM (2 years, 2 months ago) |
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I'd definitely read it. I wish someone would make mycology video lectures available online.
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rockyfungus
dirty


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Re: Mycoparasite/Mycovirus Discussion [Re: Stipe-n Cap] 1
#27526825 - 11/01/21 07:36 PM (2 years, 2 months ago) |
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Yeetusdeetus



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sandman420
Saint PP



Registered: 06/17/04
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Alright scooters thanks to good ol p9s library link I found some new interesting shits.
From this study
Basically they find that mycogone will germinate on nearly anything from the order Agaricale (20 of 28 tested showed favorable results)
Cubensis and most of our mushrooms we grow are of the fungal order Agaricales.
Quote:
Results are shown in Tables 1 and 2. Unfortunately, because of the time-consuming nature of bioassay procedures and the varying availability of material, complete data relating to tissues, extracts and mycelia (set out in Table 1) were obtained for only 10 species from 9 families. Germination was induced by contact with basidiome tissues of most fungi used here, although germination levels were usually significantly below those found for Agaricw brunnescens. The single exception was a species of Cortinarius on which germination was comparable with that on A. brunnescens. Basidiomes of some species failed to activate conidia and there was additionally a wide variation in effects within most families. Extracts of Agaricw brunnescens basidiomes induced germination, as did those from several other species, although to a lesser extent, and again some extracts failed to activate conidial cells. With the exception of Agaricus species and Coprinus cornatus, germination levels on extracts were below those occurring on corresponding whole tissues. Most mycelia tested induced germination, levels for Agaricus brunnescens being similar to those occurring on its basidiome tissues and extracts. For other species the levels were also relatively high. For instance, in C. comafw, hccaria laccafa (Scop.: Fr.) Cooke, Armillaria bulbosa (Barla) Kile & Watling and Phallus impudicus Pers. they either approached or exceeded those for basidiome tissues. Germination occurred on mycelia of Amanita nrbescens (Pers.: Fr.) S. F. Gray and Sclerodema citrinum Pers., yet not on their basidiomes or basidiome extracts.
Quote:
DISCUSSION Sometimes severe technical difficulties attended preparation of tissues and extracts for bioassay and attempts to obtain mycelial cultures from basidiomes. Therefore the data presented here are incomplete. Nevertheless, the breadth of the survey allows some firm conclusions to be drawn concerning induction of germination in thick-walled conidial cells of M. pemiciosa and permits speculation on the ecology of this mycopathogen. Turning first to Agaricales, basidiomes of 28 wild species were bioassayed to 20 of which M. perniciosa conidia responded positively, with two further species inducing just below 1 % germination. Germination occurred on basidiomes of fungi typical of a variety of habitats and on mycorrhizal, leaf-litter decomposing and lignicolous species. Although germination levels were usually below those for Agaricus bnmnescens, they were commonly above 20% and ranged as high as 66%, which gives reason to suppose that many wild species could act as hosts for M. perniciosa. In this regard it is of interest to note that over 50% germination was recorded on basidiomes of Agaricw campestris L.: Fr., the close relative of Agaricw brunnescens. This view is reinforced by the generally high germination levels produced by contact with mycelia, since it is probable that association of M. perniciosa with undifferentiated mycelium precedes the appearance of disease on an epidemic scale during mushroom cultivation. However, there is little or no direct evidence for the existence of such disease reservoirs outside the mushroom production unit, and so far it has proved to be unfeasible to carry out pathogenicity tests on basidiomes of wild species either in the field or laboratory. A notable feature of the bioassays was the wide variation in response even to basidiomes from fungi within a single genus. Thus Panaeolw semiovatus (Sow.: Fr.) Lund stimulated over 50% germination, but for P. sphinctrinus (Fr.) Quel. this was zero. Similarly Amanita vaginafa (Bull.: Fr.) Vitt. promoted 23 % germination and A. rubescens (Pers.: Fr.) S. F. Gray zero; Lactarius rufus (Scop.: Fr.) Fr. 34% and L. furpis (Weinm.) Fr. zero. Curiously, whilst basidiomes of Hypholoma fasciculare and their extracts induced germination, contact with mycelium did not. Three of the six non-agaric Basidiomycotina investigated gave positive results; basidiomes, extracts and mycelium of C. sfercorew all inducing low levels of germination, as did mycelium of Scleroderma citrinum Pers. Results using P. impudicus were of particular interest in that contact with its exoperidium brought about 25 % germination but whole egg extracts had little effect. By contrast, contact with its mycelium induced nearly 50% germination. This survey clearly demonstrates that germination-inducing factors are widespread within Agaricales but that they are by no means confined to them. In addition, it appears that whilst these factors can sometimes be extracted in aqueous solution from basidiomes, this may not always be the case; as, for example, for L. laccafa and P. impudicus. Similar factors are present in mycelia but, by contrast, cannot be extracted from them; nor, generally, are they found in media within which mycelia have been grown (Holland, 1988). Work is now in progress to identify the compounds involved in triggering germination of dormant, thick-walled conidial cells of M. perniciosa and some preliminary findings might be mentioned here. They are ethanol-soluble and are to some extent wallbound (Holland, 1988). Furthermore, two or more groups of factors are implicated which act either synergistically or in sequence (Rawlins, 1990). It is hoped to publish details in due course.
Some other important information, mycogone makes 2 types of spores. 1 one which germinates easily on anything and one of which survives a long time but only germinates in the presence of mushroom tissue/mycelium.
Quote:
Mycogone pemiciosa sporulates heavily on Agaricus basidiomes, producing small thin-walled phialospores together with large bicellular conidia each consisting of a dark, spherical, thick-walled, vermcose apical cell and a thin-walled basal cell. After secession the latter dies, leaving the thickwalled component as the major survival propagule (Holland et al., 1985). By contrast with phialospores, which germinate freely on a variety of substrata, thick-walled conidial cells are dormant and will germinate only when activated by uncharacterized factors emanating from vegetative mycelia and basidiome tissues. As well as being present in Agaricus bmnnescens Peck, such activators have also been detected in expressed juices from Lepiofa procera QuPl., Schizophyllum commune Fr., and in single, unidentified species of Collybia, Coprinus and Hygrophorw (Vincent-Davies, 1973). This suggests at least the possibility of a potentially wide host range for M. perniciosa within Agaricales and the existence of disease foci in the field.
So it is NOT a wild theory that mycogone can infect cubensis, the study would definitely support this.
Edited by sandman420 (11/05/21 05:15 PM)
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Stipe-n Cap


Registered: 08/04/12
Posts: 7,623
Loc: Canada
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Nice find, sandman.
Here is an interesting photo posted on the shroomery discord server, the cultivator claims that it had been damaged by uv irradiation:

This occurred after 63mJ of UV-C.
Just an interesting anecdote.
I'll go over that info tomorrow, I just gave it a quick scan.
Edited by Stipe-n Cap (11/05/21 06:03 PM)
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rockyfungus
dirty


Registered: 03/01/21
Posts: 1,062
Loc: Front range
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What's the NM on that thing? UV leads to reactive oxygen and nitrogen that damages major cellular pathways. DNA and lipid membranes mainly. This leads to cytotoxicity (cell death), mutations, and alterations in cell signaling.
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Stipe-n Cap


Registered: 08/04/12
Posts: 7,623
Loc: Canada
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No idea, I don't know much about the application of UV.
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sandman420
Saint PP



Registered: 06/17/04
Posts: 5,384
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I read in another one of these studies they were using 30 watt phillips TUV lights for mutagenesis. But now were off topic again!
Edited by sandman420 (11/05/21 08:16 PM)
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Stipe-n Cap


Registered: 08/04/12
Posts: 7,623
Loc: Canada
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The only way I'm going to get any satisfaction is to purchase some scopes and then get to work.
I'd like to purchase some Trichoderma from a grow shop, inoculate some plates and watch how the mycelium interacts at the contact boundary.
I'm not sure how to get my hands on other mycoparasites but I'm sure that it's possible.
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sandman420
Saint PP



Registered: 06/17/04
Posts: 5,384
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No, the only way this is getting solved is as follows
Quote:
4.1. Fungal Isolates and Morphological Characteristics Mycogone perniciosa isolates (Table 1) were obtained from the fruiting bodies of A. bisporus showing typical symptoms of wet bubbled disease in mushroom farms located in Fujian, Gansu, Hubei, and Shandong provinces of China. The disease survey was carried out in 2014–2015. The sampled infected tissues were sterilized in 2% sodium hypochlorite (NaClO) solution for 60 secs and washed three times with sterilized deionized (DI) water, then plated on Petri dishes containing PDA (potatoes 200 g/L, glucose 20 g/L, agar 15 g/L) amended with 100mg/L Kanamycin and incubated at 25 C for 5 days in darkness. Pure cultures were subsequently obtained through single spore isolation from all colonies showing the morphological characteristic of a typical M. perniciosa and five representative purified isolates sub-cultured on PDA without antibiotics. The growth characteristics, colony morphology, and conidial characteristics—such as shape, length, width—were examined for a total of 18 representative isolates. Colony color was assessed 7–10 days after single spores were transferred to PDA. A minimum of 30 conidial characters was observed under a Leica DMR HC microscope (Leica Microsystems Imaging Solutions Ltd., Cambridge, UK) fitted with Leica DFC320. The sporulation of the isolates was estimated as described by Santos et al. [27]. Briefly, 100 mg of the fungal mycelium of each isolate was collected and transferred to an Eppendorf tube, in which it was homogenized with 1 mL of Tween 80 solution (0.05%). The conidia count of each such suspension was then determined using a Neubauer chamber. Conidia counts were performed in triplicate for each isolate. All cultures were conserved on PDA in slant tubes and deposited in the Engineering Research Center of the Chinese Ministry of Education for Edible and Medicinal Fungi of Jilin Agricultural University (HMJAU) in China.
4.2. DNA Extraction and Molecular Identification Total genomic DNA was extracted from 7-day old mycelia mat growing on PDA plates with cellophane sheets using Nuclear Plant Genomic DNA Kit of CWBIO (CWBIOTECH, Beijing) following the manufacturer’s protocol. The DNA quality and quantity were measured using a BioSpec-nano spectrophotometer (Shimadzu Biotech, Tokyo, Japan) at a wavelength of 260 and 280 nm, respectively. The DNA was stored at 80 C until required for further use. PCR amplification and sequencing of the internal transcribed spacer regions of the rDNA was performed for each isolate utilizing the primer set ITS4 and ITS5 [28]. The obtained sequences were individually checked by BLAST analysis against the NCBI GenBank (http://www.ncbi.nlm.nih.gov/) database and highly corresponding sequences were retrieved, aligned, and the phylogenetic tree constructed with the maximum likelihood method using the Tamura and Nei substitution method [29] in MEGAX [30].
Quote:
4.3.2. Inoculum Preparation and Disease Assessment The inoculum for each M. perniciosa isolates were prepared from 7-day old cultures on PDA, by washing down the pathogen conidia with sterile distilled water and sieving the deferment via six layers of sterile cheesecloth. The spore/conidial concentration was estimated using a hemocytometer. The optimal spore/conidial concentration of the suspension for the isolates to cause disease was determined by inoculating 104, 105, 5 105, and 106 conidia/ml suspension on A. bisporus strains CCMJ1020 and CCMJ1036. A spore/conidial concentration of 1 x 105 was standardized and used for disease inoculation for all the A. bisporus strains. Three days after application of casing soil and regulation of temperature and relative humidity, approximately 50 ml of inoculum (spore/ conidial concentration105/ml) were sprayed into each basket containing the cultivated button mushroom. The controls were sprayed with 50 ml of sterile distilled water. Three replications were evaluated per isolate per mushroom strain. Disease assessment was recorded for the first flush. Pathogenicity was determined by the number of A. bisporus strains on which an isolate caused wet bubble disease and the order of susceptibility of strains to individual isolates. Pathogenicity tests to confirm Koch’s postulate was assessed on A. bisporus strain CCMJ1020. Disease severity was rated on sporocarp of individual mushroom strains for 30 days after inoculation using a 0–5 visual rating scale, where 0 = no symptom; 1 = 1–10%; 2 = 11–25%; 3 = 26–50%; 4 = 51–75%; and 5 = >75% based on the number of sporocarp showing disease against the total mushrooms harvested from the baskets. Based on the rating scale, the A. bisporus strains were classified as either resistant or susceptible ( 3 = resistance (R) and >3 = susceptible (S)). The severity indexes were subjected to one-way analysis of variance, and significant mean dierences (P = 0.05) were determined with Duncan’s multiple range test using GenStat 12th Edition version 12.0.0.3033 (VSNI, Hemel Hempstead, England). The experiment was repeated three times in a completely randomized design with three replicates per M. perniciosa isolate. The same batch of compost was used for each experimental trial. Also prior to each trial, the pathogenicity of each isolate was tested on the A. bisporus caps to confirm their pathogenicity before the trial. All trials were subjected to the same environmental conditions (temperature and relative humidity) and routine rigid management was maintained in a clean environment to prevent contamination from other pathogens. M. perniciosa isolate WH001 inoculated on A. bisporus strain CCMJ1020 was used as a standard for each trial to detect the eect of variation in growth room conditions on symptom expression.
4.4. AFLP Analysis The AFLP reactions were carried out as described by Vos et al. (1995) [31] with modifications. The adapters and primers used in this study are shown in Table 3 and were purchased from Genset Oligos, France and IBB PAN, Poland. Restriction digestion and adapter ligation were performed simultaneously in a 20 L reaction volume made of 4 L (50 ng/L genomic DNA, 1 L Adapter, 2 L (5 units (U)) HindIII/MseI (New England Biolabs Inc., Ipswich, MA, USA), 2.5 L 10X Reaction buer, 2.5 L 10 mM ATP, 1 L (1 unit) T4 DNA Ligase (New England Biolabs Inc., Ipswich, MA, USA) and 7 L H2O. The reaction mixture was centrifuged for 15 s, incubated at 37 C for 5 h, held at 8 C for 4 h and stored overnight at 4 C. The quantity and quality of the digested products were observed using electrophoresis on 1.5% agarose gels stained with GelRed, visualized and photographed using Bio-Rad Gel Doc XR+ system (Bio-Rad Laboratories Inc., Hercules, CA, USA). Nonselective PCR pre-amplification was performed on the digested and ligated template DNA using non-selective primer pair HindIII/MseI in a total volume of 25 L. The PCR was performed in a T-Personal thermal cycler (Biometra, Göttingen, Germany) with the following settings: 94 C for 2 min followed by 30 cycles of 30 s at 94 C, 30 s at 56 C, and 80 s at 72 C. The final thermal cycle was followed by a 5 min extension at 72 C and (hold temperature conserved at 4 C for the moment) and stored at 20 C before gel electrophoresis. The PCR products were diluted in 20-fold with TE buer. The selective PCR amplification was performed in 25 L total volume containing eight dierent primer pairs consisting of HindIII combined with MseI (Table 3). All reactions were carried out in a T-Personal thermal cycler (Biometra, Göttingen, Germany) with the following settings; first-round amplification, 94 C for 2 min followed by 12 cycles of amplification, with a decreasing annealing temperature of 0.7 C/cycle: 94 C for 30 s, first annealing for 30 s at 65 C (the annealing temperature was influenced by primers Tm), 72 C for 80 s, and next 23 amplification cycles of 94 C for 30 s, 55 C (the annealing temperature was influenced by primers Tm) for 30 s, and 72 C for 80 s. The final thermal cycle was followed by the extension of 5 min at 72 C. The PCR yields were stored at 4 C till subsequent analysis. Five L of loading buer (GelTM Vilber Lourmat, Collégien, France) were added to 25 L of the PCR products. The mixture was loaded on 1% polyacrylamide gel in 1 TBE buer (89 mM boric acid, 89 mM Tris base, 2 mM EDTA, pH8.0) and run in the Agagel Mini, Biometra electrophoresis system (Biometra, Göttingen, Germany) was run at 200 V in TBE buer for 20 min. The gels were stained with GelRed (Biotium, Inc., Fremont, CA, USA), visualized and imaged on a UV transilluminator (Vilber Lourmat FLX-20M, Collégien, France). The DNA samples of each isolate were extracted three times from fresh fungal cultures and fingerprinted twice to estimate the reproducibility of the AFLP band patterns. The electrophoretograms were examined using GeneScan® Analysis Software (Applied Biosystems, Inc., Foster City, CA, USA). AFLP markers were physically scored as binary data for the existence or nonexistence of fragments between 70 and 500 bp. This binary data obtained was later used to estimate the Jaccard’s pairwise similarity coecients as applied in FreeTree version 0.9.1.50 program [32]. The unweighted-pair-grouping method with arithmetic average (UPGMA) dendrogram was produced from DNA band patterns using the Nei and Li correlation coecient [14]. The phylogenetic tree was viewed and edited using NTSYSpc version 2.02 (Exeter Software, Setauket, New York, USA).
From the single ultimate study I have found so far, Genetic and Pathogenic Variability of Mycogone perniciosa Isolates Causing Wet Bubble Disease on Agaricus bisporus in China
so it would seem that this needs big daddy money lab and that bodhs previous post disputing the existence of mycogone p. in cubensis is... uhh... insufficient. This requires DNA analysis not 30 minutes peeping at a few slides.
Of great note is this
Quote:
On PDA chlamydospores were not observed on Hp2 and Hp9, similarly, conidia were not observed for H2, Hp8, and Hp9 (Supplementary Table S1). The isolates with brown colony morphology produced more conidia compared to the isolates with white colony morphology.
errr duh why didn't I think if this. I'm sure it would be a lot easier for one of us to get ahold of this "M. perniciosa isolate WH001"
or any other mycogone p. isolate
and we should be able to just fuck with some healthy fruits/tubs/plates and observe.
Obs we aint doing DNA but if any readers have access...
wait..is that what you just said lmao
well I'm not allowed within 1000 feet of a school by national treaty, so one of you fuckers will have to get access to some mycogone perniciosa isolates please and thank you
Edited by sandman420 (11/06/21 06:34 AM)
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Kizzle
Misanthrope


Registered: 08/30/11
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I think you'd be better off isolating it yourself. A strain used for agriculture may not be a strain that infects mushrooms but if you get it from a mushroom substrate it's more likely it will be.
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sandman420
Saint PP



Registered: 06/17/04
Posts: 5,384
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Re: Myco Parasites [Re: Kizzle]
#27532242 - 11/06/21 08:37 AM (2 years, 2 months ago) |
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Well it would just take the guess out if we take known mycogone p. and can infect and cause the symptoms.
I don't have anything doing the bubbles right now plus we aren't in agreeance that the bubbles are even that in the first place. But we can get known mycogone p.
I'm sure there is availability in academy culture banks and it's not like regulated or anything so someone could easily snatch some worry free.
Oh snap here we go $140 euro and it's ours. This is isolated from infested mushrooms. Should do us what we need.
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Yeetusdeetus



Registered: 11/23/19
Posts: 1,242
Last seen: 3 days, 7 hours
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Re: Myco Parasites [Re: Kizzle]
#27532282 - 11/06/21 09:23 AM (2 years, 2 months ago) |
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Quote:
Kizzle said: I think you'd be better off isolating it yourself. A strain used for agriculture may not be a strain that infects mushrooms but if you get it from a mushroom substrate it's more likely it will be.
I’ve read gathering samples that come directly from the fruiting substrate is fairly important when attempting to grow the white jelly mushrooms from the wild.
https://www.mushroomcompany.com/articles/0005/tremella.pdf
The way I understand it, the mushrooms physical features are caused by a species of yeast parasitizing a dark puffball shaped mushroom.
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rockyfungus
dirty


Registered: 03/01/21
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Last seen: 19 days, 12 hours
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 Not sure if mycovirus. Hispanica I keep germing and getting a feathery appearance with zones of clearing as it grows. It never forms a nice round colony just feathers.
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sandman420
Saint PP



Registered: 06/17/04
Posts: 5,384
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I'm not familiar with that species on agar but here is a pic from workman. Looks like it may be normal for it to make weak feathery mycelium.
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QM33
(NOT A PUPPET!) ❤❤❤❤❤



Registered: 04/09/20
Posts: 4,739
Loc: Oregon
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So you guys figure anything out ha?
Sound like me a couple months ago.
Glad someone else decided to start a mycogone thread as well.
But don't forget about mine ha!
https://www.shroomery.org/forums/showflat.php/Number/27450000/page/3
-------------------- OmManiPadmeHum,OmManiPadmeHum, OmManiPadMeHum... There are known knowns, there are known unknowns, there are also unknown unknowns. With great privilege comes great responsibility.
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sandman420
Saint PP



Registered: 06/17/04
Posts: 5,384
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Re: Myco Parasites [Re: QM33]
#27560364 - 11/28/21 06:35 AM (2 years, 1 month ago) |
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Not yet but I will be ordering some Mycogone perniciosa culture soon from Germany after Christmas.
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QM33
(NOT A PUPPET!) ❤❤❤❤❤



Registered: 04/09/20
Posts: 4,739
Loc: Oregon
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Ya keep is posted.
Funny besides p9 using a link to explain his hypothesis I swear I asked alot of these questions originally and even provided one or two of these links.
Thanks P9!

-------------------- OmManiPadmeHum,OmManiPadmeHum, OmManiPadMeHum... There are known knowns, there are known unknowns, there are also unknown unknowns. With great privilege comes great responsibility.
  Quantom Qups PROOF AND Soft Drops Turn your Swab to a Syringe and Syringe to Multiple Syringes! No Pours (QuantomStyal)Magic Fruit Leather DMT for IandI
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