“Mossy ground, on a ridge, with sparse grass, under oak and hickory, is very good habitat to find King Boletes.”

-John Plischke, OMS Summer Foray 2025

Supported by SARE, the Southern Ohio Chestnut Company is getting close to a conclusion to our farmer-led research project titled “Chinese chestnut seedling mycorrhization with porcini”. This is the third research update to the public. We make these research updates when we have news to share. Here is a summary of what we have accomplished since our last update.

In summary: On March 29th, at the end of the dormant season, we planted 140 seedlings in the mother tree treatment. On May 27th, the second day of the first flush of mushrooms from the mother tree treatment area, we harvested 2 lb of mushrooms and inoculated about 200 seedlings with a spore solution. We once again sent fresh samples of mushroom to the lab to be grown out on plates, however, we were unable to complete a liquid mycelial culture treatment. Fortunately for us and anyone else seeking to follow in our footsteps, we have an extensive and interesting post-mortem on the liquid mycelial culture methods so you can avoid repeating what didn’t work for us.

Mother Tree Treatment

Image 1: mother tree treatment at conclusion of planting in late March 2026, left to right is Dietrich Epp Schmidt, Badger Baldwin Johnson, Chris Smyth.

We hand-planted 280 seed nuts in 140 planting sites (2 nuts per tree shelter) in the mother tree treatment. The tree shelters were 2′ tall, designed to keep out rabbits but maximize air flow and sunlight in these shaded, humid conditions on orchard floor. These seed nuts were all dug up by squirrels within 1 week. This high squirrel pressure was surprising to us, since there were nuts from the overstory trees still left over from last year, which would seem to indicate the pressure on the nuts was low. If we had to do this again, maybe setting traps, erecting hawk perches, setting out poison bait, or coating the seed nuts in some kind of squirrel repellent, could be tried. Time was short, so we pivoted and replanted these sites with bare root chestnut seedlings instead of seed nuts.

Image 2: Annie Hartley in the mid-ground, foreground shows the Delicate Fern Moss covering the ground around one of the “Mother Trees” that is believed to be host to the Boletus variipes fagicola we harvested for the spore solution treatment. Allegedly Amanita muscaria guessowii grows abundantly here as well.

In the two weeks after these pictures were taken, Amy Miller and Steve Larson noticed that either the humidity or perhaps the additional shade inside the tree shelter was causing defoliation or mortality to some of the seedlings, so the tree shelters were removed. The orchard is behind an 8.5′ deer fence, so deer pressure was not an issue. Some of the effected seedlings bounced back.

Spore Solution Treatments

Because the mother tree treatment had been dicey, we decided to expand our spore solution treatment into multiple different treatments so we would have ways to compare our success rate. Spore solution was prepared by taking (5 gal distilled water to 1 lb of freshly purreed King Bolete mushroom. We used the mushrooms that were at the freshly opened pore stage- the underside of the mushroom caps had visibly open, yellow pores instead of the earlier “stuffed” looking white pores, or the later green looking pores as the rot sets in.

Picture 3: freshly harvested Boletus variipes fagicola about to be pureed and added to distilled water for use in the spore spolution treatments.

Picture 4: Pureed Boletus being added to distilled water before being used in the Spore Solution treatments.

Picture 5: chestnut seedling being pulled out of a Stuewe and Sons treepot, about to be dipped into a 5 gallon bucket of distilled water + mushroom puree, (spore solution method one).

Spore solution method one: take an established 1.5 year old seedling out of its Stuewe and Sons treepot, and dip the root system and attached potting soil in a 5 gallon bucket of distilled water with 1 lb of fresh Boletus variipes fagicola slurry suspended in the water; these seedlings were repotted in same pots. We did this to about 90 seedlings. The potting soil there was straight Pro Mix.

Spore solution method two: take 1/4 cup of the same spore solution, and using a large pipette to inject this fluid into the root zone of about 100 individual seedlings in their first season of growth. 50 each in two air-pruning beds. The injection site was 3/4″ away from the stem of the seedling, and about 1″ down into the growing medium, which was straight uncomposted pine fines.

Spore solution method three: flood of 4 gallons of spore solution on 100 second year chestnut seedlings, where there was directional pour so the solution splashed from 4′ up down onto the surface of the potting medium. While less controlled than spore solutions one and two, this treatment is very quick to perform, and somewhat mimics the way spores are transported to the soil surface through air deposition. The force of poring the bucket on the surface of the Pro Mix, in the air pruning beds, imitates rain drops driving into the ground and soaking down to the tree roots.

Reflection

I got the call at 9:30pm on May 26th that the Boletus variipes fagicola was popping up in the Cooper’s chestnut orchard. I rescheduled what I was doing the day, and drove up from Athens to Carrolton at 6am to harvest the mushrooms. I made iNaturalist observations of them, and of other plants growing on the orchard floor. There was smooth carrionflower, wild yam, panicled ticktrefoil, whorled loosestrife, smooth Solomon’s-seal, violet bush clover, common haircap moss, broom forkmoss, delicate fern moss, spoon-leaved moss, poverty oatgrass, orchardgrass, and other plants growing in the orchard understory. I went to Route 9 Cooperative’s HQ and we weighed the mushrooms and over-nighted some of the white pore surface mushrooms to Thomas Lodge for tissue culture, and attempted to take spore prints. The spore prints did not work, because the mushroom caps just melted in the high humidity. Needed to use the yellow pored caps rather than the green pored caps for spore prints, I guess, when the cap isn’t rotting and the spores are still dropping- but we needed the yellow caps for the spore solution treatments. I then drove from Carrolton to Cincinnati to inoculate the seedlings at Deer Orchard’s nursery. Then drove part of the way back to Columbus and cooked up some mushrooms with white pore surface for dinner. It was a long day with a delicious conclusion at 11pm. The amount of time that this took, and the the urgency of doing it on the mushrooms timeline rather than on the timeline of your day job, childcare, other gigs, etc-, may present a bottleneck for this work being replicated! Take heed. Set up agreements and a plan so that you can responsibly pivot and stop what you’re doing, and get out and do this when the time comes. Keep an eye on local mushroom social media groups and weather conditions and iNaturalist to make sure you don’t miss your window.

Tissue Culture Attempts of Wild-Collected ***Boletus variipes*: Methods, Observations, and a Negative Result**

Author: Thomas Lodge, MidAm Mushrooms

1. Summary

In 2025 and 2026, fresh tissue of Boletus variipes was transferred onto three agar formulations: Modified Melin-Norkrans (MMN), MMN amended with oxalic acid, and malt yeast extract agar.

Round 1 (2025) produced mixed fungal and bacterial contamination by slow-growing organisms that were initially considered possible Boletus within a mixed-species matrix. Round 2 (2026) produced no visible colony formation, although a slowly expanding amber discoloration developed in the agar surrounding several tissue explants.

Amplicon sequencing of three representative cultures recovered no Basidiomycota. Assigned reads consisted of Aspergillus niger, an unidentified member of Pleosporales, and a member of Cladosporiaceae. Subsequent microscopic examination failed to detect hyphal emergence from the tissue explants after several months of incubation.

Under the conditions evaluated, no culture could be confirmed as Boletus variipes.

The purpose of this report is to document the methods, observations, and negative results in sufficient detail that they need not be unnecessarily repeated, while highlighting one observation, the amber halo, that can easily be misinterpreted as early mycelial growth.

2. Objective

Ectomycorrhizal boletes remain among the most difficult edible fungi to establish in axenic culture. Although numerous media formulations have been published for ectomycorrhizal fungi, successful isolation of Boletus species from sporocarp tissue remains inconsistent and often difficult to reproduce.

The objective of this work was to evaluate the most commonly recommended agar formulations and a standard tissue-cloning technique using freshly collected Boletus variipes.

3. Materials and Methods

3.1 Collection

Species: Boletus variipes

Collection location: ______________________

Collection date(s): Round 1: May, 2025. Round 2: May 27, 2026

Interval from collection to plating (both shipped overnight mail:

Round 1: Approximately 72 hours
Round 2: Approximately 24 hours

Storage prior to plating:

Wrapped in paper and shipped with cold packs.

3.2 Tissue Preparation

Surface sterilization methods included both:

70% ethanol
Hydrogen peroxide

No obvious difference in contamination frequency was observed between the two treatments.

Fruiting bodies were opened by hand to expose uncontaminated internal tissue. Sterile tissue was excised from the inner stipe context and inner pileus context immediately above the hymenophore using a flame-sterilized scalpel.

One tissue explant was placed on each agar plate.

3.3 Media

Modified Melin-Norkrans (MMN)

Distilled/DI water: approximately 900 mL before final volume adjustment

D-glucose: 2.5 g (10 g in some published formulations)
Malt extract: 10 g (3 g in lower-strength formulations)
Agar: 15 g
CaCl₂·2H₂O (1% stock): 1.0 mL
NaCl (1% stock): 1.0 mL
MgSO₄·7H₂O (10% stock): 10.0 mL
(NH₄)₂HPO₄ (10% stock): 0.5 mL
KH₂PO₄ (10% stock): 10.0 mL
FeCl₃·6H₂O (1% solution): 1.2 mL
Thiamine HCl (0.1 mg/mL): 1.0 mL

MMN + Oxalic Acid

Standard MMN formulation adjusted to approximately pH 5.5 using oxalic acid following the published protocol.

Malt Yeast Extract Agar

Per liter:

Malt extract: 5 g
Yeast extract: 3 g
Agar: 15 g

3.4 Incubation and Observation

Temperature: approximately 70°F (21°C)

Light conditions: dark incubation

Observation period:

Round 1: >300 days
Round 2: >60 days

Cultures were periodically examined visually and microscopically for evidence of hyphal emergence and contamination. Round 1 was subcultured 2 times over the course of the observations.

4. Observations

4.1 Round 1 (2025)

Contamination was widespread across the majority of plates. Multiple filamentous fungi and bacterial contaminants were observed.

One plate developed a contaminant morphologically consistent with Sepedonium chrysospermum (“the bolete eater”), although this isolate was not sequenced.

The dominant contaminant morphology consisted of a slow-growing filamentous fungus that was initially considered a potential Boletus isolate based on its growth rate and appearance. Colony expansion was considerably slower than the rapid growth typically observed for common laboratory contaminants, leading to the hypothesis that Boletus might be present within a mixed-species culture.

4.2 Round 2 (2026)

No visible colony formation was observed on any medium.

Tissue explants remained intact without convincing evidence of hyphal emergence.

Over approximately 60 days, a diffuse amber discoloration developed in the agar immediately surrounding several explants and slowly expanded outward.

Initially this discoloration was interpreted as possible metabolite production associated with slowly growing Boletus mycelium. However, subsequent microscopic examination failed to detect hyphae, and molecular sequencing recovered no Boletus DNA.

One plausible explanation is that the amber discoloration resulted from diffusion of oxidized phenolic compounds released from damaged bolete tissue.

Boletes contain abundant phenolic compounds together with polyphenol oxidase enzymes. When tissue is cut, these compounds undergo oxidation and the resulting pigments can slowly diffuse into the surrounding agar, producing an expanding amber or brown halo. This chemical diffusion can continue for weeks and may be mistaken for extremely slow colony expansion.

In this study, microscopic examination did not support active fungal growth associated with these halos.

5. Molecular Results

Sample	Taxonomic Assignment	Relative Abundance
A	Unassigned	0.854
A	Aspergillus niger	0.146
B	Pleosporales (unresolved below order)	1.000
C	Cladosporiaceae	0.999995
C	Capnodiales (unresolved below family)	5.2 × 10⁻⁶

5.1 Interpretation

No Basidiomycota were recovered from any sequenced culture.

Control cultures of other basidiomycete species readily colonized agar prepared from the same media batches, suggesting that the media themselves were capable of supporting fungal growth.

The most informative observation from this study may be the one that initially proved misleading.

A visible amber halo surrounding bolete tissue should not be interpreted as evidence of successful culture establishment. Such halos may occur in the absence of fungal growth and should be confirmed through microscopic examination or hyphal-tip subculture before being considered evidence of successful isolation.

6. Proposed Design for the Next Round

Tissue sourcing

Collect young, firm, unopened sporocarps whenever possible.
Plate tissue within a few hours of collection.
Maintain specimens cool and dry during transport.

Media amendments

Evaluate:

Activated charcoal (1–3 g/L) for adsorption of phenolic compounds.
Antibacterial supplements (e.g., streptomycin or chloramphenicol).
Antioxidant pretreatment of explants (ascorbic acid, cysteine, or PVPP).
Multiple oxalic acid concentrations.
Host-root infusion or root exudate supplementation.

Methodological improvements

Sequence actively growing hyphal tips rather than the original tissue explant whenever possible.
Confirm cultures microscopically before prolonged incubation.

8. Conclusion

Under the conditions evaluated, including three agar formulations commonly recommended for ectomycorrhizal fungi, no confirmed culture of Boletus variipes was established from either stipe or pileus tissue.

Amplicon sequencing of three representative cultures recovered exclusively ascomycete taxa and no detectable basidiomycete sequences, indicating that the visible growth observed during Round 1 did not represent the target organism.

The amber discoloration observed during Round 2 is most plausibly explained by diffusion of oxidized phenolic compounds released from damaged tissue rather than by early colony establishment. This observation may represent a common source of misinterpretation during attempts to culture slow-growing ectomycorrhizal fungi.

Although unsuccessful, these results provide a documented negative outcome that may help guide future efforts to culture Boletus variipes and other ectomycorrhizal boletes while avoiding repeated evaluation of the same unsuccessful approaches.

Reference

Enhancing in vitro solid culture of Boletus edulis and Boletus reticulatus through elicitation with indoleacetic acid and oxalic acid

https://doi.org/10.17660/ActaHortic.2024.1400.44

2026.08.16

Post-Script

Given the above negative result that Thomas talked about, we have been talking about other ways to kick-start growth of a Liquid Mycelial Culture that we can use for an inoculation. Dietrich came across this paper,
“First successful isolation of Entoloma clypeatum species complex from basidiospores“. If you go to section 2.2 in the Methods section, it talks about this. Rather than taking a chunk out of the stipe or cap of the mushroom and having that asexually propagate, the idea is you take part of the pore surface into a plate, have it drop the spores. Then take the basidiospores in aqueous solution and put those on other plates with Modified Melin-Norkrans (MMN). The authors claim they were successful at getting basidiospores to germinate from the target species, followed by mycelial colonization. They plated that out and incubated it for two months. Maybe this spore-based method is a better path forward for producing Liquid Mycelial Culture. Getting the mushroom stem or cap to turn into mycelium again, to grow out in bulk, just wasn’t working for our mycorrhizal species- but that might just be the wrong approach!