Piptoporus betulinus

[Leo van Griensven]

Dear collegues and fellow admirers of the small world under the microscope,

Here I show some of the results I obtained from a well known mushroom that grows in large numbers in the woods in our climate region. Upon collection I fixated small pieces of fruiting body in 4 % buffered  formaldehyde for 16 hours, dehydrated through  ethanol and embedded in Technovit 7100. Sections of 2-4 μm were cut on a Microm HM360 microtome using Technovit histoblades, collected on water, dried at 40 oC and stained, commonly with toluidine blue of pH 4.4. For fluorescence I used Calcofluor White of Sigma (Optical brightener F6259). Microscopy was done on a Reichert-Jung Polyvar widefield microscope equipped for fluorescence with B1 cube, excitation 455-490 and barrier filter LP515. Pictures were made using a Canon 1000D cameraback.
If I made mistakes in interpretation, and I probably did as I am not a wood histologist by profession, please correct.

Enjoy this little part of the small world of fungi,

Leo van Griensven.


One of the most common mushrooms in European forests is Piptoporus betulinus  (Fig 1) that can be found growing anywhere that Birch trees grow naturally. P. betulinus is an annual saprobic mushroom that scavenges on dead or dying Birch. It was classified by Karst (1881) who also described two other well known medicinal fungi, Ganoderma lucidum  and Chaga (Inonotus obliquus). Some of its histological features ( see Figure 2) were published by Ryvarden & Gilbertson (1994). A further description of its properties can be found at <http://mushroomobserver.org/name/show_name_description/613?_js=on&_new=true>

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Figure 1. Fruiting body of P.betulinus growing on Birch   The white part is tramal tissue and carries the pores in which the basidiospores are formed. The stipe is 1-2 cm long and grows into the bark of the tree. Piptoporus degrades cellulose and leaves lignin intact. For that reason it is described as a brown rotter.

Figure 2:   Piptoporus betulinus (RLG 14364). a, tramal generative hyphae; b, contextual skeletal hyphae; c, inflated contextual skeletal hyphae; d, tramal binding hyphae; e, basidia; f, basidiospores.

Piptoporus is a well known medicinal mushroom Piptoporus betulinus (birch polypore), was already used by the ice man (Ötzi), probably as anti-inflammatory agent. Natural compounds and preparations of the fungus sole host genus – Betula species – are known for their activity in skin related disorders (Harms et al. 2013) and are even discussed for their potency in cancer treatment. Triterpenoid compounds like betulin and betulinic acid are assigned to be responsible for these effects.

, Pores of P. betulinus vary widely in size from 100 to 250 um.  They are surrounded by tramal tissue consisting of thin walled generative hyphae intermingled with thick walled structural hyphae. Structural hyphae could be  made visible under polarized light suggesting the presence of a high concentration of crystalline cellulose in their cell walls   The staining by toluidine blue at pH 4, which does not stain crystalline cellulose,  shows only thin walled hyphae ,

Crystalline cellulose is double breaking and can be made visible in polarized light. The picture shows the presence of crystalline material along the wall of the pores, rather suggestive of cellulose enforcement of the pore structure. It cannot be excluded however that  these big crystals were linked to the pore wall after sectioning and are not representing the thick walled hyphae.



I have not seen any basidiospores, but studying larger magnification of the polarized light pictures, one can see small tetrades suggestive of the 4 sterigmata on the basidia that are normally connected to the 4 spores. It is remarkable that these sterigmatal traces are found in the tramal region and not in the hymenium where the basidia are located.

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Slides were then stained by calcofluor white which binds to β-glucans and to chitin of fungal cell walls. The basidia measure appr. 3-5 x 10-15 μm.  Some of the thin walled generative hyphae carry  clamp connections indicative of their heterothallic nature. The thick walled hyphae are the structural hyphae. They measure 4-6 μm.

Piptoporus is a brown rotter because it degrades cellulose and leaves lignin almost intact causing the wood of the infected Birch to gradually turn brown. Crystalline cellulose is rather resistant to the hydrolytic action of Piptoporus (Valaskova & Baldrian, 2006). In the stipe by which the mushroom is connected to the host tree, I found pieces of the bark, like the sclereids shown here. The structure of the sclereids seems strongly affected by the hydrolytic action of Piptoporus enzymes . The image made of the same slide using polarized light shows the presence of double-breaking crystals . These crystals could be derived from the crystalline cellulose of the degraded sclereids. Higher magnification   under polarized light showed fracturing of the sclereid structure .
For comparison I have added a picture of  Birch bark infected by Chaga (Inonotus obliquus) another Polypore fungus. Inonotus causes severe brown rot of Birch and induces formation of big tumors (conks) on the tree that consist of both bark and fungal tissue and are strongly melanized. Sclerenchym and sclereids are affected by the action of the fungus. Chaga is a well known medicinal fungus, its extracts are sold for high prices.

References:
Piptoporus betulinus (Bull.) P. Karst., Revue Mycologique Toulouse 3 (9): 17 (1881) [MB#100963].                                                                                                                                                 
Ryvarden, L.; Gilbertson, R.L. 1994. European polypores. Part 2. 7:394-743.                                 
Harms M, Lindequist U, Al-Resly Z, Wende K. Influence of the mushroom Piptoporus betulinus on human keratinocytes. Planta Med 2013; 79 - PC4 DOI: 10.1055/s-0033-1351998.                                                                                         
Valaskova V, Baldrian P. 2006. Degradation of cellulose and hemicelluloses by the brown rot fungus Piptoporus betulinus–production of extracellular enzymes and characterization of the major cellulases. Microbiol 152:3613-3622.

[Peter Reil]

Hallo Leo,

schöne Darstellung, besten Dank!

Freundliche Grüße
Peter R.

[koestlfr]

Hey Leo!

Very exciting presentation, especially the slides of the staining with Calcofluor - superb optical quality!

Kind regards
Franz

[beamish]

Hello Leo,

very nice and interesting documentation!

Regards
Martin

[Bernhard Lebeda]

Hi Leo

very nice documentation indeed!

I´m a bit confused about the fluorescence pics: I thought Calcofluor white requires UV excitation 


Keep them coming!

Best regards

Bernhard

[Leo van Griensven]

Thank you for careful reading.
There is different opinions concerning the use of Calcofluor White. It is clear that it can be used with UV excitation at 295 nm and emission at 340 nm.
Apparently the excitation spectrum of CFW is very broad as well as the emission spectrum. Playing with filtercombinations makes the use of CFW exciting.
Read the Nikon Microscopy leaflet: http://www.microscopyu.com/articles/fluorescence/filtercubes/blueviolet/bv2a/bv2aindex.html
which advises among many others for CFW to use blue-violet excitation (400-440 nm) and a longpass barrier filter for 470 nm cut-on with a dichromatic mirror.
Also interesting: < http://aem.asm.org/content/79/13/3943.full.pdf+html > on double staining fungal parasites of phytoplankton by CFW and Sytox.

I promise I will also look by UV excitation. I have to repair my Zeiss Photomic first and that may require some time.

[Rene]

Great stuff Leo, thanks for showing!

Thanks for the link on CW and phytoplankton parasites. I'm using Calcofluor White myself routinely on dinoflagellates, it also stains cellulose. Not sure whether it has a preference for chitin though. CW fades pretty quickly by UV excitation, blue excitation is better in that respect. My supply works better by UV excitation, apparently there are several derivatives with different excitation/emission spectra on the market.

Have you seen the work by Hoch: http://www.mycologia.org/content/97/3/580.full.pdf+html? I've tried solophenyl flavin, but it wasn't as clean as CW on dinoflagellates, so went back to CW. If you like, I can send you a sample for you to play with.

Best wishes/beste wensen voor 2014,

René

[Leo van Griensven]

Dear Rene,

Nice article, well done and well explained. Of course I would like the stains to try. I did not know them. I have lots of fungal samples to try.
As I work beta glucans, it would be nice to test the stains to bind to glucan and then feed the complex to (human) cells to see whether they stay outside or are phagocytosed.
If you give me your e-mail address, I will send my university mail address.
If you want some non treated slides in return, let me know.
Thank you very much.

Leo