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Flavipin production by Epicoccum spp.
[ 354 ] Trans. Brit. mycol. Soc. 44 (3), 354-356 (1961).
FLAVIPIN PRODUCTION BY EPICOCCUM SPP. By P. C. BAMFORD, G. L. F. NORRIS AND G. WARD Imperial Chemical Industries Ltd., Akers Research Laboratories, Welwyn, Herts. An antibiotic produced by Epicoccum nigrum Link and E. andropogonis (Ces.) Schol-Schwarz has been isolated and shown to be flavipin (3,4,5-trihydroxy6-methylphthalaldehyde), a known metabolite of Aspergillus flavipes and A. terreus. It is an antifungal substance, only stable in aqueous solution at low pH.
Campbell (1956) has shown that when Epicoccum purpurascens and Helminthosporium sativum were grown side by side on agar media, the former fungus produced a diffusible substance which restricted hyphal growth of H. sativum. We have confirmed the production of an antifungal antibiotic by E. nigrum (= E. purpurascens) and by E. andropogonis. This has been isolated and shown to be identical with flavipin (3,4,5-trihydroxy-6methylphthalaldehyde) previously obtained from Aspergillus flavipes and A. terreus by Raistrick & Rudman (1956). MATERIALS AND METHODS Methods of surface culture and assay of antibiotic activity have been described previously (Curtis, Hemming & Unwin, 1951). Stirred cultures were grown in conventional glass and stainless steel fermenters containing 61. medium, stirred at 700 r.p.m. and with an air flow of 3 l.fmin. All cultures were grown at 25° C. The media used were Czapek-Dox and Raulin-Thom (Brian, Curtis & Hemming, 1946) and medium T, containing Bacto-Tryptone as nitrogen source (Brian, Curtis & Hemming, 1947). Difco yeast extract (0'01 %) was added to all media. Pea-stem section extension and cress-root growth tests have also been described elsewhere (Brian & Hemming, 1958; Brian, Radley & Hemming, 1955). The fungi used included three received from the Commonwealth Mycological Institute, Kew, as E. purpurascens (CM!. 45171, 61336 and 68797), four received from the Centraalbureau voor Schimmelcultures, Baarn, as E. nigrum ('Portugal C', 'Mulder', 724 and 'E. neglectum') and four more, from the same source, as E. andropogonis. RESULTS Preliminary experiments Cultures were grown on 30 m1. lots of Czapek-Dox and Raulin-Thom media in 100 m1. conical flasks and assayed for antifungal activity at intervals for a total of 28 days. All grew vigorously on both media producing dark-red, reddish brown or orange-brown culture filtrates. The filtrates were noticeably viscous in most cases. They were in all cases antifungal, particularly on Raulin-Thorn, the most active filtrates being
Flavipin production. P. C. Bamford et aI.
355
produced by E. purpurascens (CMI, 68797). On Raulin-Thom medium this strain produced filtrates inhibiting germination of conidia of Botrytis allii at dilutions of I in 32. Two strains of E. nigrum received from Baarn (their no. 724 and' E. neglectum') were only slightly less active. All strains grew very prolifically in stirred culture, the cultures being highly pigmented (orange-brown) and porridge-like in consistency. They were only filterable with difficulty and filtrates commonly set to a gel on standing. Production of antifungal material was also greater under these conditions; E. purpurascens (ACC. 1336), when grown on medium T with 10 % glucose, produced filtrates inhibiting Botrytis allii at I in 512. Two strains were then selected for further study: E. purpurascens CMI. 68797 (ACC. 1336 in our culture collection) and one of the strains of E. andropogonis received from Baarn (ACC. 1334 in our collection). Because of the difficulty experienced in filtering stirred cultures, surface cultures were used in most work on the isolation of the active material, and in further studies on medium composition it was found that, for ACC. 1336, medium T with 5 %glucose (' Dextrosol', Corn Products Ltd.) was most productive, and, for ACC. 1334, Czapek-Dox or Raulin-Thom with 5 % glucose ('Dextrolact') was best.
Isolation of active material The active principle was extracted from the surface culture fluids of both E. purpurascens (ACC. 1336) and E. andropogonis (ACC. 1334). After removal of the mycelium, the culture filtrate was adjusted to pH 2'0 and extracted with charcoal (15 g.jl.), The charcoal was removed by centrifuging, air dried at room temperature and extracted with acetone. In a typical batch from ACC. 1336 on medium T, culture filtrate (421.) was extracted with charcoal (630 g.), Elution with acetone (500 m1.) gave at first an inactive dark solid (26'1 g.). Further elution with acetone (21.) removed the active compound, which separated as a yellow crystalline solid (33'2 g.). A further amount ofless pure active compound (II'3 g.) was obtained by concentration of the eluates. The active compound crystallized from aqueous dioxan in yellow needles, m.p. 228-229° (dec.). (Found: C, 55'1; H, 4'2; C-Me, 5,8 %. Calc. for CgHsOs, C, 55'1; H, 4'1; C-Me, 7'1 %.) The literature m.p. for flavipin is 233-234° (dec.) (Raistrick & Rudman, 1956). The molecular weight determination (crystals from acetone) showed space group Fdd s and unit cell dimensions of a = 10'37, b = 10'56; c = 3°'76 A, The crystals had d 1'56 g.kx., giving cell weight 3184'7 and molecular weight 199'0 ± 2 % (16 molecules per unit cell). (Calc. for CgHsOS' M, 196'2.) The penta-acetate hydrate and trimethoxyphthalide were both prepared as described (Raistrick & Rudman, 1956) and found to be identical with authentic specimens by mixed m.p. and comparison of the infrared spectra. Biological activiry offlavipin At pH 3'5, flavipin almost completely prevented germination of B. allii conidia at 12'5 f£g./m1.; this is in agreement with the observations of 23-2
356
Transactions British Mycological Society
Raistrick & Rudman (1956). Almost all other fungi tested were less sensitive to flavipin. At pH 3'5, aqueous solutions retained their antifungal activity for 22 days; at pH 4'5 or above, antifungal activity was rapidly lost. This instability may account for its negligible antibacterial activity in vitro in media at pH 7'0. Flavipin reduces the extension of pea stem sections at 100 /kg. 1m!. but not at IO /kg. 1m!. It reduces cress root growth severely at IOO /kg. 1m!. but scarcely at all at 10 /kg. 1m!. It has no effect on the growth of pea seedlings when sprayed on, at 100 /kg. 1m!. Thus it is actively antifungal but only slightly phytotoxic. DISCUSSION
Many species of Epicoccum have been described, but recent opinion favours the view that most of them really fall into the single species, E. nigrum Link (Ellis, 1956; Schol-Schwartz, 1959). Thus of our cultures, those received as E. purpurascens and E. nigrum all represent this one species. Recently, Schol-Schwarz (1959) has transferred the species previously known as Cerebella andropogonis Ces. to Epicoccum, as E. andropogonis (Ces.) Schol-Schwarz. It is interesting to note that we have found that this species, like E. nigrum, produces flavipin, confirming their close relationship. In surface culture one of our organisms (ACC. 1336) produced three times the best yield of flavipin obtained by Raistrick & Rudman (1956) from Aspergillus spp. We are indebted to Prof. J. H. Birkinshaw and Dr C. E. Stickings, London School of Hygiene and Tropical Medicine, for making available authentic specimens offlavipin as prepared by Prof. H. Raistrick, F.R.S., and Dr P. Rudman; to Dr P. G. Owston for the X-ray data; and to several colleagues for technical assistance. REFERENCES BRIAN, P. W., CURTIS, P.]. & HEMMING, H. G. (1946). A substance causing abnormal development of fungal hyphae produced by Penicillium [anczeioskii Zal. Trans. Brit. mycol. Soc. 29, 173-187. BRIAN, P. W., CURTIS, P.]. & HEMMING, H. G. (1947). Glutinosin: a fungistatic metabolic product of the mould Metarrhizium glutinosum S. Pope. Proc. Roy. Soc. B, 135, 106- 1 32• BRIAN, P. W. & HEMMING, H. G. (1958). Complementary action of gibberellic acid and auxins in pea internode extension. Ann. Bot., Lond., 22, 1-17. BRIAN, P. W., RADLEY, M. & HEMMING, H. G. (1955). A physiological comparison of gibberellic acid with some auxins. Physiol. Plant. 8, 899-912. CAMPBELL, W. P. (1956). The influence ofassociated microorganisms on the pathogenicity of Helminihosporium sativum. Ganad.]. Bot. 34, 865-874. CURTIS, P.]., HEMMING, H. G. & UNWIN, C. H. (1951). Albidin, an antibiotic red pigment from Penicillium albidum. Trans. Brit. mycol. Soc. 34, 332-339. ELLIS,].]. (1956). A note on Epicoccum. Proc. Iowa Acad. Sci. 63, 3°7-310. RAISTRICK, H. & RUDMAN, P. (1956). Studies in the biochemistry of micro-organisms. 97. Flavipin, a crystalline metabolite of Aspergillusflavipes and A. terreus. Biochem.]. 63, 395-406. SCHOL-SCHWARZ, M. B. (1959). The genus Epicoccum Link. Trans. Brit. mycol. Soc. 42, 149- 173.
(Accepted for publication
I
October
1960)
FLAVIPIN PRODUCTION BY EPICOCCUM SPP. By P. C. BAMFORD, G. L. F. NORRIS AND G. WARD Imperial Chemical Industries Ltd., Akers Research Laboratories, Welwyn, Herts. An antibiotic produced by Epicoccum nigrum Link and E. andropogonis (Ces.) Schol-Schwarz has been isolated and shown to be flavipin (3,4,5-trihydroxy6-methylphthalaldehyde), a known metabolite of Aspergillus flavipes and A. terreus. It is an antifungal substance, only stable in aqueous solution at low pH.
Campbell (1956) has shown that when Epicoccum purpurascens and Helminthosporium sativum were grown side by side on agar media, the former fungus produced a diffusible substance which restricted hyphal growth of H. sativum. We have confirmed the production of an antifungal antibiotic by E. nigrum (= E. purpurascens) and by E. andropogonis. This has been isolated and shown to be identical with flavipin (3,4,5-trihydroxy-6methylphthalaldehyde) previously obtained from Aspergillus flavipes and A. terreus by Raistrick & Rudman (1956). MATERIALS AND METHODS Methods of surface culture and assay of antibiotic activity have been described previously (Curtis, Hemming & Unwin, 1951). Stirred cultures were grown in conventional glass and stainless steel fermenters containing 61. medium, stirred at 700 r.p.m. and with an air flow of 3 l.fmin. All cultures were grown at 25° C. The media used were Czapek-Dox and Raulin-Thom (Brian, Curtis & Hemming, 1946) and medium T, containing Bacto-Tryptone as nitrogen source (Brian, Curtis & Hemming, 1947). Difco yeast extract (0'01 %) was added to all media. Pea-stem section extension and cress-root growth tests have also been described elsewhere (Brian & Hemming, 1958; Brian, Radley & Hemming, 1955). The fungi used included three received from the Commonwealth Mycological Institute, Kew, as E. purpurascens (CM!. 45171, 61336 and 68797), four received from the Centraalbureau voor Schimmelcultures, Baarn, as E. nigrum ('Portugal C', 'Mulder', 724 and 'E. neglectum') and four more, from the same source, as E. andropogonis. RESULTS Preliminary experiments Cultures were grown on 30 m1. lots of Czapek-Dox and Raulin-Thom media in 100 m1. conical flasks and assayed for antifungal activity at intervals for a total of 28 days. All grew vigorously on both media producing dark-red, reddish brown or orange-brown culture filtrates. The filtrates were noticeably viscous in most cases. They were in all cases antifungal, particularly on Raulin-Thorn, the most active filtrates being
Flavipin production. P. C. Bamford et aI.
355
produced by E. purpurascens (CMI, 68797). On Raulin-Thom medium this strain produced filtrates inhibiting germination of conidia of Botrytis allii at dilutions of I in 32. Two strains of E. nigrum received from Baarn (their no. 724 and' E. neglectum') were only slightly less active. All strains grew very prolifically in stirred culture, the cultures being highly pigmented (orange-brown) and porridge-like in consistency. They were only filterable with difficulty and filtrates commonly set to a gel on standing. Production of antifungal material was also greater under these conditions; E. purpurascens (ACC. 1336), when grown on medium T with 10 % glucose, produced filtrates inhibiting Botrytis allii at I in 512. Two strains were then selected for further study: E. purpurascens CMI. 68797 (ACC. 1336 in our culture collection) and one of the strains of E. andropogonis received from Baarn (ACC. 1334 in our collection). Because of the difficulty experienced in filtering stirred cultures, surface cultures were used in most work on the isolation of the active material, and in further studies on medium composition it was found that, for ACC. 1336, medium T with 5 %glucose (' Dextrosol', Corn Products Ltd.) was most productive, and, for ACC. 1334, Czapek-Dox or Raulin-Thom with 5 % glucose ('Dextrolact') was best.
Isolation of active material The active principle was extracted from the surface culture fluids of both E. purpurascens (ACC. 1336) and E. andropogonis (ACC. 1334). After removal of the mycelium, the culture filtrate was adjusted to pH 2'0 and extracted with charcoal (15 g.jl.), The charcoal was removed by centrifuging, air dried at room temperature and extracted with acetone. In a typical batch from ACC. 1336 on medium T, culture filtrate (421.) was extracted with charcoal (630 g.), Elution with acetone (500 m1.) gave at first an inactive dark solid (26'1 g.). Further elution with acetone (21.) removed the active compound, which separated as a yellow crystalline solid (33'2 g.). A further amount ofless pure active compound (II'3 g.) was obtained by concentration of the eluates. The active compound crystallized from aqueous dioxan in yellow needles, m.p. 228-229° (dec.). (Found: C, 55'1; H, 4'2; C-Me, 5,8 %. Calc. for CgHsOs, C, 55'1; H, 4'1; C-Me, 7'1 %.) The literature m.p. for flavipin is 233-234° (dec.) (Raistrick & Rudman, 1956). The molecular weight determination (crystals from acetone) showed space group Fdd s and unit cell dimensions of a = 10'37, b = 10'56; c = 3°'76 A, The crystals had d 1'56 g.kx., giving cell weight 3184'7 and molecular weight 199'0 ± 2 % (16 molecules per unit cell). (Calc. for CgHsOS' M, 196'2.) The penta-acetate hydrate and trimethoxyphthalide were both prepared as described (Raistrick & Rudman, 1956) and found to be identical with authentic specimens by mixed m.p. and comparison of the infrared spectra. Biological activiry offlavipin At pH 3'5, flavipin almost completely prevented germination of B. allii conidia at 12'5 f£g./m1.; this is in agreement with the observations of 23-2
356
Transactions British Mycological Society
Raistrick & Rudman (1956). Almost all other fungi tested were less sensitive to flavipin. At pH 3'5, aqueous solutions retained their antifungal activity for 22 days; at pH 4'5 or above, antifungal activity was rapidly lost. This instability may account for its negligible antibacterial activity in vitro in media at pH 7'0. Flavipin reduces the extension of pea stem sections at 100 /kg. 1m!. but not at IO /kg. 1m!. It reduces cress root growth severely at IOO /kg. 1m!. but scarcely at all at 10 /kg. 1m!. It has no effect on the growth of pea seedlings when sprayed on, at 100 /kg. 1m!. Thus it is actively antifungal but only slightly phytotoxic. DISCUSSION
Many species of Epicoccum have been described, but recent opinion favours the view that most of them really fall into the single species, E. nigrum Link (Ellis, 1956; Schol-Schwartz, 1959). Thus of our cultures, those received as E. purpurascens and E. nigrum all represent this one species. Recently, Schol-Schwarz (1959) has transferred the species previously known as Cerebella andropogonis Ces. to Epicoccum, as E. andropogonis (Ces.) Schol-Schwarz. It is interesting to note that we have found that this species, like E. nigrum, produces flavipin, confirming their close relationship. In surface culture one of our organisms (ACC. 1336) produced three times the best yield of flavipin obtained by Raistrick & Rudman (1956) from Aspergillus spp. We are indebted to Prof. J. H. Birkinshaw and Dr C. E. Stickings, London School of Hygiene and Tropical Medicine, for making available authentic specimens offlavipin as prepared by Prof. H. Raistrick, F.R.S., and Dr P. Rudman; to Dr P. G. Owston for the X-ray data; and to several colleagues for technical assistance. REFERENCES BRIAN, P. W., CURTIS, P.]. & HEMMING, H. G. (1946). A substance causing abnormal development of fungal hyphae produced by Penicillium [anczeioskii Zal. Trans. Brit. mycol. Soc. 29, 173-187. BRIAN, P. W., CURTIS, P.]. & HEMMING, H. G. (1947). Glutinosin: a fungistatic metabolic product of the mould Metarrhizium glutinosum S. Pope. Proc. Roy. Soc. B, 135, 106- 1 32• BRIAN, P. W. & HEMMING, H. G. (1958). Complementary action of gibberellic acid and auxins in pea internode extension. Ann. Bot., Lond., 22, 1-17. BRIAN, P. W., RADLEY, M. & HEMMING, H. G. (1955). A physiological comparison of gibberellic acid with some auxins. Physiol. Plant. 8, 899-912. CAMPBELL, W. P. (1956). The influence ofassociated microorganisms on the pathogenicity of Helminihosporium sativum. Ganad.]. Bot. 34, 865-874. CURTIS, P.]., HEMMING, H. G. & UNWIN, C. H. (1951). Albidin, an antibiotic red pigment from Penicillium albidum. Trans. Brit. mycol. Soc. 34, 332-339. ELLIS,].]. (1956). A note on Epicoccum. Proc. Iowa Acad. Sci. 63, 3°7-310. RAISTRICK, H. & RUDMAN, P. (1956). Studies in the biochemistry of micro-organisms. 97. Flavipin, a crystalline metabolite of Aspergillusflavipes and A. terreus. Biochem.]. 63, 395-406. SCHOL-SCHWARZ, M. B. (1959). The genus Epicoccum Link. Trans. Brit. mycol. Soc. 42, 149- 173.
(Accepted for publication
I
October
1960)