Crystalline Metabolites of the Tinder Polypore (Fomes fomentarius)
Abstract
In 2005, author Paul Stamets detailed a novel fungal antimicrobial mechanism in his book “Mycelium Running.” According to Stamets' observations, microscopic octahedral crystals produced by Fomes fomentarius “stunned” surrounding E. coli bacteria. In light of the lack of published peer-reviewed evidence, the present study was conducted to evaluate Stamets' claims. Specifically, the claim that large “secondary” crystals only occur in the presence of E. coli was addressed. Fruiting bodies of F. fomentarius were collected from the Montreat Wilderness Area (Buncombe County, North Carolina) and tissue culture techniques were used to cultivate mycelia on malt-agar plates. Successful cultures were randomly assigned to two treatment groups: F. fomentarius grown in isolation (n = 6) and F. fomentarius grown in the presence of E. coli (n = 6). After 28 days of growth and 10 days of E. coli exposure, the periphery of each mycelium was observed using 400–1,000× Differential Interference Contrast (DIC) microscopy. None of the interspecies frontiers and one of the isolated F. fomentarius cultures contained “secondary” crystals. Samples were then selected from the center of each dish, where hyphae had been established between 28 and 58 days. High magnification microscopy (400–1,000× DIC) revealed that five out of six interspecies dishes and six out of six isolated F. fomentarius dishes contained “secondary” crystals. While these observations do confirm the presence of secondary crystals documented by Stamets, they do not support his hypothesis that they occur only in the presence of E. coli. In light of this new evidence, Stamets' proposed antibacterial mechanism is improbable. It is suggested that future chemical tests evaluate whether or not F. fomentarius crystals are composed of calcium oxalate (an unlikely antibacterial agent).
INTRODUCTION
Fomes fomentarius is a type of white-rot fungus that inhabits the forests of the northern hemisphere. As a xylem endophyte, F. fomentarius infiltrates the wood of living and dead trees where it acts as both a parasite and decomposer (Sieber 2007; Schwarze 2007). Beech (Fagus sp.) and birch (Betula sp.) trees are preferred hosts, though F. fomentarius can be found inhabiting a range of other species (National Audubon Society 2001; Sieber 2007).
Uses of F. fomentarius go far beyond its immediate ecological niche. The hard fruiting bodies of the fungus have been used by humans for millennia, with archaeological traces of their use dating as far back as 11,555 ± 100 years before present (Peintner et al. 1998). Since ancient times, the tough fibers from the fruiting body of F. fomentarius have been used to make tinder and prolong the life of embers (Pegler 2001; Peintner et al. 1998). These same fibers have traditionally been processed to produce felted clothing and compresses, acting as a highly absorbent styptic when applied to wounds (Pegler 2001; Stamets 2002).
F. fomentarius is recognized as having medicinal properties in traditional East Asian and Slavic medicine (Beketova et al. 2011; Goldsmith et al. 2009). Different preparations of the fungus have been used to treat “oral ulcers, gastroenteric disorders, hepatocirrhosis, inflammation, and various cancers” (Chen et al. 2008). Recent in vitro and in vivo studies have confirmed some of the antibiotic, immuno-stimulating, antioxidant, diuretic, anti-tumor, and anti-inflammatory properties of F. fomentarius implied by traditional practices (Beketova et al. 2011; Choi et al. 2004; Lee 2005; Chen et al. 2008).
In light of this growing evidence of efficacy, clinical case studies involving F. fomentarius are imminent (An and Jiang 2000; Beketova et al. 2011; Chen et al. 2008; Choi et al. 2004; Lee 2005; Wasser 2011). However, there remains a need to identify the active compounds and mechanisms behind the observed effects of F. fomentarius treatments (Beketova et al. 2011; Wasser 2011).
One such mechanism has been proposed by author Paul E. Stamets in his book Mycelium Running (2005). Having demonstrated that cultures of F. fomentarius were able to completely inhibit the growth of E. coli bacteria, Stamets suggested that F. fomentarius could be producing an “extracellular antibiotic” (Stamets 2002). Upon closer examination, Stamets found that F. fomentarius produced microscopic octahedral crystals at the leading edge of its mycelia (2005). Termed “primary crystals,” Stamets proposed that these crystals served as chemical “messengers,” releasing a “chemical scent trail” upon contact with E. coli (Stamets 2005). Triggered by the dissolution of “primary crystals,” larger “secondary crystals” were produced in cultures of F. fomentarius exposed to E. coli (Stamets 2005). Unlike their smaller counterparts, Stamets concluded that these “secondary crystals” “stunned” encroaching E. coli, allowing the “mother mycelium” to devour the pathogenic bacteria in direct proximity (Stamets 2005). Although Stamets does not provide evidence for a “scent trail” or a “stunning” mechanism, the fact that the crystals appeared to be associated with the inhibition of E. coli is compelling. Others have previously reported the observation of such fungal crystalline metabolites (Cheng et al. 2004; Choi et al. 2003; Nakata 2003) but Stamets was the first to observe them in cultures of F. fomentarius and speculate on their potential antibacterial properties. Clearly, as a potential source of novel antibiotics for medicine and bioremediation, these larger “secondary crystals” are of enormous interest.
Citation: Journal of the North Carolina Academy of Science 130, 1; 10.7572/2167-5880-130.1.16
Unfortunately, no description of experimental design is provided in Mycelium Running, presumably because the book is geared toward a general audience. The little information given is that the original study was conducted during Stamets' work with the Battelle mycoremediation team (Stamets 2005). Based on extensive literature searches, this study has not been published in any peer-reviewed journal. Similar articles paralleling or supporting Stamets' observations were not found in published literature.
Thus, in the wake of such intriguing descriptions, there remains a real need to replicate and review Stamets' work. This study investigates the antibacterial mechanism proposed by Stamets, approximating his experimental design and producing a second set of observations using microscopy.
Citation: Journal of the North Carolina Academy of Science 130, 1; 10.7572/2167-5880-130.1.16
Citation: Journal of the North Carolina Academy of Science 130, 1; 10.7572/2167-5880-130.1.16
Citation: Journal of the North Carolina Academy of Science 130, 1; 10.7572/2167-5880-130.1.16
Citation: Journal of the North Carolina Academy of Science 130, 1; 10.7572/2167-5880-130.1.16
Citation: Journal of the North Carolina Academy of Science 130, 1; 10.7572/2167-5880-130.1.16
Citation: Journal of the North Carolina Academy of Science 130, 1; 10.7572/2167-5880-130.1.16
METHODS
Preliminary Study
Wild specimens of F. fomentarius fruiting bodies were collected from the Montreat Wilderness area (Buncombe County, North Carolina) with permission from the Montreat Wilderness Committee. Specimens were identified in the field using the National Audubon Society Field Guide to North American Mushrooms (2001). Exposure to a strong base (1M KOH) indicated that fomentariol was present in the fruiting bodies, confirming sample identity as F. fomentarius (Peintner et al. 1998).
Fruiting bodies were then broken open under sterile lab conditions, exposing internal tissue. This tissue was transferred to plates of malt extract agar prepared in accordance with manufacturer instructions (Difco™, 2009). Ten out of 16 initial tissue cultures were discarded because of contamination.
In vitro mycelia were identified as F. fomentarius by observing growth patterns (Campbell 1938). Subsequent to identification, successful mycelia were cloned to new plates of malt agar by transplanting plugs of hyphae into the new dishes (Rodriguez et al. 2008). Dishes were sealed with Parafilm™ and stored at room temperature (∼25°C) in the dark (Chen et al. 2007). Non-pathogenic E. coli from Carolina Biological™ was stored under identical conditions on plates of tryptic soy and malt agar.
Microscopic observations of the E. coli and fungal cultures were made using an Olympus BX60 compound microscope at 40×, 100×, 200×, 400×, and 1,000× magnification. Bright Field, Phase Contrast, and Differential Interference Contrast (DIC) forms of photomicroscopy were used for all initial observations. Photographs were collected using CapturePro2.8.8 software. All subsequent measurements were made using this software.
Slides were prepared from pure F. fomentarius (n = 1 dish), pure E. coli (n = 1 dish), and interspecies frontiers between the two organisms (n = 7 dishes) to determine whether crystals could be found under any conditions. These cultures were viewed at irregular intervals over the course of several weeks using a complete range of microscopy methods.
Frequency Comparison
After the preliminary study confirmed that crystals could be found using the aforementioned set of methods, a second study was launched to test Stamets' hypothesis that “secondary crystals” only occur when F. fomentarius is in the presence of E. coli.
New cultures of F. fomentarius were cloned onto plates of solid growth medium (10 g tryptic soy agar per liter, 26 g malt agar per liter, 5 g yeast extract per liter, and 5 g D-(+)-glucose per liter). Using a random number generator, uncontaminated cultures of F. fomentarius were assigned to two treatments: F. fomentarius grown in isolation (n = 6 plates) and F. fomentarius grown in the presence of E. coli (n = 6 plates). A plate of sterile growth medium (same as treatments) and a plate of E. coli served as controls. Plates were sealed with Parafilm™ and stored at room temperature (∼25°C) in the dark (Chen et al. 2007).
After 28 days of mycelial growth and 10 days of E. coli exposure, cross-section samples were taken from the periphery of each plate using a sterile glass pipette. It should be noted that sample sites were not randomly selected; because of limited and irregular mycelial growth, samples had to be taken from the leading edges of hyphae wherever sizeable new growth could be found. In the case of the interspecies treatment, samples were taken from wherever new hyphal growth was in direct contact with bacterial colonies.
Immediately following collection, each sample was transferred to a glass slide and viewed under 400–1,000× Differential Interference Contrast (DIC) microscopy. “Secondary crystals” were recorded as either present or absent from each plate according to sample results. Based on Stamets' pictures in Mycelium Running (2005), “secondary crystals” were defined as bi-pyramidal octahedral crystals whose side lengths exceeded 4 µm.
Samples were also collected and evaluated from the center of each mycelial mat. Presumably, these sites contained the oldest hyphae within each plate, having been transferred from clones dating back to the preliminary study. This would make the hyphae in these samples between 28 and 58 days old. Note that none of the cloned parent tissue had been exposed to E. coli prior to this study of crystal frequency. Pseudoreplication is present as parent tissue for both treatments came from three plates of isolated F. fomentarius.
Observed “secondary crystal” frequencies were compared to Stamets' expected “secondary crystal” frequencies. According to Stamets, “secondary crystals” are produced as a response to the presence of E. coli (2005). The presence of any “secondary crystals” in isolated cultures of F. fomentarius would contradict Stamets' proposed antibacterial mechanism.
RESULTS
Preliminary Study
Day 5
Samples were prepared from one dish of isolated F. fomentarius, one dish of isolated E. coli, and seven dishes containing both species. No crystals matching Stamets' description were found.
Day 14
Crystals roughly matching Stamets' description of “secondary crystals” were observed in one out of seven interspecies dishes. Within this dish, only one sample site out of nine contained crystals. Hyphae and an unknown bacterial contaminant were present in this site.
Day 23
Octahedral, bipyramidal crystals were found in the same site as on day 14. These were an exact match to Stamets' descriptions of “secondary crystals.” In a separate interspecies dish, oblong disk shaped crystals were discovered, constituting a new and unexpected crystal morphology.
Day 24
In an effort to inundate the fungi with a more thorough bacterial exposure, fresh scrapings of E. coli were taken from tryptic soy plates and transferred directly onto the mycelial mats of the seven interspecies dishes. For record keeping, these dishes were labeled ‘A’ through ‘G’.
Day 27.—Octahedral crystals were observed in dish ‘B’. The majority of crystals were visible using 400× magnification. However, 1,000× magnification was required for a complete inventory of crystals as they varied considerably in size and apparent development (<1 µm to 7 µm in length). Crystals appeared to be clustered on the surfaces of certain hypha.
Day 36
Octahedral crystals were observed in all seven interspecies dishes (‘A’ through ‘G’). Crystals were found almost exclusively within clumps of hyphae. Some strands of hyphae appeared to have crystals lining their entire surface. A new crystal morphology was observed exclusively with dish “A.” These large rhombus shaped crystals measured ∼20 µm in length.
Frequency Comparison
DISCUSSION
This study confirms Stamets' original observation of crystalline metabolites of F. fomentarius, which has not previously been reported in the peer-reviewed literature. Additionally, while the preliminary study was originally meant to establish and refine methods for such observations, it ultimately led to several unexpected discoveries. Before the experiment, it was expected that crystals would either appear exactly as described by Stamets or be completely absent. It was not anticipated that a range of crystal shapes and sizes would exist. Similarly, because Stamets distinguished between “primary crystals” and “secondary crystals” on the basis of size, it was expected that these bipyramidal octahedral crystals would display a clear size dichotomy. Instead, an apparent continuum of sizes was observed (but not statistically evaluated). These observations suggest that a size-based distinction between “primary crystals” and “secondary crystals” is inadequate.
For the sake of comparing crystal frequency between treatments, “secondary crystals” were defined as octahedral crystals with side lengths greater than 4 µm. This definition was based on an image of “secondary crystals” provided by Stamets in Mycelium Running (2005). The image, which showed “secondary crystals” surrounded by bacteria (presumably E. coli), lacked any indication of scale. Crystal size had to be estimated using bacteria in the picture as primitive scale-bars. The resulting definition constitutes the best characterization of “secondary crystals” currently available (K. Brownson, pers. comm. 2 April 2012).
Using this definition, “secondary crystals” were identified at the center of all six (100%) cultures of F. fomentarius grown in isolation. According to Stamets' proposed antibacterial mechanism, none of these plates should have contained “secondary crystals.” Following Stamets' hypothesis, “secondary crystals” were only predicted in cases where F. fomentarius encountered E. coli. Clearly, the results of this study do not support Stamets' predictions; rather, they contradict a key component of his proposed antibacterial mechanism by showing that “secondary crystals” can occur in the absence of any pathogen.
Note that the chemical composition of these crystals remains unknown and the question as to whether or not they contain antibacterial compounds remains unanswered. However, similarities in crystal shape, size, and circumstance are highly suggestive of calcium oxalate.
A host of journal articles have documented calcium oxalate forming bipyramidal, octahedral crystals (Cheng et al. 2004; Choi et al. 2003; Nakata 2003). The size of these crystals spans the same range as those described by Stamets and the present study (Cheng et al. 2004; Choi et al. 2003; Nakata 2003). The fact that calcium oxalate crystals are commonly found among higher fungi further suggests that the “primary” and “secondary crystals” described by Stamets are likely composed of calcium oxalate (Aragno et al. 2011; Larsson 1994).
The considerable variation of crystal morphology observed herein can also be explained by the calcium oxalate hypothesis. According to Larsson's examination of the Trechispora genus of fungi, calcium oxalate crystal morphology varies considerably across gradients of temperature, chemical environment, and host genetics (1994). Time is also a significant factor, causing “aberrant growth and corrosion” and ultimately “recrystallization” into morphologically unique derivatives (Larsson 1994). Thus, under the calcium oxalate hypothesis, time alone could account for changes in crystal morphology.
Larsson also notes how “in hyphae, young crystals are seen as blisters on the hyphal surface” and that “heavily encrusted parts” sometimes develop (Larsson 1994). These descriptions are consistent with observations made here of encrusted hyphae bearing a spectrum of “developing” crystals. Given these similarities, it is possible that Fomes fomentarius develops calcium oxalate crystals in a similar way as the genus Trechispora.
Notably, while no calcium oxalate article specifically addresses F. fomentarius, other white-rot fungi have been documented producing calcium oxalate crystals when grown on malt agar medium (Aragno et al. 2011). In all cases of crystal production, the oxalate is biosynthesized and the calcium taken from surroundings (Nakata 2003). Within malt agar medium, residual levels of calcium are present from the initial processing of barley grains (Aragno et al. 2011). This means that, provided F. fomentarius can biosynthesize oxalic acid, all of the reactants needed for calcium oxalate crystal production were present.
Evaluating this “calcium oxalate” hypothesis will require chemical testing. Using methods adapted from this study, F. fomentarius could be cultivated on malt agar growth medium. After 10 or more days of growth, various HPLC methods could be used to determine oxalic acid concentrations within cultures of F. fomentarius and compare these levels to controls (Aragno et al. 2011). Provided access to the necessary instruments, energy-dispersive X-ray microanalysis could be used to determine whether or not crystals contain high concentrations of calcium (Larsson 1994). The most direct method for analyzing calcium oxalate content may be X-ray powder diffraction (as cited by Larsson 1994). This list of potential methods is not exhaustive and will, no doubt, be expanded in preparation for future studies.
Photographs of the crystals observed on day 14. Photographs were taken at 1,000× magnification using DIC photomicroscopy. The edge length of each crystal is approximately 15 µm.
Bipyrimidal octahedral crystals with edges measuring approximately 3–7 µm in length, as observed on day 23 (1,000× DIC photomicroscopy).
New crystal morphology observed on day 23 (400× DIC photomicroscopy). Note that the crystals are flat, oblong polygons roughly 15 µm in length.
Picture from dish ‘B’ sample, taken on day 27 using 1,000× DIC. At the center, a single bipyramidal, octahedral crystal can be seen surrounded by hyphae. With a side length of ∼5 µm, this crystal appears to be an exact match to Stamets' description of a “secondary crystal.” Note the range of smaller crystals and particulates in the upper right corner.
Picture from dish ‘B’, taken on day 27 using 1,000× DIC. Note the range of crystal development and size as they follow along the length of a single hypha.
Picture from dish ‘B’, taken on day 36 using 1,000× DIC. Note the dense clusters of crystals forming along the two central hyphae. Note the apparent range of crystal size and development.
Picture from dish ‘A’, taken on day 36 using 200× DIC. Note the new rhombus morphology of crystal, measuring ∼ 20 µm in length.
Contributor Notes
Warren Wilson College undergraduate researcher
