- Email: [email protected]
A Cercospora isolate from soybean roots produces cebetin B and cercosporin
0031 9422/93 $6.00+0.00 ,Q 1993 Pergamon Press Ltd
Vol. 33, No. 6, pp. 1546-1548, 1993 Pnnted IIIGreat Britain.
Phytochemistry,
A CERCOSPORA
ISOLATE FROM SOYBEAN ROOTS PRODUCES CEBETIN B AND CERCOSPORIN DAVID J. ROBESON and MAHBUBUL A. F. JALAL*
The Plant Cell Research
Institute,
Inc., 6560 Trinity
Court,
Dublin,
CA 94568, U.S.A.
(Received 3 September 1992)
Key Word Index-Cercospora; toxin; phototoxin.
Dematiaceae;
Glycine max; soybean; cercosporin;
cebetin; CBT; phyto-
Abstract-A Cercospora isolate (CE14) was isolated from roots of soybean seedlings germinated which appeared red, contained cercosporin which was isolated and quantified. In vitro cultures cebetin B, previously known as CBT (C. b&cola toxin).
in vitro. The roots, of CE14 produced
INTRODIJCTION
Collectively, members of the genus Cercospora are pathogenic to a very broad taxonomic range of plants spanning at least 43 families, including representatives of the Monocotyledonae, Dicotyledonae and Gymnospermae [l]. Many important crop plants are severely attacked by various Cercospora species [2, 31. Two morphologically distinct species, C. kikuchii and C. sojina are known to attack soybean [Glycine max] and to infect the seed [4]. Cercospora spp. attack the aerial parts of plants, typically causing necrotic leaf spots [2, 31. The production of the red perylenequinone derivative, cercosporin, is a characteristic feature of the genus [3,5, 63. This metabolite is well known as a photoactivated phytotoxin [3]. A second class of photoactivated phytotoxins, previously known as ‘CBT’ [7], has only recently been characterized and its two representatives assigned the trivial names of cebetin A and B [S]. In comparison with cercosporin, the known distribution of the cebetins is restricted to just two species [9]. Herein we report the in vitro production of cebetin B (1) by a Cercospora isolate from soybean seedlings, and demonstrate cercosporin production in soybean roots. RESULTS AND DISCUSSION
Soybean seedlings, the seeds of which had been surfacesterilized before germination on a dilute nutrient medium, showed signs of purple pigmentation, primarily on the seed coat and roots and, to a lesser extent, in the surrounding agar. Patches of fungal growth were present on the agar surface. Subsequently, necrotic lesions developed on stems and cotyledons and, in addition, many
*Author to whom correspondence should be address& Pan-Agricultural Labs, Inc.. 32380 Avenue 10, Madera, CA 93638, U.S.A.
of the roots appeared distinctly red. Pigmented root tissue gave a red/orange extract when homogenized in ethanol. The clarified extract exhibited UV/visible absorption virtually identical to that of cercosporin. TLC of the same extract gave a red pigment which co-chromatographed with cercosporin. The pigment from soybean roots was bright red on TLC and fluoresced orange under long wavelength UV, as observed for cercosporin. Mass spectral analysis of the red band confirmed its identity with cercosporin. No other pigments were detected on the chromatogram. The average concentration of cercosporin in the pigmented root tissue was 328 pg g-’ fr. wt. Plating of infected leaf and pigmented root tissue on potato dextrose agar (PDA) gave fungal colonies of uniform gross morphology. They appeared orange from below and were surrounded by a yellow diffusible pigment. Fungal colonies cultured on V8 agar or SH agar, in contrast, showed red or purple pigmentation. The fungal isolate from infected seedlings, designated CE14, was grown in potato dextrose broth (PDB). The yellow-green mycelium was extracted with methanol and
1546
Short Reports the extract was chromatographed with an authentic sample of cebetin B [S]. The major pigment in the extract co-migrated with cebetin B and had the same appearance in both visible light (bright yellow) and long wavelength UV (pale yellow fluorescence). The major yellow compound was purified by TLC for spectroscopic analysis. The identity of this metabolite with cebetin B was demonstrated by W/visible and ’ H NMR spectroscopy [S]. Microscopic examination of conidia of isolate CE14 confirmed that it was a Cercospora sp. Isolation of the fungus from roots, which contained relatively high concentrations of cercosporin, indicates that CE14 is capable of infecting soybean roots grown in vitro. Cercospora kikuchii and C. sojina are both known to attack seeds, pods, stems and leaves of soybean [4]. To date, however, infection of plant roots by a member of the genus Cercospora has not been reported. Cercosporin is a lightactivated phytotoxin which has been strongly implicated in causing disease [3, lo]. Whether Cercospora can infect roots in the field, under conditions where light is largely excluded from these organs, is open to investigation. From the scientific viewpoint, the susceptibility of roots to attack by Cercospora may be further examined using in oitro root cultures or hydroponically grown plants. The isolation of cercosporin from soybean seeds infected with C. kikuchii [S] and from host plants infected with other Cercospora spp. has been described previously [lo, 111. Cebetin B, like cercosporin, is also a photoactivated phytotoxin [S]. In contrast to cercosporin, the former metabolite was not detected in soybean root tissue, nor has it been described as a vivotoxin. The metalfree tautomer of cebetin B, known as cebetin A [8], was not encountered in our investigation of CE14. ‘CBT’ was reported previously from cultures of C. beticola and C. bertoreae during a survey of 61 Cercospora spp. for secondary metabolites [9]. Other than isolate CE14, Phoma musae as well as three randomly selected C. beticola isolates also gave indications of cebetin production. The production of cebetins by a wider range of Cercospora isolates is predictable in view of the apparent synonymy which exists in some common ‘species’ of Cercospora [12, 133. EXPERIMENTAL
Culture of soybean seedlings. Seeds were surface-sterilized with Cl, gas, and germinated on hormone-free SH medium [14], with a l/10 diln of the major nutrients, on 150 mm diam. Petri plates, 7 seeds per plate, at 25” with a 16 hr photoperiod for 9 days. Cercospora isolate CE14. Small sections (ca 3 mm) of leaf tissue bearing necrotic lesions, or of pigmented roots, were excised, immersed with 1% aq. NaOCl for a few set, and rinsed in sterile H,O. Leaf tissue (3 pieces) and 3 root segments were plated on FDA containing 100 pg ml- ’ kanamycin sulphate. Five of the 6 explants each gave rise to a fungal colony. Extraction of cercosporin from soybean roots. Roots were sepd from the medium and the red-brown segments excised and weighed (547 mg fr. wt). The tissue was frozen
1547
at -20” for 15 min and then extracted with EtOH. The extract was filtered and centrifuged to provide a supernatant (20 ml) which showed UV/visible absorption the same as that of cercosporin [lS]; A,,, “,=0.458. The extract was dild with l/2 vol H,O, and EtOH removed in uacuo prior to extraction with EtOAc (x 2). The organic phase was dried (MgS04), and after removal of solvent, a small aliquot of the residue was used for cochromatography with cercosporin (TLC, silica gel, CHCl,-MeOH-H,O, 35: 12:2; R, 0.70). The bulk (90%) of the residue (0.5 mg) was subjected to prep. TLC (silica gel, 0.25 mm, solvent as above) to give a major red band detected under visible light which was eluted with CHCl,-EtOH (1: 1). For MS data of cercosporin see ref. [15]. Preparation of cebetin B from in vitro cultures. Inoculum of isolate CE14 from PDA+ 100 ,ug ml- ’ kanamycin sulphate was transferred to 250 ml PDB in 1 I conical flasks and incubated under continuous illumination at 25”, 150 rpm. After 9 days the mycelium was shaken with MeOH overnight, solvent was removed in oacuo and the aq. material partitioned against EtOAc. After drying and evapn of the organic phase, the yellow residue was dissolved in MeOH, and co-chromatographed with an authentic sample of cebetin B (TLC, silica gel, CHCl-MeOH-H,O, 35 : 12: 2; R, 0.69). Prep. TLC (silica gel, 0.25 mm) of the dried residue (55 mg) with the above solvent gave a bright yellow band which was eluted with CHCl,-MeOH (1: 1); the ‘H NMR corresponded with cebetin B [S]. UV/visible IzE$ nm (log E): 213.4 (4.3843), 261sh (4.0500), 287sh (3.9049), 346 (4.18603, 386sh (4.0350), 434 (4.0616), 457sh (3.9568); l;EH +NaOH nm: 204, 285sh, 348, 457. Trace amounts of a second yellow compound, with fluorescence similar to that of 1, ran at slightly lower R, (0.62). Acknowledgements-We thank Dr G. K. Eigendorf (British Columbia Regional Mass Spectrometry Centre, University of British Columbia, Vancouver, Canada) for MS analysis. REFERENCES
1. Chupp, C. (1953) A Monograph of the Fungus Genus Cercospora. Cornell University Press, Ithaca, New York. 2. Smith, G. A. and Ruppel, E. G. (1973) Can. J. Plant Sei. 53, 695. 3. Daub, M. E. (1987) Light-Activated Pesticides (Heitz, J. R. and Downum, K. R., eds), pp. 271-280. American Chemical Society, Washington, DC. 4. Sinclair, J. B. (ed.) (1982) Compendium of Soybean Diseases. American Phytopathological Society Press, St Paul, Minnesota. 5. Kuyama, S. and Tamura, T. (1957) J. Am. Chem. Sot. 79, 5725.
6. Lynch, F. J. and Geoghegan, M. J. (1977) Trans. Br. Mycol. Sot. 69, 496.
7. Sclosser, E. (1971) Phytopath. Medit. 10, 154. 8. Jalal, M. A. F., Hossain, M. B., Robeson, D. J. and
1.548
Short Reports
van der Helm, D. (1992) J. Am. Chem. Sot. 114,5967. 9. Assante, G., Locci, R., Camarda, L., Merlini, L. and Nasini, G. (1977) Phytochemistry 16, 243. 10. Venkataramani, K. (1967) Phytopathol. Zeitschrift ss, 379. 11. Duvick, J. (1987) Phytopathology 77, 1754 (Abstr.). 12. Johnson, E. M. and Valleau, W. D. (1949) Phytopathology 39, 763.
13. Ellis, M. B. (1971) Dematiuceous Hyphomycetes, p. 275. CAB, Kew. 14. Schenk, R. V. and Hildebrant, A. C. (1972) Con. J. Botany 50, 199. 15. Yamazaki, S. and Ogawa, T. (1972) Agric. Biol. Chem. 36, 1707.
Vol. 33, No. 6, pp. 1546-1548, 1993 Pnnted IIIGreat Britain.
Phytochemistry,
A CERCOSPORA
ISOLATE FROM SOYBEAN ROOTS PRODUCES CEBETIN B AND CERCOSPORIN DAVID J. ROBESON and MAHBUBUL A. F. JALAL*
The Plant Cell Research
Institute,
Inc., 6560 Trinity
Court,
Dublin,
CA 94568, U.S.A.
(Received 3 September 1992)
Key Word Index-Cercospora; toxin; phototoxin.
Dematiaceae;
Glycine max; soybean; cercosporin;
cebetin; CBT; phyto-
Abstract-A Cercospora isolate (CE14) was isolated from roots of soybean seedlings germinated which appeared red, contained cercosporin which was isolated and quantified. In vitro cultures cebetin B, previously known as CBT (C. b&cola toxin).
in vitro. The roots, of CE14 produced
INTRODIJCTION
Collectively, members of the genus Cercospora are pathogenic to a very broad taxonomic range of plants spanning at least 43 families, including representatives of the Monocotyledonae, Dicotyledonae and Gymnospermae [l]. Many important crop plants are severely attacked by various Cercospora species [2, 31. Two morphologically distinct species, C. kikuchii and C. sojina are known to attack soybean [Glycine max] and to infect the seed [4]. Cercospora spp. attack the aerial parts of plants, typically causing necrotic leaf spots [2, 31. The production of the red perylenequinone derivative, cercosporin, is a characteristic feature of the genus [3,5, 63. This metabolite is well known as a photoactivated phytotoxin [3]. A second class of photoactivated phytotoxins, previously known as ‘CBT’ [7], has only recently been characterized and its two representatives assigned the trivial names of cebetin A and B [S]. In comparison with cercosporin, the known distribution of the cebetins is restricted to just two species [9]. Herein we report the in vitro production of cebetin B (1) by a Cercospora isolate from soybean seedlings, and demonstrate cercosporin production in soybean roots. RESULTS AND DISCUSSION
Soybean seedlings, the seeds of which had been surfacesterilized before germination on a dilute nutrient medium, showed signs of purple pigmentation, primarily on the seed coat and roots and, to a lesser extent, in the surrounding agar. Patches of fungal growth were present on the agar surface. Subsequently, necrotic lesions developed on stems and cotyledons and, in addition, many
*Author to whom correspondence should be address& Pan-Agricultural Labs, Inc.. 32380 Avenue 10, Madera, CA 93638, U.S.A.
of the roots appeared distinctly red. Pigmented root tissue gave a red/orange extract when homogenized in ethanol. The clarified extract exhibited UV/visible absorption virtually identical to that of cercosporin. TLC of the same extract gave a red pigment which co-chromatographed with cercosporin. The pigment from soybean roots was bright red on TLC and fluoresced orange under long wavelength UV, as observed for cercosporin. Mass spectral analysis of the red band confirmed its identity with cercosporin. No other pigments were detected on the chromatogram. The average concentration of cercosporin in the pigmented root tissue was 328 pg g-’ fr. wt. Plating of infected leaf and pigmented root tissue on potato dextrose agar (PDA) gave fungal colonies of uniform gross morphology. They appeared orange from below and were surrounded by a yellow diffusible pigment. Fungal colonies cultured on V8 agar or SH agar, in contrast, showed red or purple pigmentation. The fungal isolate from infected seedlings, designated CE14, was grown in potato dextrose broth (PDB). The yellow-green mycelium was extracted with methanol and
1546
Short Reports the extract was chromatographed with an authentic sample of cebetin B [S]. The major pigment in the extract co-migrated with cebetin B and had the same appearance in both visible light (bright yellow) and long wavelength UV (pale yellow fluorescence). The major yellow compound was purified by TLC for spectroscopic analysis. The identity of this metabolite with cebetin B was demonstrated by W/visible and ’ H NMR spectroscopy [S]. Microscopic examination of conidia of isolate CE14 confirmed that it was a Cercospora sp. Isolation of the fungus from roots, which contained relatively high concentrations of cercosporin, indicates that CE14 is capable of infecting soybean roots grown in vitro. Cercospora kikuchii and C. sojina are both known to attack seeds, pods, stems and leaves of soybean [4]. To date, however, infection of plant roots by a member of the genus Cercospora has not been reported. Cercosporin is a lightactivated phytotoxin which has been strongly implicated in causing disease [3, lo]. Whether Cercospora can infect roots in the field, under conditions where light is largely excluded from these organs, is open to investigation. From the scientific viewpoint, the susceptibility of roots to attack by Cercospora may be further examined using in oitro root cultures or hydroponically grown plants. The isolation of cercosporin from soybean seeds infected with C. kikuchii [S] and from host plants infected with other Cercospora spp. has been described previously [lo, 111. Cebetin B, like cercosporin, is also a photoactivated phytotoxin [S]. In contrast to cercosporin, the former metabolite was not detected in soybean root tissue, nor has it been described as a vivotoxin. The metalfree tautomer of cebetin B, known as cebetin A [8], was not encountered in our investigation of CE14. ‘CBT’ was reported previously from cultures of C. beticola and C. bertoreae during a survey of 61 Cercospora spp. for secondary metabolites [9]. Other than isolate CE14, Phoma musae as well as three randomly selected C. beticola isolates also gave indications of cebetin production. The production of cebetins by a wider range of Cercospora isolates is predictable in view of the apparent synonymy which exists in some common ‘species’ of Cercospora [12, 133. EXPERIMENTAL
Culture of soybean seedlings. Seeds were surface-sterilized with Cl, gas, and germinated on hormone-free SH medium [14], with a l/10 diln of the major nutrients, on 150 mm diam. Petri plates, 7 seeds per plate, at 25” with a 16 hr photoperiod for 9 days. Cercospora isolate CE14. Small sections (ca 3 mm) of leaf tissue bearing necrotic lesions, or of pigmented roots, were excised, immersed with 1% aq. NaOCl for a few set, and rinsed in sterile H,O. Leaf tissue (3 pieces) and 3 root segments were plated on FDA containing 100 pg ml- ’ kanamycin sulphate. Five of the 6 explants each gave rise to a fungal colony. Extraction of cercosporin from soybean roots. Roots were sepd from the medium and the red-brown segments excised and weighed (547 mg fr. wt). The tissue was frozen
1547
at -20” for 15 min and then extracted with EtOH. The extract was filtered and centrifuged to provide a supernatant (20 ml) which showed UV/visible absorption the same as that of cercosporin [lS]; A,,, “,=0.458. The extract was dild with l/2 vol H,O, and EtOH removed in uacuo prior to extraction with EtOAc (x 2). The organic phase was dried (MgS04), and after removal of solvent, a small aliquot of the residue was used for cochromatography with cercosporin (TLC, silica gel, CHCl,-MeOH-H,O, 35: 12:2; R, 0.70). The bulk (90%) of the residue (0.5 mg) was subjected to prep. TLC (silica gel, 0.25 mm, solvent as above) to give a major red band detected under visible light which was eluted with CHCl,-EtOH (1: 1). For MS data of cercosporin see ref. [15]. Preparation of cebetin B from in vitro cultures. Inoculum of isolate CE14 from PDA+ 100 ,ug ml- ’ kanamycin sulphate was transferred to 250 ml PDB in 1 I conical flasks and incubated under continuous illumination at 25”, 150 rpm. After 9 days the mycelium was shaken with MeOH overnight, solvent was removed in oacuo and the aq. material partitioned against EtOAc. After drying and evapn of the organic phase, the yellow residue was dissolved in MeOH, and co-chromatographed with an authentic sample of cebetin B (TLC, silica gel, CHCl-MeOH-H,O, 35 : 12: 2; R, 0.69). Prep. TLC (silica gel, 0.25 mm) of the dried residue (55 mg) with the above solvent gave a bright yellow band which was eluted with CHCl,-MeOH (1: 1); the ‘H NMR corresponded with cebetin B [S]. UV/visible IzE$ nm (log E): 213.4 (4.3843), 261sh (4.0500), 287sh (3.9049), 346 (4.18603, 386sh (4.0350), 434 (4.0616), 457sh (3.9568); l;EH +NaOH nm: 204, 285sh, 348, 457. Trace amounts of a second yellow compound, with fluorescence similar to that of 1, ran at slightly lower R, (0.62). Acknowledgements-We thank Dr G. K. Eigendorf (British Columbia Regional Mass Spectrometry Centre, University of British Columbia, Vancouver, Canada) for MS analysis. REFERENCES
1. Chupp, C. (1953) A Monograph of the Fungus Genus Cercospora. Cornell University Press, Ithaca, New York. 2. Smith, G. A. and Ruppel, E. G. (1973) Can. J. Plant Sei. 53, 695. 3. Daub, M. E. (1987) Light-Activated Pesticides (Heitz, J. R. and Downum, K. R., eds), pp. 271-280. American Chemical Society, Washington, DC. 4. Sinclair, J. B. (ed.) (1982) Compendium of Soybean Diseases. American Phytopathological Society Press, St Paul, Minnesota. 5. Kuyama, S. and Tamura, T. (1957) J. Am. Chem. Sot. 79, 5725.
6. Lynch, F. J. and Geoghegan, M. J. (1977) Trans. Br. Mycol. Sot. 69, 496.
7. Sclosser, E. (1971) Phytopath. Medit. 10, 154. 8. Jalal, M. A. F., Hossain, M. B., Robeson, D. J. and
1.548
Short Reports
van der Helm, D. (1992) J. Am. Chem. Sot. 114,5967. 9. Assante, G., Locci, R., Camarda, L., Merlini, L. and Nasini, G. (1977) Phytochemistry 16, 243. 10. Venkataramani, K. (1967) Phytopathol. Zeitschrift ss, 379. 11. Duvick, J. (1987) Phytopathology 77, 1754 (Abstr.). 12. Johnson, E. M. and Valleau, W. D. (1949) Phytopathology 39, 763.
13. Ellis, M. B. (1971) Dematiuceous Hyphomycetes, p. 275. CAB, Kew. 14. Schenk, R. V. and Hildebrant, A. C. (1972) Con. J. Botany 50, 199. 15. Yamazaki, S. and Ogawa, T. (1972) Agric. Biol. Chem. 36, 1707.