Bioregulators for Crop Protection and Pest Control - ACS Publications

Chapter 11

Inhibition of Phytotoxin (Viridiol) Biosynthesis in the Biocontrol Agent Gliocladium virens

Downloaded by UNIV OF ARIZONA on August 18, 2012 | http://pubs.acs.org Publication Date: May 27, 1994 | doi: 10.1021/bk-1994-0557.ch011

R. D. Stipanovic and C. R. Howell Southern Crops Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, College Station, TX 77845

Triazole fungicides that inhibit cytochrome P-450 enzymatic 14-α -demethylation of sterols also inhibit the biosynthesis of the phytotoxin viridiol produced by the biocontrol agent Gliocladium virens. Concentrations (0.5 - 1 ppm) of fungicides required to inhibit viridiol biosynthesis do not inhibit growth of the biocontrol agent. The plant pathogens Pythium ultimum and Rhizoctonia solani cause significant losses to cotton and other economically important agricultural crops. These pathogens generally attack the plant during the early stages of growth and are thus termed "seedling diseases." Until recently, various agrochemicals applied as a seed treatment were the only methods available to protect plants from these pathogens. The current emphasis on reducing the use of agrochemicals has led to a search for alternative means of controlling the plant pathogens that cause seedling diseases, and the exploitation of beneficial microbes as biocontrol agents has received increased scrutiny. Gliocladium virens as a Biocontrol Agent The fungus, Gliocladium virens, appears to offer a viable alternative or enhancement to agrochemicals in controlling seedling diseases (1). The biocontrol efficacy of G. virens has been attributed to both mycoparasitism and antibiosis (2,3) but the former appears to play only a minor role as a control mechanism (4). Rather, the production of antibiotics appears to be the principal method by which G. virens controls diseases incited by R. Solani or P. ultimum. Antibiotics. Four antibiotics isolated from G. virens have been identified: gliotoxin (5,6), gliovirin (7), viridin (8), and heptelidic acid (9-11). Of these, gliotoxin and gliovirin are the most active against R solani and P. ultimum, respectively (Table I) (12). This chapter not subject to U.S. copyright Published 1994 American Chemical Society In Bioregulators for Crop Protection and Pest Control; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.


11.

STIPANOVIC & HOWELL

Inhibition of Phytotoxin Biosynthesis

Gliotoxin

137

Gliovirin

Table I. Minimum Inhibitory Concentration fog/ml) of antibiotics from Gliocladium virens Antibiotic

Rhizoctonia solani

Gliotoxin Viridin Gliovirin Heptelidic Acid

Conidia 0.5 50 50 25

Mvcelia 5 >30 >30 >30

Pvthiwn ultimum Oospores Mvcelia 1 30 >30 25 0.5 0.05 20 10

Gliotoxin. Gliotoxin is classified as an epidithiodiketopiperazine. It shows a broad spectrum of activity against fungi, bacteria, and viruses (1) and inhibits farnesyl-protein transferase (13). A bridged disulfide appears to be essential for antimicrobial activity in gliotoxin (14) and other epidithiodiketopiperazines (15). Gliotoxin is highly toxic to R. solani but shows less activity against P. ultimum. Gliovirin. Gliovirin also contains a diketopiperazine ring and a disulfide group (8). However, unlike gliotoxin and related compounds, the disulfide group does not bridge directly across the diketopiperazine ring. Its activity appears to be more specific than gliotoxin and is active only against the Oomycetes, including P. ultimum (16).

In Bioregulators for Crop Protection and Pest Control; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

BIOREGULATORS FOR CROP PROTECTION AND PEST CONTROL

138


G. virens Strain Differences. Strains of G. virens show significant differences in their activity against P. ultimum and R. solani. Weindling (17) divided G. virens into two groups (i.e. P- and Q- strains) based on their pigment production on specific media. We find P-strains control P. ultimum more effectively than Qstrains, and Q-strains control Κ solani more effectively than P-strains (Howell, C. R., unpublished data). HPLC analysis of extracts from P-strains show that gliovirin, but not gliotoxin, is produced by these strains, whereas Q-strains produce gliotoxin but not gliovirin. Both strains produce viridin and the related steroid, viridiol. Thus, gliovirin in the P-strains and gliotoxin in the Q-strains appear to play pivotal roles in the biocontrol efficacy of these respective biocontrol agents. Phytotoxicity. The use of G. virens as a biocontrol agent has been plagued by the phytotoxicity of some of its metabolites. Both viridin and gliotoxin have been shown to inhibit seedling root growth of wheat, white mustard and red clover (18). More importantly, however, when cotton seed was coated with high concentrations of G. virens/rice preparations, they did not emerge, and the radical tips were necrotic (12). This indicated a more serious phytotoxic activity. In addition to the steroid viridin, the related compound viridiol has also been isolated from G. virens (19). GV-P strains of G. virens, when grown on rice, produce significant quantities of viridiol but only small quantities when grown on potato-dextrose agar (PDA). Seed treated with extracts from the latter produced healthy seedlings, while those from the former produced necrotic radicals (20). Indeed, G. virens grown on rice was found to be an effective pigweed herbicide. When viridiol was purified, it was found to be phytotoxic (20).

Viridiol

Dernemoxyviridin R ^ O H , R =H Dehya^oxydemetfK)xyviridin R^ R =H 2

2

Viridiol Biosynthesis. A number of publications indicate that viridiol is a byproduct of the biosynthetic conversion of lanosterol to ergosterol (Figure 1). Hanson, et. al. (21) have shown that G. deliquescens converts both demethoxyviridin and dehydroxydemethoxyviridin into viridiol. Jones and Hancock (22) found viridin was irreversibly reduced to viridiol and that viridin, but not viridiol, was rapidly taken up by G. virens mycelium from liquid culture.




11. STIPANOVIC & HOWELL


139

140


ο

HO'

ο' Virone

Wortmannobne


14

13

Biosynthetic studies with C-mevalonate (23) and C-acetate have shown the steroidal origin of viridin, demethoxyviridin (24), and wortmannin (25). The conversion of lanosterol to viridin (26) and squalene and lanosterol to viridiol (21) have been reported. Based on the various metabolites identified from the viridiol series, Hanson, et. al. (21) proposed that oxidative modification of ring A probably occurs in the later stages of biosynthesis. Based on the isolation of various precursors including virone and wortmannolone, they propose that earlier stages involve the formation of the furan ring, removal of the side chain, loss of the C18 methyl and aromatization of ring C. These observations lead one to conclude that viridiol is the end product in a series of steroid modifying steps which branch off from the main biosynthetic pathway of lanosterol to ergosterol. A teleologist would recognize that reduction of the ketone in viridin to an alcohol in viridiol would increase the water solubility of this compound and allow its diffusion into the surrounding soil. Its ability to act as a phytotoxin could aid survival of the microorganism by destroying root tissue and thus providing nutrients for the organism. Viridin exhibits some antibiotic activity (Table I), and thus probably increases the biocontrol efficacy of G. virens. It would, therefore, appear advantageous to retain this metabolite but eliminate viridiol. One may also conclude that the conversion of viridin to viridiol may be accomplished by a nonspecific alcohol dehydrogenase. Thus, attempts to selectively inhibit this conversion may be futile. Therefore, the usefulness of G. virens as a biocontrol agent could probably be improved by interrupting the viridiol pathway at an earlier stage, even though this would result in the loss of viridin production by the fungus. Inhibition of Viridiol Biosynthesis. There are a number of commercially available fungicides that operate by interrupting the conversion of lanosterol to ergosterol in fungi. These compounds have been shown to inhibit the cytochrome P-450 responsible for the 14a-demethylation of sterols. We found compounds such as propiconazole and flusilazole to effectively inhibit the production of viridin and viridiol at very low concentrations.



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Treatment of G. virens cultures with 0.5 to 1 ppm of these fungicides inhibited viridin and viridiol biosynthesis (Table II) without adverse effects on Table II. Effects of Fungicides on the Production of Viridiol and Other Metabolites of Gliocladium virens in culture 3

Strain (Treatment)

Viridiol

Viridin

Gliotoxin

Gliovirin

26.8 16.8 25.6 28.3

b

Ρ strains G-4(NT) G-8(NT) G-4(PC) G-8(PC)

15.1 12.5 -

11.0 7.0 4.0 5.5

-

Ο strains G-6 (NT) G-ll(NT) G-6 (FL) G-ll(FL)

24.1 31.0 -

3.0 4.0 -

7.7 9.8 6.3 7.3

Concentration of metabolite expressed in μg/100ml of culture medium. N T = No treatment; PC = Propiconazole; F L = Flusilazole.

b

the growth of the organism or the production of the nonsteroid antibiotics gliotoxin and gliovirin (Howell, C. R. and Stipanovic, R. D., unpublished data). Cottonseed treated with G. virens and a sterol inhibitor produced healthy and normal radicals, while those treated with G. virens alone produced radicals that were stunted and apical meristems that were necrotic.



142


The site of inhibition of these fungicides in the viridiol pathway is not known. As indicated above, the conversion of lanosterol to viridin (26) and viridiol (21) has been documented. Thus, viridin is a side product of the main lanosterol to ergosterol pathway (Figure 1). Fungicides that inhibit 14ademethylase effectively block viridiol biosynthesis, but the fungicide tolnaftate which inhibits squalene synthase does not effectively inhibit viridiol production. Blockage of the 14cc-demethylase reaction is unlikely since the organism appears to grow normally. Rather, these fungicides appear to be acting on another enzyme which is exquisitely sensitive to these compounds. Hanson's (21) proposed stages in the biosynthesis of viridiol led us to conclude that a likely site of action may be the cytochrome P-450 enzyme responsible for C-18 demethylation (i.e. the C-ring aromatase enzyme). This enzyme apparently operates after formation of the furan ring and removal of the side chain. Experiments to probe the validity of this hypothesis are presently under investigation. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

Jones, R. W.; Hancock, J. G. J. Gen. Microbiol. 1988, 134, 2067. Tu, J.C.;Vaartaja, O. Can. J. Bot. 1980, 59, 22. Howell, C. R.; Phytopathology 1982, 72, 496. Howell, C. R.; Phytopathology 1987, 77, 992. Weindling, R.; Emerson, Ο. H. Phytopathology 1936, 26, 1068. Weindling, R.; Phytopathology 1941, 31, 991. Stipanovic, R. D.; Howell, C. R. J. Antibiotics 1982, 35, 1326. Grove, J. F.; McCloskey, P.; Moffatt, J. S. J. Chem. Soc. (C) 1966, 1966, 743. Arigoni, D. Pure App. Chem. 1975, 41, 219. Itoh, Y.; Takahshi, S.; Haneishi, T.; Arai, M. J. Antibiotics 1980, 33, 468. Stipanovic, R. D.; Howell, C. R. Tetrahedron 1983, 39, 1103. Howell, C. R. Phytopathology 1991, 81, 738. Van der Pyl, D.; Inokoshi, J.; Shiomi, K.; Yang, H.; Takeshima, H.; Omura, S. J. Antibiotics 1992, 45, 1802. Cavillito, J. C.; Bailey, J. H.; Warner, W. F. J. Amer. Chem. Soc. 1946, 68, 715. Michel, K. H.; Chaney, M. O.; Jones, N. D.; Hoehn, M. M.; Nagarajan, R. J. Antibiotics 1974, 27, 57. Howell, C. R.; Stipanovic, R. D. Can. J. Microbiol. 1983, 29, 321. Weindling, R.; Phytopathology 1934, 24, 1153. Wright, J. M. Ann. Bot. 1951, 60, 493. Moffatt, J. S.; Bu'Lock, J. D.; Tse Hing Yuen, T.L.S. J. Chem. Soc. (D), 1969, 1969, 839. Howell, C. R.; Stipanovic, R. D. Phytopathology 1984, 74, 1346. Hanson, J. R.; O'Leary, Μ. Α.; Wadsworth, H. J.; Yeoh, B. L. Phytochemistry 1988, 27, 387. Jones, R. W.; Hancock, J. G. Can. J. Microbiol. 1987, 33, 963.


11. STIPANOVIC & HOWELL 23. 24. 25. 26.

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Grove, J. F.; J. Chem. Soc. (C) 1969, 1969, 549. Hanson, J. R.; O'Leary, Μ. Α.; Wadsworth, H . J. J. Chem. Soc. Perkin Trans. I 1983, 1983, 867. Simpson, T. J.; Lunnon, M . W.; MacMillan, J. J. Chem. Soc. Perkin Trans. I. 1979, 1979, 931. Golden, W. S.; Watson, T. R. J. Chem. Soc. Perkin Trans. I 1980, 1980, 422. December 6, 1993


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