Extracts of Cortinarius sanguineus (Wulf: Fr.) S.F. Gray - containing anthraquinones - and C. rubellus Cooke - containing orellanine - have been examined for their ability to suppress the toxicity of aluminium ions by a bioassay experiment using the saprotrophic basidiomycete Mycena septentrionalis Maas G. as the test organism. The aluminium concentrations used were: 0, 0.5, 0.5e (1.36), 0.5e▓ (3.69), 0.5e^3 (10.04), and 0.5e^4 (27.30) mM Al. All were given as Al sulphate, with or without the extract of Cortinarius sanguineus or C. rubellus. The extract of C. sanguineus suppressed the toxic effect of Al up to 0.5e^3 mM. The C. rubellus extract inhibited growth of the test organism up to 0.5e mM Al and suppressed the toxic effect of Al up to 0.5e^3 mM Al. Mycena septentrionalis grew rapidly in 0.5e▓ and 0.5e^3 mM Al. Chromatographical investigations indicated that Al was bound to anthraquinone-glycosides, but not to anthraquinone aglycones in the C. sanguineus extract. Corresponding procedures for the C. rubellus extract revealed that Al was bound to orellanine (and orelline).
The bioassey shows that both Cortinarius sanguineus and C. rubellus contain substances which are able to suppress the toxic effects of soluble aluminium. The observed increase of these two species in acidified forests may thus be given a plausible explanation. In this respect it is worth noting that both species are generally found in soils with pH ranging from 3.5-4. At these values aluminium has been reported to dissolve into soil-water.
The pH measurements indicate that the extracts supress the release of aluminium ions up to 0.5e^3 mM total concentration. The major part of aluminium is probably bound to organic molecules in these solutions. The growth of the test organism, therefore, continues up to 0.5e^3 mM, but is retarded at 0.5e4 mM where the level of free aluminium ions may equal those in media without extracts.
Possible detoxification pathways
The chromatographical results show that aluminium may bind to anthraquinone-glycosides, but not to the anthraquinone aglycones. The suggestion that ligand-forming groups are established solely at the anthraquinone molecules therefore gains no support. Possible ligands able to bind aluminium may be a combination of hydroxy-groups at the anthraquinone and at the sugar moieties. From the chromatograms it seems that dermocybin-1-▀-D-glucopyranoside has the strongest ability to bind aluminium. This may be because the dermocybin moiety has a higher number of ligandforming hydroxy-groups than the corresponding emodin and dermoglaucin moieties.
Judged from the growth measurements, I may conclude that neither the anthraquinones themselves nor the anthraquinoneglycoside/aluminium complexes have any toxic effect on fungi.
In the tentative orellanine/aluminium complex the metal is probably bound to one of the negatively charged oxygen atoms (at position 1) and its neighbouring hydroxy-group (at position 3') making a bidentate ligand at the dipyridine structure. Since the orellanine molecule possesses two identical bidentate ligands, it has the capacity to bind two aluminium atoms. Another possible orellanine/aluminium complex is a polymer where the metal is bound to the negatively charged oxygen atom at position 1 in the first orellanine molecule and to the corresponding oxygen atom at position 1' in the next molecule, giving rise to a repeated chain forming process. In the suggested polymer complex, each orellanine structure binds only one aluminium atom. Therefore it may evolve at moderate aluminium levels. At higher levels, the chelate (with two aluminium atoms) is probably formed. This may explain the strong turbidity phenomenon in the medium with 0.5e^3 mM aluminium, but not in the medium with 0.5e^4 mM. Excess aluminium ions may break the polymer chain into single orellanine/aluminium chelate molecules with two aluminium atoms, thus bringing the sediment into solution.
Judged from the growth measurements, I may conclude that orellanine is toxic to other fungi, but that the orellanine/aluminium complexes are nontoxic. This may be the reason why the test organism only grew in the moderately strong aluminium concentrations under the influence of the extract. The toxicity of the orellanine molecule is probably due to a special molecular configuration with a negatively charged nitrogen and a positively charged oxygen atom at each pyridine-ring. A disruption of this electrochemical balance by aluminium ions may be the reason for the nontoxic property of the complex molecule - it may have either a chelate or a polymer structure.
Other substances, e.g. polyols, sugars, amino acids, phenolic acids, and steroids, in the extracts may also form aluminium complexes. However, since all the other spots on the chromatograms remained unchanged after the treatment with aluminium sulphate, the main complexing effect may best be ascribed to the anthraquinone-glycosides or orellanine (and orelline).
In ectomycorrhizal systems soluble aluminium binds phosphate and induces phosphorus deficiency by precipitation in the roots, interferes with phosphorus metabolism, binds to the polar region of the phospholipids, and inhibits the conversion of orthophosphate to polyphosphate. In phosphorus-poor soils increased levels of soluble aluminium may be harmful to mycorrhizal fungi. Only species which are able to detoxify aluminium may function normally and outcompete less tolerant species over time. In mycorrhizal organs or outer mycelia with anthraquinone-glycosides or orellanine, aluminium may be bound either as soluble complexes or insoluble polymers. The soluble complexes are mobile and are possibly translocated to the basidiocarp primordia. By the maturing process the metal complexes may be directed into the vacuolar sap of the inflating hyphae making up the trama. Basidiocarps are terminal structures which in this way may act as repository organs for toxic metals. If these suggestions are valid, basidiocarps may withdraw, accumulate, and detoxify aluminium, and possibly other toxic metals taken up by tolerant ectomycorrhizal fungi.
Top: Structure of orellanine (after Antkowiak & Gessner). Bottom: Structure of a possible orellanine/mono-aluminium-polymer.
H°iland, K. 1994. Suppression of the toxic effect of soluble aluminium on fungi by dermocybin-1-▀-D-glucopyranoside and orellanine from Cortinarius sanguineus and C. orellanoides. - Nord. J. Bot. 14: 221-228.