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Toxigenic potential of Alternaria macrospora pathogenic to cotton
Microbiol. Res. (1996) 151,239-241
Microbiological Research © Gustav Fischer Verlag Jena
Toxigenic potential of Alternaria macrospora pathogenic to cotton M. Vijayalakshmi Department of Botany, Nagarjuna University, Guntur 522510, India Accepted: October 16, 1995
Abstract Alternaria macrospora Zimm. causing leaf spots on cotton, was grown on three culture media viz. CzapekDox liquid medium (CD), CD with 0.5% peptone and CD with 0.5% yeast extract for testing its toxigenic potential. The culture filtrates of the pathogen exhibited phytotoxicity against cotton (host), tomato and bajra (non-hosts) as well as antibacterial activity. The culture filtrates collected from CD with 0.5% peptone were more toxic to the test plants than the other two. The toxic effects of culture filtrates gradually increased with aging of the cultures in the three cases. Analysis of the crude toxin revealed the presence of two phytotoxic compounds, of which one was identified as tenuazonic acid. Key words: Alternaria macrospora - cotton - tenuazonic acid
Introduction Alternaria macrospora Zimm. causing leaf spots, is one of the important foliar pathogens of cotton. Several metabolites produced by Alternaria species have been associated with phytotoxicity (Harvan and Pero 1976; Cotty et al. 1983) and some of them play an important role in symptom production (Templeton 1972; Siler and Gilchrist 1983). However, regarding the toxins of A. macrospora, less data are available. Balasubramanian and Bhama (1977) suggested that the toxic compound produced by A. macrospora might be of phenolic nature. Padmanaban and Narayanasamy (1978) reported that the toxin of A. macrospora inhibited seed germination and was not host-specific. The toxin produced by the same fungus was reported to induce leaf spot symptoms in cotton (Krishnamohan and Vidhyasekaran 1989). The present study has been aimed at assessing
the toxigenic potential of A. macrospora and to identify the metabolites responsible for phytotoxicity.
Materials and Methods Three culture media viz., Czapek-Dox liquid medium (CD), CD with 0.5% peptone and CD with 0.5% yeast extract inoculated with mycelial discs of 5 mm diameter taken from the periphery of actively growing culture of A. macrospora were incubated at room temperature (28 ± 4°C) as stationary cultures for 40 days. Phytotoxicity against seed germination, root elongation and detached leaves of cotton, tomato and bajra was tested with the culture filtrates collected aseptically at 10-day intervals up to 40 days as described earlier (Vijayalakshmi and Rao 1985). Concentrated chloroform fractions extracted from the culture filtrates were employed for testing antibacterial activity against Bacillus megaterium and B. subtilis (Burmeister and Hesseltine 1966). The culture filtrates of the fungus cultured on yeast extract-sucrose broth for 30 days were screened for known Alternaria toxins (Rosett et al. 1957, Templeton 1972, Meronuck et al. 1972, Schade and King 1984). Culture filtrates acidified to pH less than 2.0 with conc. hydrochloric acid were extracted twice with ethyl acetate. Pooled extracts were concentrated and used for TLC analysis of tenuazonic acid employing toluene - ethyl acetate-90% formic acid (5: 4: 1) as the developing solvent system. Both fluorescing and non-fluorescing compounds on TLC plates were marked after exposing the plates to UV light. These compounds were eluted separately in ethanol and tested for phytotoxicity using detached leaf bioassay method against cotton. Selected toxins Microbiol. Res. 151 (1996) 3
239
basing on colour, Rf values and fluorescence of spots were cochromatographed with authentic samples of known Alternaria toxins. Confirmatory testes with ethanolic ferric chloride (Rosett et al. 1957), panisaldehyde (Scott and Kanhere 1980) and UV spectral chracteristics for tenuazonic acid (Stickings 1959) were carried out.
Results and Discussion The effects of culture filtrates of A. macrospora on seed germination and root elongation of cotton, tomato and bajra are presented in Figure 1. Germination of seeds and elongation of roots in the germinated seeds were adversely effected by the culture filtrates. Among the three culture media tested, culture filtrates collected from CD with 0.5% peptone were highly toxic followed by CD with 0.5% yeast extract and CD. No seed germination was found in the test plants when treated with culture filtrates from CD with 0.5% peptone collected after 20 days of incubation period. The toxic effects of culture filtrates gradually increased with aging of the cultures in all the cases. The inhibitory effects of culture filtrates seemed to be relatively more on root elonga-
100
tion than on seed germination in the three test plants. Brown necrotic lesions appeared on leaves of cotton and tomato when treated with concentrated culture filtrates. The metabolites produced by A. macrospora were also inhibitory to test bacteria. The inhibition zones recorded were 2.1 and 2.8 cm 2 in plates seeded with B. megaterium and B. subtilis, respectively. Among the eight compounds detected on TLC plates, ethanolic extracts of two compounds produced necrotic symptoms on cotton leaves. Of the two, one is a tan spot under visible light with an Rf value extending from 0.25 to 0.38 and the other one with an Rf 0.75 emitted blue fluorescence when exposed to UV ligth. Co-chromatography with authentic samples of some of known Alternaria toxins revealed the identity of tan spot as tenuazonic acid. Spraying the plates with ethanolic ferric chloride resulted in deep reddish spots for tenuazonic acid. The than spot when sprayed with acidic anisaldehyde emitted green fluorescence when viewed under long wave UV light. These two tests further confirmed its identity as tenuazonic acid. UV spectral characteristics of this compound with peaks at 239 and 279 nm in water and 217 and 277 nm in ethanol coincided with those of authentic tenuazonic acid sample. TLC characteri-
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Microbiol. Res. 151 (1996) 3
PERIOD (DAYS)
Fig. 1. Phytotoxicity of culture filtrates of A. macrospora grown on three culture media against cotton, tomato and bajra. · - - . Czapek-Dox broth (CD) · - _ - . CD with 0.5% peptone CD with 0.5% yeast extract •
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stics of the other toxic compound did not coincide with the known Alternaria toxins and its identity is not known. Krishnamohan and Vidhyasekaran (1989) reported that the yellowish brown residue obtained after extracting the acidified culture filtrate of A. macrospora with diethyl ether, induced leaf spot symptoms in cotton. Tenuazonic acid production by A. macrospora reported in the present study is new to literature, though isolates of A. alternata pathogenic to several crop plants are known to produce it (Kinoshita et al. 1972). Cultures of A. alternata and A. tenuissima obtained from cotton seeds and bolls were reported to produce tenuazonic acid (Davis et al. 1977). Attempts are in progress to extract and establish mycotoxic nature of tenuazonic acid in cotton seeds since A. macrospora was seed-borne. Acknowledgements The author wishes to thank Dr. P. M. Scott, Health Protection Branch, Canada for providing authentic samples of Alternaria toxins. She wishes to express her sincere gratitude to Prof. A S. Rao for his valuable suggestions.
References Balasubramanian, R., Bhama, K. S. (1977): Phytotoxic metabolite from Alternaria macrospora. Curro Sci. 46, 426. Burmeister, H. R., Hesseltine, C. W. (1966): Survey of sensitivity of microorganisms to aflatoxin. Appl. MicrobioI. 14, 403 - 404. Cotty, P. J., Misaghi, I. J., Hine, R. B. (1983): Production of zinniol by Alternaria tagetica and its phytotoxic effect on Tagetes erecta. Phytopath. 73, 1326 -1328. Davis, N. D., Diener, V. L., Morgan-Jones, G. (1977): Tenuazonic acid production by Alternaria alternata and Alternaria tenuissima isolated from cotton. Appl. Environ. Microbiol. 34, 155 -157.
Harvan, D. J., Pero, R. W. (1976): The structure and toxicity of Alternaria metabolites. In: Mycotoxins and other fungal related food problems (Ed. Rodricks, J. V.), Adv. Chem. Ser. 149, ACS, Washington, 344-355. Kinoshita, T., Renbutsu, Y., Khan, I. D., Kohmoto, K., Nishimura, S. (1972): Distribution of tenuazonic acid production in the genus Alternaria and its pathological evaluation. Ann. Phytopath. Soc. Japan, 38, 397-404. Krishnamohan, G., Vidhyasekaran, P. (1989): Possible involvement of toxin(s) in Alternaria leaf spot development in cotton. Indian Phytopath. 42, 99 -102. Meronuck, R. A, Steele, J. A, Mirocha, C. J., Christensen, C. M. (1972): Tenuazonic, acid, a toxin produced by Alternaria alternata. Appl. Microbiol. 23, 613 - 617. Padmanaban, P., Narayanasamy, P. (1978): Characterization of toxin produced by Alternaria macrospora zimm. Madras Agric. J. 65, 33-35. Rosett, T., Sankhala, R. H., Sticking, C. E., Taylor, M. E. 0., Thomas, R. (1957): Studies on the biochemistry of microorganisms 103. Metabolites of Alternaria tenuis Auct: culture filtrate products. Biochem. J. 67, 390-400. Schade, J. E., King, A D. (1984): Analysis of the major Alternaria toxins. J. Food Prot. 47, 978-995. Scott, P. M., Kanhere, S. R. (1980): Liquid chromatographic determination oftenuazonic acid in tomato paste. J. Assoc. Off. Anal. Chem. 63, 612 - 621. Siler, D. J., Gilchrist, D. G. (1983): Properties of hostspecific toxins produced by Alternaria alternata f. sp. lycopersici in culture and in tomato plants. Physiol. plant pathol. 23, 265 - 274. Stickings, C. E. (1959): Studies in the biochemistry of microorganisms. 106. Metabolites of Alternaria tenuis Auct. : The structure of tenuazonic acid. Biochem. J. 72, 332-340. Templeton, G. E. (1972): Alternaria toxins related to pathogenesis in plants. In: Microbial Toxins vol. VIII. Fungal Toxins. (Eds. Kadis et al.) Academic Press, New York. Vijayalakshmi, M., Rao, A. S. (1985): Toxigenic fungi associated with sunflower seeds during development. In: Trichothecenes and other Mycotoxins. (Ed. Lacey, J.), John Wiley, New York, 3-45.
Microbiol. Res. 151 (1996) 3
241
Microbiological Research © Gustav Fischer Verlag Jena
Toxigenic potential of Alternaria macrospora pathogenic to cotton M. Vijayalakshmi Department of Botany, Nagarjuna University, Guntur 522510, India Accepted: October 16, 1995
Abstract Alternaria macrospora Zimm. causing leaf spots on cotton, was grown on three culture media viz. CzapekDox liquid medium (CD), CD with 0.5% peptone and CD with 0.5% yeast extract for testing its toxigenic potential. The culture filtrates of the pathogen exhibited phytotoxicity against cotton (host), tomato and bajra (non-hosts) as well as antibacterial activity. The culture filtrates collected from CD with 0.5% peptone were more toxic to the test plants than the other two. The toxic effects of culture filtrates gradually increased with aging of the cultures in the three cases. Analysis of the crude toxin revealed the presence of two phytotoxic compounds, of which one was identified as tenuazonic acid. Key words: Alternaria macrospora - cotton - tenuazonic acid
Introduction Alternaria macrospora Zimm. causing leaf spots, is one of the important foliar pathogens of cotton. Several metabolites produced by Alternaria species have been associated with phytotoxicity (Harvan and Pero 1976; Cotty et al. 1983) and some of them play an important role in symptom production (Templeton 1972; Siler and Gilchrist 1983). However, regarding the toxins of A. macrospora, less data are available. Balasubramanian and Bhama (1977) suggested that the toxic compound produced by A. macrospora might be of phenolic nature. Padmanaban and Narayanasamy (1978) reported that the toxin of A. macrospora inhibited seed germination and was not host-specific. The toxin produced by the same fungus was reported to induce leaf spot symptoms in cotton (Krishnamohan and Vidhyasekaran 1989). The present study has been aimed at assessing
the toxigenic potential of A. macrospora and to identify the metabolites responsible for phytotoxicity.
Materials and Methods Three culture media viz., Czapek-Dox liquid medium (CD), CD with 0.5% peptone and CD with 0.5% yeast extract inoculated with mycelial discs of 5 mm diameter taken from the periphery of actively growing culture of A. macrospora were incubated at room temperature (28 ± 4°C) as stationary cultures for 40 days. Phytotoxicity against seed germination, root elongation and detached leaves of cotton, tomato and bajra was tested with the culture filtrates collected aseptically at 10-day intervals up to 40 days as described earlier (Vijayalakshmi and Rao 1985). Concentrated chloroform fractions extracted from the culture filtrates were employed for testing antibacterial activity against Bacillus megaterium and B. subtilis (Burmeister and Hesseltine 1966). The culture filtrates of the fungus cultured on yeast extract-sucrose broth for 30 days were screened for known Alternaria toxins (Rosett et al. 1957, Templeton 1972, Meronuck et al. 1972, Schade and King 1984). Culture filtrates acidified to pH less than 2.0 with conc. hydrochloric acid were extracted twice with ethyl acetate. Pooled extracts were concentrated and used for TLC analysis of tenuazonic acid employing toluene - ethyl acetate-90% formic acid (5: 4: 1) as the developing solvent system. Both fluorescing and non-fluorescing compounds on TLC plates were marked after exposing the plates to UV light. These compounds were eluted separately in ethanol and tested for phytotoxicity using detached leaf bioassay method against cotton. Selected toxins Microbiol. Res. 151 (1996) 3
239
basing on colour, Rf values and fluorescence of spots were cochromatographed with authentic samples of known Alternaria toxins. Confirmatory testes with ethanolic ferric chloride (Rosett et al. 1957), panisaldehyde (Scott and Kanhere 1980) and UV spectral chracteristics for tenuazonic acid (Stickings 1959) were carried out.
Results and Discussion The effects of culture filtrates of A. macrospora on seed germination and root elongation of cotton, tomato and bajra are presented in Figure 1. Germination of seeds and elongation of roots in the germinated seeds were adversely effected by the culture filtrates. Among the three culture media tested, culture filtrates collected from CD with 0.5% peptone were highly toxic followed by CD with 0.5% yeast extract and CD. No seed germination was found in the test plants when treated with culture filtrates from CD with 0.5% peptone collected after 20 days of incubation period. The toxic effects of culture filtrates gradually increased with aging of the cultures in all the cases. The inhibitory effects of culture filtrates seemed to be relatively more on root elonga-
100
tion than on seed germination in the three test plants. Brown necrotic lesions appeared on leaves of cotton and tomato when treated with concentrated culture filtrates. The metabolites produced by A. macrospora were also inhibitory to test bacteria. The inhibition zones recorded were 2.1 and 2.8 cm 2 in plates seeded with B. megaterium and B. subtilis, respectively. Among the eight compounds detected on TLC plates, ethanolic extracts of two compounds produced necrotic symptoms on cotton leaves. Of the two, one is a tan spot under visible light with an Rf value extending from 0.25 to 0.38 and the other one with an Rf 0.75 emitted blue fluorescence when exposed to UV ligth. Co-chromatography with authentic samples of some of known Alternaria toxins revealed the identity of tan spot as tenuazonic acid. Spraying the plates with ethanolic ferric chloride resulted in deep reddish spots for tenuazonic acid. The than spot when sprayed with acidic anisaldehyde emitted green fluorescence when viewed under long wave UV light. These two tests further confirmed its identity as tenuazonic acid. UV spectral characteristics of this compound with peaks at 239 and 279 nm in water and 217 and 277 nm in ethanol coincided with those of authentic tenuazonic acid sample. TLC characteri-
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Microbiol. Res. 151 (1996) 3
PERIOD (DAYS)
Fig. 1. Phytotoxicity of culture filtrates of A. macrospora grown on three culture media against cotton, tomato and bajra. · - - . Czapek-Dox broth (CD) · - _ - . CD with 0.5% peptone CD with 0.5% yeast extract •
-
0
-
•
stics of the other toxic compound did not coincide with the known Alternaria toxins and its identity is not known. Krishnamohan and Vidhyasekaran (1989) reported that the yellowish brown residue obtained after extracting the acidified culture filtrate of A. macrospora with diethyl ether, induced leaf spot symptoms in cotton. Tenuazonic acid production by A. macrospora reported in the present study is new to literature, though isolates of A. alternata pathogenic to several crop plants are known to produce it (Kinoshita et al. 1972). Cultures of A. alternata and A. tenuissima obtained from cotton seeds and bolls were reported to produce tenuazonic acid (Davis et al. 1977). Attempts are in progress to extract and establish mycotoxic nature of tenuazonic acid in cotton seeds since A. macrospora was seed-borne. Acknowledgements The author wishes to thank Dr. P. M. Scott, Health Protection Branch, Canada for providing authentic samples of Alternaria toxins. She wishes to express her sincere gratitude to Prof. A S. Rao for his valuable suggestions.
References Balasubramanian, R., Bhama, K. S. (1977): Phytotoxic metabolite from Alternaria macrospora. Curro Sci. 46, 426. Burmeister, H. R., Hesseltine, C. W. (1966): Survey of sensitivity of microorganisms to aflatoxin. Appl. MicrobioI. 14, 403 - 404. Cotty, P. J., Misaghi, I. J., Hine, R. B. (1983): Production of zinniol by Alternaria tagetica and its phytotoxic effect on Tagetes erecta. Phytopath. 73, 1326 -1328. Davis, N. D., Diener, V. L., Morgan-Jones, G. (1977): Tenuazonic acid production by Alternaria alternata and Alternaria tenuissima isolated from cotton. Appl. Environ. Microbiol. 34, 155 -157.
Harvan, D. J., Pero, R. W. (1976): The structure and toxicity of Alternaria metabolites. In: Mycotoxins and other fungal related food problems (Ed. Rodricks, J. V.), Adv. Chem. Ser. 149, ACS, Washington, 344-355. Kinoshita, T., Renbutsu, Y., Khan, I. D., Kohmoto, K., Nishimura, S. (1972): Distribution of tenuazonic acid production in the genus Alternaria and its pathological evaluation. Ann. Phytopath. Soc. Japan, 38, 397-404. Krishnamohan, G., Vidhyasekaran, P. (1989): Possible involvement of toxin(s) in Alternaria leaf spot development in cotton. Indian Phytopath. 42, 99 -102. Meronuck, R. A, Steele, J. A, Mirocha, C. J., Christensen, C. M. (1972): Tenuazonic, acid, a toxin produced by Alternaria alternata. Appl. Microbiol. 23, 613 - 617. Padmanaban, P., Narayanasamy, P. (1978): Characterization of toxin produced by Alternaria macrospora zimm. Madras Agric. J. 65, 33-35. Rosett, T., Sankhala, R. H., Sticking, C. E., Taylor, M. E. 0., Thomas, R. (1957): Studies on the biochemistry of microorganisms 103. Metabolites of Alternaria tenuis Auct: culture filtrate products. Biochem. J. 67, 390-400. Schade, J. E., King, A D. (1984): Analysis of the major Alternaria toxins. J. Food Prot. 47, 978-995. Scott, P. M., Kanhere, S. R. (1980): Liquid chromatographic determination oftenuazonic acid in tomato paste. J. Assoc. Off. Anal. Chem. 63, 612 - 621. Siler, D. J., Gilchrist, D. G. (1983): Properties of hostspecific toxins produced by Alternaria alternata f. sp. lycopersici in culture and in tomato plants. Physiol. plant pathol. 23, 265 - 274. Stickings, C. E. (1959): Studies in the biochemistry of microorganisms. 106. Metabolites of Alternaria tenuis Auct. : The structure of tenuazonic acid. Biochem. J. 72, 332-340. Templeton, G. E. (1972): Alternaria toxins related to pathogenesis in plants. In: Microbial Toxins vol. VIII. Fungal Toxins. (Eds. Kadis et al.) Academic Press, New York. Vijayalakshmi, M., Rao, A. S. (1985): Toxigenic fungi associated with sunflower seeds during development. In: Trichothecenes and other Mycotoxins. (Ed. Lacey, J.), John Wiley, New York, 3-45.
Microbiol. Res. 151 (1996) 3
241