Mycologia
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Services
Right arrow Similar articles in this journal
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Koller, G. EB.
Right arrow Articles by Størmer, F. C.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Koller, G. EB.
Right arrow Articles by Størmer, F. C.
Agricola
Right arrow Articles by Koller, G. EB.
Right arrow Articles by Størmer, F. C.
Mycologia, 94(5), 2002, pp. 752-756.
© 2002 by The Mycological Society of America

The presence of orellanine in spores and basidiocarp from Cortinarius orellanus and Cortinarius rubellus


Gry EB. Koller

     Division of Molecular Cell Biology,

Klaus Høiland

     Division of Botany and Plant Physiology, Department of Biology, University of Oslo, P.O. 1045 Blindern, 0316 Oslo, Norway

Karel Janak
Fredrik C. Størmer 1

     Department of Environmental Medicine, Norwegian Institute of Public Health, P.O. 4404 Nydalen, 0403 Oslo, Norway

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

This is the first report quantifying the orellanine content in basidiospores. The toxin content and tissue distribution of orellanine were determined from Cortinarius orellanus (Fr.) Fr. and Cortinarius rubellus Cooke. Basidiospores, the basidiocarp, divided into cap and stem, and mycorrhiza roots were analyzed to determine the amount of orellanine by reversed phase high performance liquid chromatography and thin layer chromatography. The orellanine contents in spores were 0.31% (C. orellanus) and 0.09% (C. rubellus). In caps, we found the toxin content to be 0.94% (C. orellanus) and 0.78% (C. rubellus), in stems 0.48% (C. orellanus) and 0.42% (C. rubellus) and in mycorrhiza roots from C. rubellus we determined the orellanine contents to 0.03%. In addition, extracts from the different structures of the basidiocarp of C. orellanus and C. rubellus, with an orellanine content corresponding to 25 nmol, inhibited the growth of Bacillus subtilis.

Key words: Basidiospores, Cortinarius orellanus, Cortinarius rubellus, mushroom poisoning, nephrotoxicity, orellanine


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Several mycotoxins have been detected in the conidia of molds. Aflatoxins have been determined in conidia from toxigenic strains of Aspergillus flavus and A. parasiticus (Wicklow and Shotwell 1983Citation). Conidia from A. niger and A. fumigatus contain significant concentrations of the toxins aurasperone C and fumigaclavine (Palmgren and Lee 1986Citation); and conidia of Stachybotrys atra contain trichothecene mycotoxins (Sorenson et al 1987). Fumonisins and AAL-toxin have been determined in conidia of Alternaria alternata (Abbas and Riley 1996Citation), and citrinin and minor amounts of ochratoxin A have been found in spores of Penicillium verrucosum (Størmer et al 1998Citation).

Mycotoxins may have different functions for spore germination and survival. Based on the presence of large amounts of the UV absorbing mycotoxin citrinin in the outer layer of spores of P. verrucosum (Størmer et al 1998Citation), it was suggested that the toxin may function as a sun protectant in addition to creating favorable conditions during the initial stages of germination. An additional function of citrinin in spores could be to affect the uptake of iron in other competing microorganisms (Størmer and Høiby 1996Citation).

The presence of ochratoxin A in dust collected from households and from cowsheds (Richard et al 1999, Skaug et al 2001) indicates that fungal spores containing mycotoxins may pose a respiratory problem for humans as well as for animals. A similar issue may also arise from mushrooms if their basidiospores contain toxins.

To our knowledge no reports have described the quantification of toxin in basidiospores from species belonging to the Basidiomycota. Orellanine, (2,2'-bipyridine)-3,3', 4,4'-tetrol-1, 1'-dioxide, is the toxin responsible for the lethal nephrotoxicity of C. orellanus (Fr.) Fr. and C. rubellus Cooke (Schumacher and Høiland 1983Citation), but prior to this study it has not been isolated from spores. Therefore, we have determined the concentration of orellanine in spores from these two species and compared it with the concentration of the toxin in caps and stems.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Collection of fungal material and spore isolation – Cortinarius orellanus was collected in six different localities in Arendal and Grimstad, Aust-Agder County, at the Norwegian south coast in 1999. The more common Cortinarius rubellus was collected in three different localities in Nannestad, Akershus County, SE Norway, in autumn 1999 and 2000, together with soil samples containing ectomycorrhiza, close to the same localities. The stems were removed from the mushrooms, and the caps were placed onto glass plates for 24 h to collect the spores. The caps from these mushrooms were dry, and no moisture was observed during the spore drop. The sampled spores contained approximately 95% basidiospores, as evaluated by microscopic examination. The caps, stems, and spores were dried and stored at -80 C. The mycorrhiza roots were selected on roots of Picea abies by following the hyphae from the fruit bodies of C. rubellus. All soil materials were removed, and mycorrhiza roots and the spores were dried at 60 C before use. Spore volume can be calculated as that of an ellipsoid, (Gross 1972Citation, Meerts 1999Citation), with spores of these Cortinarius species having a spore volume of approximately 300 µm3. Assuming a density of 1 g/cm3, this corresponds to a spore weight of 300 pg.

Extraction of orellanine – The dried caps and stems were powdered in liquid N2, and fatty material and non polar pigments were removed through two 30-min extractions with diethylether (Prast et al 1988Citation, Holmdahl et al 1987Citation). This was followed by five extractions for 30 min with methanol-4% KCl in water (4:1 v/v). After centrifugation for 15 min at 15 000 g, the extract was evaporated to dryness at 60 C. Before analysis, the sample was dissolved in 1% trifluoroacetic acid (TFA) in water. With the exception of liquid N2 powdering, the extraction of spores and mycorrhiza were carried out in the same way.

Orellanine standard – The orellanine was a generous gift from Jean-Michel Richard, Université J. Fourier de Grenoble, France.

Chromatographic conditions – High pressure liquid chromatography (HPLC). The HPLC equipment used for analysis consisted of a Perkin-Elmer series 4 HPLC pump, a Hewlett Packard 11040 photodiode array detector, a 7125-075 Rheodyne injector with a variable volume loop, and a Waters (115 x 13 mm) C-18 preparative column. The mobile phase, flow rate of 1 mL/min, consisted of acetonitrile-water (5:95 v/v) acidified to pH 1 with 1% TFA. All analyses were carried out at laboratory temperature. Orellanine in extracts was tentatively confirmed by retention of standard at 6.5 min, and the amounts of orellanine in the sample were determined by comparison with a standard curve. The concentration of the standard and extracts was calculated using a molecular extinction coefficient of 9100 M-1cm-1 at 288 nm (Cantin et al 1989Citation). The fractions were dried in a rotavapor at 50 C, and the material was dissolved in 1% TFA in water and subjected to thin-layer chromatography.

Thin-layer chromatography (TLC). The samples were applied on silica or cellulose plates. Silica glass plates and aluminum sheets with cellulose without fluorescent indicator, and cellulose glass plates with fluorescent indicator, were used with two different solvent systems. The first system consisted of n-butanol-acetic acid-water (BAW) (3:1:1 v/v/v) (Keller-Dilitz et al 1985Citation, Kürnsteiner and Moser 1981Citation), and the second was n-butanol-TFA-water (BTW) (3:1:1 v/v/v), with pH adjusted to 2 and 0, respectively. Orellanine and orelline were identified by exposing the plates to UV-light from a transilluminator with an intensity of 6000 W/cm2 at a wavelength of 365 nm. The amount of orellanine in the spots was determined by comparison with standard solutions.

Liquid chromatography-Mass spectrometry (LC-MS). Mass spectra were obtained with a VG Platform quadruple mass spectrometer (Fisons Instruments, VG Biotech, Altrincham, UK) equipped with an atmospheric pressure electrospray ionization source. Samples were directly introduced into the MS source at a flow of 10 µL/min from a 200 µL loop. Total ion current mass spectra were measured in both positive and negative ion detector modes for orellanine and orelline standards at a concentration of 0.5 µg/mL, and for extracts of caps and of the spores. Acetonitrile-water (1:1 v/v) was used for preparation of solutions. The pH of solutions used for positive ionization was adjusted to 2.0 with 1% formic acid, while the pH of solutions used for negative ionization was adjusted between 5.0–6.0 with ammonium/ammonium acetate.

Effects of spore-, cap- and stem extracts upon growth of Bacillus subtilis – Cultures of Bacillus subtilis ATCC 6633 were grown on a minimal medium containing per 1000 mL: KH2PO4 3 g; MgSO4·7H2O 1 g; (NH4)2SO4 1 g; and glucose 10 g (Davies and Mingioli 1950Citation). The medium was prepared in plates of 4 mm depth, with the pH of the medium adjusted to 7.0. Paper discs, 6 mm in diameter, were purchased from AB Biodisk, Solna, Sweden. They were placed on the plates after being impregnated with extracts from caps, stems, or spores containing 25 nmol orellanine dissolved in methanol or 1% TFA. The zones given as the diameter (mm) with complete inhibition were measured after 24 h of incubation at 37 C. The discs were dried before application. Controls with methanol or 1% TFA gave no inhibition. The inhibition zones are presented as an average of two different experiments (Table II).


View this table:
[in this window]
[in a new window]
  		TABLE II. Effect upon growth of Bacillus subtilis by the amount of extracts of cap, stem, and spores containing 25 nmol orellanine from Cortinarius orellanus and C. rubellus

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
The amount of orellanine in spores of C. orellanus was determined to be 0.31 % ± 0.07 corresponding to 0.93 pg/spore by HPLC, and this was confirmed by TLC. The latter showed the amount of toxin to be in the range 0.2–0.3% as determined by image analysis. In C. rubellus spores, the toxin content was found to be 0.09% ± 0.02 corresponding to 0.27 pg/spore by HPLC, and this is also consistent with the findings with TLC. The amounts of orellanine in dried C. orellanus caps were 0.9% and in stem 0.5%. In C. rubellus caps and stems, we found the orellanine content to be 0.8% and 0.4%, respectively. Thus, C. orellanus contains a higher level of orellanine than C. rubellus, and toxin content varies among different structures of the basidiocarp with twice as much toxin in the cap as in the stem. The orellanine content in C. rubellus mycorrhizal roots was determined to be 0.03% (Table I).


View this table:
[in this window]
[in a new window]
  		TABLE I. Amount of orellanine in cap, stem and spores from Cortinarius orellanus and C. rubellus, and the amount of orellanine in mycorrhiza from C. rubellus, calculated as percentage weight of dried material for their orellanine contents by HPLC. Samples were taken from the dried material and four parallels from each sample were analyzed

 
In UV-light, orellanine is easily decomposed into its photodegradation product orelline, observed as a delayed, bright turquoise-blue fluorescence (Kürnsteiner and Moser 1981Citation). This phenomenon is characteristic of 2,2'-bipyridines (Schumacher and Høiland 1983Citation). In acid solution of FeCl3, orellanine forms a violet Fe(III)-complex (Kürnsteiner and Moser 1981Citation, Schumacher and Høiland 1983Citation). A cellulose TLC layer gave the required separation of orellanine and orelline in both TLC eluent systems, but BTW allowed a quicker and even better separation than BAW, with clear spots of both orellanine and orelline. The pKa1 of orellanine is 1.5 (Richard et al 1991Citation); therefore, a low pH is required for the fully protonated form to dominate. This could explain why the best separation was achieved with the BTW-system. Based on visible color, color of fluorescence, and Rf -values of a co-chromatographed reference sample, spots of orellanine and orelline were detected with Rf-values 0.64 and 0.24, respectively, in the BAW-system. In the TFA-containing system (BTW), orellanine had a Rf-value of 0.63 and orelline a Rf-value of 0.16. Orellanine and orelline have both been reported to form stable violet complexes with Fe3+ and were, thus, also identified by their reaction with 3% FeCl3 in 0.5 M HCl. By comparison with a dilution series of standards on cellulose glass plates with fluorescent indicator, the approximate quantities of orellanine in spores from C. orellanus and C. rubellus were found to be 0.2–0.3% and 0.1% of the dry weight, respectively, in good agreement with the results found by HPLC. Orellanine was not determined in the extracts of C. rubellus mycorrhiza with these chromatographic systems. The detection limit was found to be 0.2 nmol orellanine image processing

LC-MS experiments were performed to confirm the findings of orellanine in the different tissues in C. orellanus and C. rubellus. In accordance with literature data (Antkowiak et al 1994Citation), orellanine was found to be more stable in acidic solution than in neutral solution. Low pH supports positive ionization, while basic solutions support generation of negative ions in electrospray MS. By positive ionization in acidic solutions, similar ions were found in spectra of orellanine standard, in cap and spore extracts of the C. orellanus and extract of the cap from C. rubellus. The most intense ion was triple loaded molecular ion (m/z = 85.0) in both standard and samples. Mono-loaded molecular ion (M+H)+ with m/z = 253.0 was also detected in standard and samples. However, while this was the next most intense ion in the standard, two other ions had higher responses in extracts of basidiocarps from both species and spores from C. orellanus. These ions corresponded to oxidized and/or decomposed products of orellanine. As a result of orellanine oxidation, quinone (MW 250) with ion at m/z = 83.2 was also detected in both basidiocarp and spore extracts. Furthermore, decomposition products 3,3', 4,4'-tetrahydroxy-2, 2'-bypiridine (orelline, MW 220) giving ions at m/z = 3.2 and m/z = 111.0, and 3,3', 4,4'-tetrahydroxy-2, 2'-bypiridine-N-oxide (MW 236) giving ion at m/z = 78.3 were detected only in extracts of basidiocarps. It was not possible to obtain negative ion mass spectra of the orellanine standard at low pH. Only at pH above 4.8 a spectrum for orellanine standard was recovered. However, at this low pH, most was in the form of the oxidation product (quinone, MW 250) and only small amounts of the orellanine M-1-ion could be detected. Good sensitivity was obtained at pH 12, but then only the product of oxidation and its adduct with acetonitrile were detected, in extracts of basidiocarps and of spores. No decomposition products were found in the standard, but high concentrations of mono-N-oxide and somewhat lower concentrations of orelline were found in extracts of basidiocarp, similar to the analysis using positive ionization.

We measured the effect of C. orellanus and C. rubellus extracts upon growth of B. subtilis. Extracts from cap, stem or spores from the two species containing equal amounts of orellanine (25 nmol) inhibited the growth of B. subtilis differently (Table II). No inhibition zone was observed from extract of caps from C. orellanus (6 mm, i.e., disc diameter 6 mm), whereas spore extract from C. orellanus revealed an inhibition zone of 25 mm as compared to standard orellanine (15 mm). Dose-response curves for the standard orellanine showed a reasonably linear relationship between concentration of the toxin and the diameter of the inhibition zones (not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
There have been many attempts to quantify the amount of orellanine in C. orellanus and C. rubellus. Prast et al (1988)Citation found the orellanine content of dried carpophores from these mushrooms to be 1.4% and 0.9%, respectively, by TLC. Cantin et al (1989)Citation quantified the toxin content to 1.2% of dried whole carpophores of C. orellanus by HPLC. Using electrophoresis, Oubrahim et al (1997)Citation determined the amount of orellanine of dried carpophores to be 1.4% (C. orellanus) and 0.6% (C. rubellus). Correspondingly, applying electron spin resonance (ESR), the amounts were found to be 1.1% and 0.5%, respectively. They also found that the toxin seemed to be unequally distributed in the mushroom, with the toxin content in the cap being 2 to 3 times of that in the stem.

Our results are in good agreement with those findings, showing the toxin content of C. orellanus cap to be 0.9% and that of stem to be 0.5%, and the toxin content of C. rubellus cap to be 0.8% and that of stem to be 0.4%. (Table I). These contents are slightly lower than those found by others, but the differences might be due to sample variation. It is known that the phallotoxin content of Amanita phalloides varies with carpophore development stage and with altitude. An unequal distribution of toxin among the different structures of the A. phalloides basidiocarp was also observed (Enjalbert et al 1989Citation).

To our knowledge there have been no reports of the orellanine content in Cortinarius basidiospores. The orellanine content of basidiospores of C. orellanus was determined to 0.3% and that of C. rubellus to be 0.1% of the spore weight by HPLC and TLC. One C. orellanus spore thus contains approximately 0.9 pg of orellanine, while one C. rubellus spore contains about 0.3 pg of orellanine. By comparison, the smaller conidia of Penicillium verrucosum (average diameter 3.2 µm) have been found to contain citrinin in the range 1.4–4.1 pg/spore, or 8–24% of the spore weight (Størmer et al 1998Citation). In fungal spores, there are substances which could be important for spore survival, activation, and ultimately germination. The amount of orellanine in spores from the Cortinarius species, in particular C. rubellus, is low compared to those of the whole basidiocarp.

In 1987 Rapior et al isolated mycelium from C. orellanus grown on an agar medium and showed the presence of orellanine by TLC. The orellanine content of the mycelium was much lower than in the basidiocarp but no further quantification was done. Cortinarius species are important fungal partners in ectomycorrhiza, particularly in arctic, boreal, and nemoral regions. The ectomycorrhizal association has been shown to increase the growth rate and biomass production of the host plant, and to influence the development of the root system. The fungal component may contribute 25% or more to the dry weight of an infected root (Isaac 1992Citation). We found that the orellanine content in mycorrhizal roots, the fungus partner being C. rubellus, was quite low. Assuming 25% fungal component, the orellanine content was determined to be only 0.03% of the dry weight of the mycorrhizal root. This is even lower than what was found for the spores, and may therefore be regarded as a contamination rather than an actively metabolized secondary metabolite in this tissue.

The extracts from various parts of the two species containing the same amount of orellanine as the standard (25 nmol) inhibited the bacterial growth from no inhibition 6 mm (C. orellanus cap), to 25 mm (C. orellanus spores) as compared to 15 mm (orellanine standard). The reason for this discrepancy could be that the extracts contain various other compounds that could effect bacterial growth, indicating that the C. orellanus spores contains additional substances toxic to the bacterium. These toxins could yield a beneficial effect for the spores during the initial spore germination, possibly by inhibiting the growth of competing microorganisms. A basidiocarp is usually short-lived; it forms a special structure separated from the rest of the fungus individual, and it has a distinct and important role in producing and distributing spores. Therefore, the metabolites in the fruit body may be formed for completely different reasons than those found in spores and mycelium (Stradler and Sterner 1998Citation).


   ACKNOWLEDGMENTS
 
The authors would like to thank Jean-Michel Richard, Université J. Fourier de Grenoble, France for providing samples of orellanine, and Inger Lise Fonneland, Grimstad, Norway for providing the C. orellanus material.


   FOOTNOTES
 
1 Corresponding author, Tel.: +4722042200, Fax: +4722353605, Email: fredrik.stormer{at}fhi.no Back

Accepted for publication May 3, 2002.


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Abbas HK, Riley RT., 1996 The presence of phytotoxicity of fumonisins and AAL-toxin in Alternaria alternata. Toxicon 34:133-136[Medline]

Antkowiak WZ, Antkowiak R, Wyrzykiewicz E, Czerwinski G., 1994 Mass spectral fragmentation of orellanine and its tetramethyl ether with regard to their facile thermal and photochemical deoxygenation. Heterocycles 39:477-490

Cantin D, Ricard JM, Alray J., 1989 Chromatographic behaviour and determination of orellanine, a toxin from the mushroom Cortinarius orellanus. J Chromatogr 478:231-237[Medline]

Davies BD, Mingioli ES., 1950 Mutants of Escherichia coli requiring methionine or B12. J Bacteriol 60:17-28[Free Full Text]

Enjalbert F, Cassanas G, Andary C., 1989 Variation in amounts of main phallotoxins in Amanita phalloides. Mycologia 81:266-271

Gross G., 1972 Kernzahl und sporenvolumen bei einigen Hymenogasterarten. Z Pilzk 38:109-158

Holmdahl J, Ahlmén J, Bergek S, Lundberg S, Persson S., 1987 Isolation and nephrotoxic studies of orellanine from the mushroom Cortinarius speciosissimus. Toxicon 25:195-199[Medline]

Isaac S., 1992 Fungal-plant interactions. London: Chapman & Hall. 417 p

Keller-Dilitz H, Moser M, Ammirati JF., 1985 Orellanine and other fluorescent compounds in the genus Cortinarius, section Orellani. Mycologia 77:667-673

Kürnsteiner H, Moser M., 1981 Isolation of a lethal toxin from Cortinarius orellanus Fr. Mycopathol 74:65-72

Meerts P., 1999 The evolution of spores in Agarics: do big mushrooms have big spores?. J Evolution Biol 12:161-165

Oubrahim H, Ricard JM, Cantin-Esnault D, Seigle-Munrandi F, Trécourt F., 1997 Novel methods for identification and quantification of the mushroom nephrotoxin orellanine. Thin layer chromatography and screening of mushrooms with electron spin resonance determination of the toxin. J Chromatogr A 758:145-157[Medline]

Palmgren MS, Lee LS., 1986 Separation of mycotoxin containing sources in grain dust and determination of their mycotoxin potential. Environ Health Persp 66:105-108[Medline]

Prast H, Werner ER, Pfaller W, Moser M., 1988 Toxic properties of the mushroom Cortinarius orellanus. Arch Toxicol 62:81-88[Medline]

Rapior S, Andary C, Mousain D., 1987 Cortinarius section Orellani: isolation and culture of Cortinarius orellanus. Trans Brit Mycol Soc 89:41-44

Richard JM, Cantin-Esnault D, Dumont C, Benoit-Guyod JL., 1991 Détermination des pK et des équilibres acido-basique d’une toxine d’origine fongique, l’orellanine. Analusis 19:236-243

Richard JL, Plattner RD, May J, Liska SL., 1999 The occurrence of ochratoxin A in dust collected from a problem household. Mycopathol 146:99-103

Schumacher T, Høiland K., 1983 Mushroom poisoning caused by species of the genus Cortinarius Fries. Arch Toxicol 53:87-106[Medline]

Skaug MA, Eduard W, Størmer FC., 2001 Ochratoxin A in airborne dust and fungal conidia. Mycopathol 151:93-98

Sorenson WG, Frazer DG, Jarvis BB, Simpson J, Robinson VA., 1987 Trichothecene mycotoxins in aerosolized conidia of Stachybotrys atra. Appl Environ Microbiol 53:1370-1375[Abstract/Free Full Text]

Stradler M, Sterner O., 1998 Production of bioactive secondary metabolites in the fruit bodies of macrofungi as a response to injury. Phytochemistry 49:1013-1019

Størmer FC, Høiby EA., 1996 Citrinin, ochratoxin A and iron. Possible implications for their biological function and induction of nephropathy. Mycopathol 134:103-107

———, Sandven P, Huitfeldt HS, Eduard W, Skogstad A., 1998 Does the mycotoxin citrinin function as a sun protectant in conidia from Penicillium verrucosum?. Mycopathol 142:43-47

Wicklow DT, Shotwell OL., 1983 Intrafungal distribution of aflatoxins among conidia. Can J Microbiol 29:1-5[Medline]





This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Services
Right arrow Similar articles in this journal
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Koller, G. EB.
Right arrow Articles by Størmer, F. C.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Koller, G. EB.
Right arrow Articles by Størmer, F. C.
Agricola
Right arrow Articles by Koller, G. EB.
Right arrow Articles by Størmer, F. C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS