production of the phytotoxic metabolite, ferricrocin, by the fungus colletotrichum...

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This article was downloaded by: [University of Windsor] On: 17 November 2014, At: 12:02 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Bioscience, Biotechnology, and Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbbb20 Production of the Phytotoxic Metabolite, Ferricrocin, by the Fungus Colletotrichum gloeosporioides Junko Ohra a , Kenji Morita a , Yasuko Tsujino a , Hiroyuki Tazaki a , Takane Fujimori a , Matt Goering ab , Steve Evans ab & Paul Zorner ab a Applied Plant Research Laboratory, Yokohama Center, JAPAN TOBACCO INC., 6-2 Umegaoka, Aoba-ku, Yokohama, Kanagawa 227, Japan b Mycogen Corporation, 4980 Carroll Canyon Road, San Diego, CA 92121, U.S.A. Published online: 12 Jun 2014. To cite this article: Junko Ohra, Kenji Morita, Yasuko Tsujino, Hiroyuki Tazaki, Takane Fujimori, Matt Goering, Steve Evans & Paul Zorner (1995) Production of the Phytotoxic Metabolite, Ferricrocin, by the Fungus Colletotrichum gloeosporioides, Bioscience, Biotechnology, and Biochemistry, 59:1, 113-114, DOI: 10.1271/bbb.59.113 To link to this article: http://dx.doi.org/10.1271/bbb.59.113 PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions

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Page 1: Production of the Phytotoxic Metabolite, Ferricrocin, by the Fungus               Colletotrichum gloeosporioides

This article was downloaded by: [University of Windsor]On: 17 November 2014, At: 12:02Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: MortimerHouse, 37-41 Mortimer Street, London W1T 3JH, UK

Bioscience, Biotechnology, and BiochemistryPublication details, including instructions for authors and subscription information:http://www.tandfonline.com/loi/tbbb20

Production of the Phytotoxic Metabolite, Ferricrocin,by the Fungus Colletotrichum gloeosporioidesJunko Ohraa, Kenji Moritaa, Yasuko Tsujinoa, Hiroyuki Tazakia, Takane Fujimoria, MattGoeringab, Steve Evansab & Paul Zornerab

a Applied Plant Research Laboratory, Yokohama Center, JAPAN TOBACCO INC., 6-2Umegaoka, Aoba-ku, Yokohama, Kanagawa 227, Japanb Mycogen Corporation, 4980 Carroll Canyon Road, San Diego, CA 92121, U.S.A.Published online: 12 Jun 2014.

To cite this article: Junko Ohra, Kenji Morita, Yasuko Tsujino, Hiroyuki Tazaki, Takane Fujimori, Matt Goering, Steve Evans& Paul Zorner (1995) Production of the Phytotoxic Metabolite, Ferricrocin, by the Fungus Colletotrichum gloeosporioides,Bioscience, Biotechnology, and Biochemistry, 59:1, 113-114, DOI: 10.1271/bbb.59.113

To link to this article: http://dx.doi.org/10.1271/bbb.59.113

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) containedin the publications on our platform. However, Taylor & Francis, our agents, and our licensors make norepresentations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose ofthe Content. Any opinions and views expressed in this publication are the opinions and views of the authors,and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be reliedupon and should be independently verified with primary sources of information. Taylor and Francis shallnot be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and otherliabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to orarising out of the use of the Content.

This article may be used for research, teaching, and private study purposes. Any substantial or systematicreproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in anyform to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions

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Biosci. Biotech. Biochem., 59 (1), 113-114, 1995

Note

Production of the Phytotoxic Metabolite, Ferricrocin, by the Fungus Colletotrichum gloeosporioides

lunko OHRA, Kenji MORITA, Yasuko TSUJINO, Hiroyuki TAZAKI, Takane FUJIMORI, Matt GOERING,*

Steve EVANs, * and Paul ZORNER *

Applied Plant Research Laboratory, Yokohama Center, JAPAN TOBACCO INC, 6-2 Umegaoka, Aoba-ku, Yokohama, Kanagawa 227, Japan * Mycogen Corporation, 4980 Carroll Canyon Road, San Diego, CA 92121, U.S.A. Received June 29, 1994

A siderophore was isolated as a non-specific phytotoxic compound from a culture of Colletotrichum gloeosporioides isolated from infected blackberry. This siderophore was identified as ferricrocin by NMR, IR, MS, and CD spectra. The phytotoxic activities of ferricrocin and deferriferricrocin were compared.

Since it has been reported that many phytopathogens produce phytotoxins,1) we have been searching for fungal phytotoxins which could be of potential bioherbicidal use. Colletotrichum gloeosporioides was isolated from an infected blackberry (Rubus spp.) stem from North Carolina, U.S.A. Rubus spp. is known as a host of C. gloeosporioides Penzig. 2

) The metabolites of phytotoxins by C. gloeosporioides, whose host is the olive (Olea europaea L.), has been reported, 3) but the production of phytotoxic compounds by this fungus was not described.

C. gloeosporioides was grown on a solid meidum and extracted with Me2CO. We observed that C. gloeosporioides produced non-selective phytotoxic substances from results obtained by a whole-plant assay on 7 different weeds, the results being as follows: jointvech was the most severely damaged, pigweed and florida beggarweed were severely burned and would not grow out of the damage, andjohnsongrass was stunted but not killed by the extract. The Me2CO extracts showed further phytotoxic activity from a leaf-wounding assay, and an active phytotoxic fraction was purified. The Me2CO extracts (221.4 g) were fractionated into the H 20 phase and EtOAc phase. The H 20 phase (199.5 g) was purified by HP-20, silica gel column chromatography and by HPLC, and compound 1 (111.5 mg) was isolated as a phytotoxin. Compound 1 was found to contain iron when treated with color-producing iron ammonium thiocyanate and potassium ferrocyanide indicators.

The FAB-MS data for compound 1 showed a mass peak at m/z 771 [M + H] + .4) An amino acid analysis of compound 1 detected ornithine, glycine, and serine in a 3: 2 : 1 ratio. These data indicated compound 1 to be ferricrocin.

Since the iron existing in compound 1 caused severe line broadening of the NMR signals, compound 3 was derived from compound 1 by the Uinas method 5) in order to elucidate the structure of compound 1 by NMR. 1 H-NMR chemical-shift assignments of compound 3 were confirmed by 1 H_1 H COSY as described in the experimental section,6) while its 13C-NMR signal assignments were confirmed by DEPT, 13C_1 H COSY and HMBC as also described. 7

) The six carbonyl groups of amino acids in the 13C-NMR spectrum were assigned by HMBC. The 1H_ and 13C-NMR signals of three ornithines became distinguishable by COSY, HMBC, and NOESY, and the sequence of the six amino acids, three ornithines, two glycines, and serine was first examined by NOESY. It was confirmed that the six amino acids were connected in a sequence (Fig.). The ligand chirality around the

metal center of compound 1 was determined by its CD spectrum, which exhibited a positive CD band at 450 nm, so that it must have had a A configuration. 8) Compound 1 was thereby confirmed to be ferricrocin from its NMR and CD spectra.

While ferricrocin has been isolated from Neurospora, Aspergillus and Epicoccum cultures,9 11) this is the first description of the production of siderophores from Colletotrichum.

Compound 1 was tested against cotyledons, the results of this assay, shown in Table I, giving a phytotoxicity from 10 - 2 M to 1/64 X 10- 2 M.

Compounds 1 and 2 derived were examined for phytotoxic activity by a leaf-wounding bioassay. The results are shown in Table II, compound 2 exhibiting stronger phytotoxicity and faster appearance of the effect than compound 1.

® Orn2 co- NH-CII co

I I I Glyl IH~ (CH2h CH3 NH

N 1/1 I~H3VN, . 0#'N-(CH2h-CH co ~ 0" / I 0- - _M2t. -0. Orol

Ser CHCH:zC)H / ... I -0 0

( ~H IJ co NL "'c CH2 (C~2h H3

Gly2 I I NH- CO-CH NH--CO

~ Glyl -Ser -Gly2 -Orn3 - Orol-Orn2

Fig. Structures of Compounds 1, 2, and 3 (1, M=Fe; 2, M=Free; 3, M=AI).

Arrows represent observed NOE in the NOESY spectrum of compound 3.

Table I. Results of the Cotyledon Assay on Velvet leaf

Concentration 1/2 1/4 1/8 1/16 1/32 1/64 1/128

Effect on 1 + + + + + + + + + + + + +

Effect: no symptoms; +, slight; + +, moderate; + + +, severe; + + + +, death.

This compound was dissolved in H 20 and then diluted with H 20. Concentration: 1, 10- 2 M; 1/2, 1/2 x 10- 2 M; 1/4, 1/4 x 10- 2 M; 1/8,

1/8 x 10- 2 M; 1/16, 1/16 x 10- 2 M; 1/32, 1/32 x 10- 2 M; 1/64, 1/64 x 10- 2 M;

and 1/128,1/128 x 1O- 2 M.

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114 J. OHRA et al.

Table II. Phytotoxicity of Compounds 1 and 2 by a Leaf-wounding Assay

Concentration 1/2 1/4 1/8 1/16 1/32

Effect on compound 1 + Effect on compound 2 + + + + + + + + Effect on control*

Effect: no symptoms; +, 0-5 mm zone of necrosis; + +, 6-10 mm zone; + + +, 11-15 mm zone.

Concentration: 1, lO- z M; 1/2, 1/2 x lO- z M; 1/4, 1/4 x 10- 2 M; 1/8, 1/8xl0- 2 M; 1/16, 1/16xlO- 2M,and 1/32, 1/32xl0- 2 M.

Compounds 1 and 2 were dissolved in H 2 0 and then diluted with HzO. * The control was H 20.

Siderophores produced by microorganisms have been used to scavenge iron from environments suffering from iron defi­ciency.12.13) Siderophores have also been reported to have many biological functions,14.15) but there are no reports on their phytotoxic activity. Since the activity of compound 1 was enhanced by the removal of iron, this suggests that phytotoxic activity has some relation with chelating activity.

Experimental Culture. Colletotrichum gloeosporioides was grown at 22°C for 7-10 days

in 3-liter flasks containing a Shredded Wheat-based medium, composed of the following compounds: Shredded Wheat (Nabisco spoonsize), 100 g; sucrose, 40 g; yeast extract (Difco Laboratories), 4 g; mycological broth (Difco Laboratories), 10 g; and distilled H 20, 200 ml.

Whole plant assay. Whole plant tests were done on florida beggarweed (Desmodium tortuosum), dandelion (Taraxacum vulgare), pigweed (Amaranthus retrofiexus), johnsongrass (Sorghum hale pense), tall fescue (Festuca arundinacea) , velvetleaf (Abutilon theophrasti), and jointvetch (Aeschynomene spp.). These plants were treated with a 2-ml solution at I g/4 ml, and phytotoxicity was assessed 7 days after the treatment.

Leaf-wounding assay. Cowpea plants (2 weeks old) were grown in a greenhouse. The toxin solution to be tested was dissolved in 20% MeOH-H20, and true leaves of cowpea were injured with 2 J.tl of this solution. The control used was a 20% MeOH-H 20 solution, and phytotoxicity was assessed 5 days after the treatment.

Cotyledon assay. Velvetleaf plants (7 days old) were grown in a greenhouse, and the cotyledons were removed. The stems were trimmed, leaving 3 cm of hypocotyl below the point of cotyledon attachment plus the shoot, which included two primary leaves. The cuttings were immersed in I-ml vials containing 750 J.tl of the toxic solution which had been dissolved in H20. The reSUlting phytotoxicity was observed 3 days after the treatment.

Compound 1 (ferricrocin). Red amorphous substance; UV Amax (H20) nm (e): 422.8 (2058); CD nm (L1e): 289 (- 3.07), 360 (-1.32), 450 ( + 1.74); FAB-MS mlz: 771 [M+HJ+. FAB-HR-MS mlz: [M+HJ+: ca1cd. for C28H4SN9013Fe, 771.2486; found, 771.2590. IRvmax (KBr)cm- 1

: 3339 br,2940, 1653, 1581, 1524.

Compound 2 (deferriferricrocin). Crude compound 2 was derived from

compound 1 (15mg), and then purified by HPLC in a YMC C-18 AQ reverse-phase column (30% MeOH-HzO) to give 12 mg of pure compound 2. FAB-MS mlz: 718.3 [M+HJ+. FAB-HR-MS mlz: [M+HJ+: calcd. for C28H48N9013, 718.3372; found, 718.3369.

Compound 3 (alumicrocin). Compound 3 (12 mg) was derived from crude compound 2, and then purified by HPLC in a YMC C-18 AQ reverse-phase column (30% MeOH-H20) to give 14mg of pure compound 3. FAB-MS mlz: 742.3 [M+HJ+. FAB-HR-MS mlz: [M+HJ+: calcd. for C2sH4sN9013Al, 742.2952; found, 742.3004. 300MHz IH-NMR (DMSO-d6) 15: 1.08 (Orn3 CpH), 1.71 (Orn l CpH), 1.75 (Ornz CpH), 2.03, 2.06, 2.07 (CO = CH 3 x 3), 2.80 (Orn l CpH), 3.41 (Gly I C~H), 3.50 (Ser CpH), 3.73 (GlY2 C~H), 3.80 (GlYI C",H), 4.00 (Ser CaH), 4.12 (Orn l CaH), 4.20 (Om3 C~H), 4.73 (Orn2 C~H), 5.25 (Ser CpOH), 6.40 (Om3 NH), 6.86 (GlY2 NH), 7.95 (Orn2 NH), 8.74 (Ser NH), 9.08 (GlYI NH), 10.3 (Om I NH). 75.5 MHz 13C-NMR (DMSO-d6) <5: 19.3, 19.8, 20.2 (hydroxamic acyl CH3), 23.4, 25.0 (Om C y), 28.3 (Orn l Cp), 28.6 (Orn2 Cp), 29.5 (Orn 3 Cp), 30.1 (Om C y), 45.2 (GlY2 C,,), 47.0 (GlYl C.), 51.3, 52.3, 52.5 (Om Co), 55.6 (Om3 CIl)' 55.8 (Orn2 Ca), 61.6 (Ser Ca), 61.6 (Om! Ca), 64.2 (Ser Cp), 165.3, 165.4, 165.7 (hydroxamic acyl C = 0), 171.7 (GlYl C=O), 172.1 (GIY2 C=O), 173.2 (Orn2 C=O), 173.9 (Orn3 C=O), 175.4 (Ser C=O), 178.4 (Orn l C=O).

Acknowledgments. We are grateful to Toray Research Center Inc. for conducting the amino acid analysis and measuring the CD spectral data. We thank Dr. Masayoshi Kusama, Mrs. Junko Iseda, and Mr. Tetsuya Tobita (Tobacco Science Research Laboratory of JAPAN TOBACCO INC.) for mass, JR, and NMR measurements.

References 1) D. J. Robeson and G. A. Strobel, Phytochemistry, 23, 1597-1599

(1984). 2) In "Fungi on Plants and Plant Products in the United States," ed.

by D. F. Farr, G. F. Bills, G. P. Chamuris, and A. Y. Rossman, APS Press, 1989, p. 638.

3) A. Ballio, A. Bottalico, V. Buonocore, A. Carilli, V. Di Vittorio, and A. Graniti, Phytopathol. Mediter., 8, 187-196 (1969).

4) A. Dell, R. C. Hider, M. Barber, R. S. Bordoli, R. D. Sedgwick, A. N. Tyler, and J. B. Neilands, Biomedical Mass Spectrometry, 9, 158-161 (1982).

5) M. Llinas, M. P. Klein, and J. B. Neilands, J. Mol. Bioi., 52,399-414 (1970).

6) M. Uinas, M. P. Klein, and J. B. Neilands, J. Mol. Bioi., 68,265-284 (1972).

7) M. Uinas, D. M. Wilson, M. P. Klein, and J. B. Neilands, J. Mol. Bioi., 104, 853-864 (1976).

8) G. B. Wong, M. J. Kappel, K. N. Raymond, B. Matzanke, and G. Winkelmann, J. Am. Chem. Soc., 105, 810-815 (1983).

9) W. Keller-Schierlein and A. Deer, Helv. Chim. Acta, 215, 1907-1929 (1963).

10) J. B. Neilands, Science, 156, 1443-1447 (1967). 11) c. L. Barnes, D. L. Eng-Wilmot, and D. V. D. Helm, Acta Cryst.,

C40, 922-926 (1984). 12) J. B. Neilands, Annu. Rev. Biochem., 50, 715-731 (1981). 13) G. Winkelmann, in "Biotechnology," Vol. 4, ed. by H. Pape and H.

J. Rehm, VCH, Weinheim, 1986, pp. 215-243. 14) H. Anke, J. Kinn, K. Bergquist, and O. Sterner, Bioi. Metal., 4,

176-180 (1991). 15) J. B. Neilands and S. A. Leong, Annu. Rev. Plant Physiol., 37, 187-

208 (1986).

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