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Pigment production by an isolate of epicoccum nigrum
145 2. The second method was basically the same except that glass Petri dishes were employed. The dishes were taped to the cylinders before sterilization and the whole autoclaved, thus reducing the risk of contamination. Liquid medium was poured into the dish through the cylinder. Care was required not to splash medium on the cylinder walls and the temperature must not be too high on pouring or condensation will form within the cylinder. Should a spore harvest be required with either of these methods, the fruit bodies are left until the caps begin to darken, indicating spore maturation and the SUbsequent onset of autolysis. The caps are then aseptically removed from their stipes using a pair of forceps and placed in a sterile Petri dish. As autolysis proceeds, spores are liberated into the dish leaving spore prints. These can be stored dry for several months until required. Quantitative viability tests have not been carried out but spores from prints up to 12 months' old have been germinated successfully. For immediate use, 10 cm 3 of sterile distilled water or sterile quarter-strength Ringers solution can be added to the dish, and the spores stirred into this. The remains of the gills adhering to the base of the Petri dish are usually easily removed in one piece. If however, any gill material should remain the suspension can be filtered through sterile glass wool. This technique requires only basic materials which should be available in most laboratories. It is simpler, cleaner and more efficient than methods employing dung and narrow necked bottles, and has been used to provide regular supplies of material for use in our laboratories over a number of years. References ANDERSON, C.E. (1971). The Life History and Genetics ofCoprinus lagopus. Harris Biological Supplies. KEMP, R.F.O., & WATLING, R. (1977). Coprinus Workshop. Bulletin of the British Mycological Society. 11,24-27.
PIGMENT PRODUCTION BY AN ISOLATE OF EPICOCCUM NIGRUM Hugo J. Fletcher & Y.S. Kwong School of Pharmacy, Robert Gordon's Institute of Technology, Aberdeen AB9 1FR An organism which produces dark red mycelium and yellow diffusate into the medium which was erroneously the subject of a poster at the 'Fusarium Meeting' (Fletcher, 1983) has since been identified as Epicoccum nigrum Link by members of the C.M.I. staff. Epicoccum has been known for over seventy years to produce a wide range of pigments. These have been reviewed by Burge et al. (1976) and by Stricker et al, (1981). The latter authors suggested that a 'new' yellow substance isolated by them could be suitable for colouring food. Amongst the pigments isolated by Burge et al, (1976) were two with antibiotic properties, latterly named epirodins by Ikawa et al. (1978). The chemistry of both of these pigments has still to be elucidated fully. Our isolate (lMI 271436) from rose twigs in Aberdeen possessed characteristics that justified further investigation. On potato dextrose (C.M.I. 1980), Sabouraud maltose, and Czapek-Dox agars it produced a dark-red pigment in the mycelium when viewed from the back of the plate. When plugs of the
146 red mycelium were plated on to Czapek-Dox agar, an intense yellow pigment diffused into the agar before the growth of the mould was fully established. On potato dextrose agar the yellow exudate followed the development of a colony containing red mycelium. In Czapek-Dox broth a yellow pigment is similarly produced early in the culture period, but the colour usually fades to be replaced by further yellow-orange materials after a few weeks. Cultures were grown on the colourless Czapek-Dox broth for about four weeks to investigate this phenomenon. A spectrum was obtained from the newly-harvested culture filtrate that was similar to that of the subsequent yellow extracts in various solvents at pH 8. This had a peak between 428430 nm and a 'shoulder' at 450 nm. At pH <2, two distinct spectra were obtained depending on solvent. The culture medium and other aqueous (orange) solutions had one distinct peak between 395-398 nm. In ethanol at this pH there was a peak at 436 with 'shoulders' both at 410 and 460 nm. Both epirodins and 'Stricker's pigment' have a peak at 429 nm at neutral and alkaline pH. Epirodins have a peak of 390 nm at pH 2, but are only slightly soluble in aqueous acid. Acetylated epirodin in cyclohexane has a peak at 437 nm with shoulders at 417 and 465 nm (Ikawa et al.., 1978). Thin layer chromatography also confirmed the presence of at least two pigments in the culture filtrate with different properties as regard to stability and fluorescence. The original culture filtrate and the ethanolic fraction were both inhibitory to the germination of the spores of Pilaira caucasica Milko, whereas the aqueous fraction had no effect. We therefore suggest that there is evidence of two distinct 'water-soluble' pigments in the culture filtrate, one of which has antibiotic properties not unlike the epirodins. Cultures were grown on PDA for about two weeks to compare extracts of the yellow exudate with that of the red mycelium. A spectrum was obtained of the yellow solutions from the agar and mycelium at pH 8 similar to those for the broths above. As with them, differences occurred at pH <2, with the spectrum for the aqueous solvent system differing from that for the ethanolic. Examination of the red hyphae under the microscope before and after extraction demonstrated that the pigment had been effectively removed by the process. Thin layer chromatography confirmed in this case that there was only a single stable pigment in the aqueous extracts from both the agar and mycelium, which was different from the unstable material in ethanol. We therefore conclude that there are two different pigments in the mycelium and diffusate and suggest that the ethanolic solution contains the epirodin and the more hydrophilic one is Stricker's pigment. Moreover it is the leaching of this pigment from the red mycelium that is so marked on neutral media such as Czapek-Dox, Finally we should point out that the isolates of Epicoccum nigrum are notoriously variable (Schol-Schwarz, 1959) and that also an entirely different source of red-yellow colouration is the presence of a range of carotenoids, including rhodoxanthin (Foppen & Gribanovski-Sassu, 1968), the cause of the red hue in the arils of Taxus baccata. Our isolate only produced minute amounts of carotenoids under the conditions used. We wish to thank Dr D.G. Durham for his helpful advice and Mrs M.T. George for technical assistance. References BURGE, W.R., BUCKLEY, L.J., SULLIVAN, J.D. Jr., McGRATTAN, C.L & IKAWA, M. (1976). Isolation and biological activity of the pigments of the mold Epicoccum nigrum. Journal ofAgricultural and Food Chemistry. 24,555-559. C.M.I. (1980) Descriptions of pathogenic fungi and bacteria No. 680 Epicoccum nigrum. FLETCHER, H.J. (1983) in The Applied Mycology of Fusarium. Bulletin of the British Mycological Society. 17, 10-15.
147 FOPPEN, F.H. & GRIBANOVSKI-SASSU, O. (1968). Lipids produced by Epicoccum nigrum in submerged culture. Biochemical Journal. 106,97-100. IKAWA, M., McGRATTAN, C.L, BURGE, W.R., IANNITELLI, R.C., UEBEL, J.J. & NOGUCHI, T. (1978). The Journal of Antibiotics. 31, 159-161. SCHOL-SCHWARZ, M.B. (1959). The genus Epicoccum Link. Transactions of the British Mycological Society. 42, 149-173. STRICKER, R., ROMAILLER, G., TURIAN, G. & TZANOS, D. (1981). Production et applications alimentaires d'un pigment jaune hydrosoluble d'origine fongique (Epicoccum nigrum Link). Lebensmittel Wissenschaftund Technologie. 14, 18-20.
QUEST FOR AN ENDOTHIA S.L. Anagnostakis The Connecticut Agricultural Experiment Station, Box 1106, New Haven, Connecticut 06504, U.S.A. On April 15, 1809, C. Lyell collected a fungus from the bark of Spanish chestnut (Castanea sativa) in the New Forest. Sowerby illustrated it in 1814 and called it Spheria fluens (Sowerby, 1814). Italian, Swiss, and American isolates of this fungus were compared with each other and with other members of the genus by Shear, Stevens & Tiller (1917). They reported that this species was abundant in the United States on dead American chestnut bark. In the northern limits of its range it was also found on the bark of exposed oak roots. This mild mannered fungus is now known as Endothia radicalis (Schw.) Ces. and DeNot, or Cryphonectria radicalis (Schw. ex Fries) Barr. I am interested in it because of its reported similarity to the chestnut blight pathogen, Endothia parasitica (Murr.) And. ", .. E. fluens and E. [luens mississippiensis, which resemble E. parasitica so closely in their morphological characteristics, and to a less degree on culture media, are almost purely saprophytic." (Shear, Stevens & Tiller, 1917). A number of years ago I began asking American and Italian mycologists for recently isolated cultures of E. radicalis, but none have been found. This aroused my curiosity even more. E. parasitica has been in the U.S.A. since at least 1906 and in Italy since the late 1930's (Anagnostakis, 1982). It is possible that this aggressive pathogen has either hybridized with its saprophytic cousin or pushed it entirely out of its favored habitat. On a recent trip to England, I was fortunate to have the help of Dr. J.G. Manners in examining Spanish chestnut trees in the New Forest. One recently cut specimen yielded orange stromata which closely resembled E. parasitica, tucked into deep crevices of the bark. The tree was estimated by Peter Hillier to be about 100 years old. Bark samples were brought back to my laboratory at The Connecticut Agricultural Experiment Station (with a U.S. Plant Quarantine Office Permit) and placed in moist chambers. Isolations produced many interesting fungi, including several cultures which were apparently a Libertella sp.. However, none of the isolates fit the description of E. radicalis given by Shear, Stevens, & Tiller (1917). My failure to find my quarry leads me to ask the help of my British colleagues. Has anyone seen an Endothia radicalis ? References ANAGNOSTAKIS, S.L. (1982). Biological control of chestnut blight. Science, 215,466-471. SHEAR, C.L., STEVENS, N.E. & TILLER, R.J. (1917). Endothia parasitica and related species. United States Dept. of Agriculture Bulletin No. 380, Washington, D.C., 82pp. SOWERBY, J. (1814). Coloured figures of English Fungi or Mushrooms. London (Sup. plate 438 which is part of plate 420, figs. 1 & 2).
PIGMENT PRODUCTION BY AN ISOLATE OF EPICOCCUM NIGRUM Hugo J. Fletcher & Y.S. Kwong School of Pharmacy, Robert Gordon's Institute of Technology, Aberdeen AB9 1FR An organism which produces dark red mycelium and yellow diffusate into the medium which was erroneously the subject of a poster at the 'Fusarium Meeting' (Fletcher, 1983) has since been identified as Epicoccum nigrum Link by members of the C.M.I. staff. Epicoccum has been known for over seventy years to produce a wide range of pigments. These have been reviewed by Burge et al. (1976) and by Stricker et al, (1981). The latter authors suggested that a 'new' yellow substance isolated by them could be suitable for colouring food. Amongst the pigments isolated by Burge et al, (1976) were two with antibiotic properties, latterly named epirodins by Ikawa et al. (1978). The chemistry of both of these pigments has still to be elucidated fully. Our isolate (lMI 271436) from rose twigs in Aberdeen possessed characteristics that justified further investigation. On potato dextrose (C.M.I. 1980), Sabouraud maltose, and Czapek-Dox agars it produced a dark-red pigment in the mycelium when viewed from the back of the plate. When plugs of the
146 red mycelium were plated on to Czapek-Dox agar, an intense yellow pigment diffused into the agar before the growth of the mould was fully established. On potato dextrose agar the yellow exudate followed the development of a colony containing red mycelium. In Czapek-Dox broth a yellow pigment is similarly produced early in the culture period, but the colour usually fades to be replaced by further yellow-orange materials after a few weeks. Cultures were grown on the colourless Czapek-Dox broth for about four weeks to investigate this phenomenon. A spectrum was obtained from the newly-harvested culture filtrate that was similar to that of the subsequent yellow extracts in various solvents at pH 8. This had a peak between 428430 nm and a 'shoulder' at 450 nm. At pH <2, two distinct spectra were obtained depending on solvent. The culture medium and other aqueous (orange) solutions had one distinct peak between 395-398 nm. In ethanol at this pH there was a peak at 436 with 'shoulders' both at 410 and 460 nm. Both epirodins and 'Stricker's pigment' have a peak at 429 nm at neutral and alkaline pH. Epirodins have a peak of 390 nm at pH 2, but are only slightly soluble in aqueous acid. Acetylated epirodin in cyclohexane has a peak at 437 nm with shoulders at 417 and 465 nm (Ikawa et al.., 1978). Thin layer chromatography also confirmed the presence of at least two pigments in the culture filtrate with different properties as regard to stability and fluorescence. The original culture filtrate and the ethanolic fraction were both inhibitory to the germination of the spores of Pilaira caucasica Milko, whereas the aqueous fraction had no effect. We therefore suggest that there is evidence of two distinct 'water-soluble' pigments in the culture filtrate, one of which has antibiotic properties not unlike the epirodins. Cultures were grown on PDA for about two weeks to compare extracts of the yellow exudate with that of the red mycelium. A spectrum was obtained of the yellow solutions from the agar and mycelium at pH 8 similar to those for the broths above. As with them, differences occurred at pH <2, with the spectrum for the aqueous solvent system differing from that for the ethanolic. Examination of the red hyphae under the microscope before and after extraction demonstrated that the pigment had been effectively removed by the process. Thin layer chromatography confirmed in this case that there was only a single stable pigment in the aqueous extracts from both the agar and mycelium, which was different from the unstable material in ethanol. We therefore conclude that there are two different pigments in the mycelium and diffusate and suggest that the ethanolic solution contains the epirodin and the more hydrophilic one is Stricker's pigment. Moreover it is the leaching of this pigment from the red mycelium that is so marked on neutral media such as Czapek-Dox, Finally we should point out that the isolates of Epicoccum nigrum are notoriously variable (Schol-Schwarz, 1959) and that also an entirely different source of red-yellow colouration is the presence of a range of carotenoids, including rhodoxanthin (Foppen & Gribanovski-Sassu, 1968), the cause of the red hue in the arils of Taxus baccata. Our isolate only produced minute amounts of carotenoids under the conditions used. We wish to thank Dr D.G. Durham for his helpful advice and Mrs M.T. George for technical assistance. References BURGE, W.R., BUCKLEY, L.J., SULLIVAN, J.D. Jr., McGRATTAN, C.L & IKAWA, M. (1976). Isolation and biological activity of the pigments of the mold Epicoccum nigrum. Journal ofAgricultural and Food Chemistry. 24,555-559. C.M.I. (1980) Descriptions of pathogenic fungi and bacteria No. 680 Epicoccum nigrum. FLETCHER, H.J. (1983) in The Applied Mycology of Fusarium. Bulletin of the British Mycological Society. 17, 10-15.
147 FOPPEN, F.H. & GRIBANOVSKI-SASSU, O. (1968). Lipids produced by Epicoccum nigrum in submerged culture. Biochemical Journal. 106,97-100. IKAWA, M., McGRATTAN, C.L, BURGE, W.R., IANNITELLI, R.C., UEBEL, J.J. & NOGUCHI, T. (1978). The Journal of Antibiotics. 31, 159-161. SCHOL-SCHWARZ, M.B. (1959). The genus Epicoccum Link. Transactions of the British Mycological Society. 42, 149-173. STRICKER, R., ROMAILLER, G., TURIAN, G. & TZANOS, D. (1981). Production et applications alimentaires d'un pigment jaune hydrosoluble d'origine fongique (Epicoccum nigrum Link). Lebensmittel Wissenschaftund Technologie. 14, 18-20.
QUEST FOR AN ENDOTHIA S.L. Anagnostakis The Connecticut Agricultural Experiment Station, Box 1106, New Haven, Connecticut 06504, U.S.A. On April 15, 1809, C. Lyell collected a fungus from the bark of Spanish chestnut (Castanea sativa) in the New Forest. Sowerby illustrated it in 1814 and called it Spheria fluens (Sowerby, 1814). Italian, Swiss, and American isolates of this fungus were compared with each other and with other members of the genus by Shear, Stevens & Tiller (1917). They reported that this species was abundant in the United States on dead American chestnut bark. In the northern limits of its range it was also found on the bark of exposed oak roots. This mild mannered fungus is now known as Endothia radicalis (Schw.) Ces. and DeNot, or Cryphonectria radicalis (Schw. ex Fries) Barr. I am interested in it because of its reported similarity to the chestnut blight pathogen, Endothia parasitica (Murr.) And. ", .. E. fluens and E. [luens mississippiensis, which resemble E. parasitica so closely in their morphological characteristics, and to a less degree on culture media, are almost purely saprophytic." (Shear, Stevens & Tiller, 1917). A number of years ago I began asking American and Italian mycologists for recently isolated cultures of E. radicalis, but none have been found. This aroused my curiosity even more. E. parasitica has been in the U.S.A. since at least 1906 and in Italy since the late 1930's (Anagnostakis, 1982). It is possible that this aggressive pathogen has either hybridized with its saprophytic cousin or pushed it entirely out of its favored habitat. On a recent trip to England, I was fortunate to have the help of Dr. J.G. Manners in examining Spanish chestnut trees in the New Forest. One recently cut specimen yielded orange stromata which closely resembled E. parasitica, tucked into deep crevices of the bark. The tree was estimated by Peter Hillier to be about 100 years old. Bark samples were brought back to my laboratory at The Connecticut Agricultural Experiment Station (with a U.S. Plant Quarantine Office Permit) and placed in moist chambers. Isolations produced many interesting fungi, including several cultures which were apparently a Libertella sp.. However, none of the isolates fit the description of E. radicalis given by Shear, Stevens, & Tiller (1917). My failure to find my quarry leads me to ask the help of my British colleagues. Has anyone seen an Endothia radicalis ? References ANAGNOSTAKIS, S.L. (1982). Biological control of chestnut blight. Science, 215,466-471. SHEAR, C.L., STEVENS, N.E. & TILLER, R.J. (1917). Endothia parasitica and related species. United States Dept. of Agriculture Bulletin No. 380, Washington, D.C., 82pp. SOWERBY, J. (1814). Coloured figures of English Fungi or Mushrooms. London (Sup. plate 438 which is part of plate 420, figs. 1 & 2).