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Interaction of the azole fungicide, flutriafol, with Leptosphaeria maculans.

Kate M. Adams1,2, Phillip A. Salisbury1,3 and Barbara J. Howlett2

1Victorian Institute for Dryland Agriculture, Horsham, 3400 VIC. 2School of Botany, the University of Melbourne, Parkville, 3052 VIC. 3Department of Crop Production, the University of Melbourne, Parkville, 3052 VIC.

ABSTRACT

The azole fungicide flutriafol (Impact in-furrow) is a highly effective chemical control agent used against Leptosphaeria maculans, the cause of blackleg disease in oilseed crops such as canola (Brassica napus). Flutriafol is capable of increasing yields many fold and is currently the fungicide of choice in Australia, particularly in high rainfall areas,or when varieties with low resitance are planted. Despite overall success, significant losses following flutriafol application have been observed and relatively little is known about its mechanism of action in L. maculans. In other fungal species flutriafol appears to act by binding to 14α-methylase (a member of the Cytochrome P450 superfamily) an enzyme involved in the ergosterol biosynthesis pathway. Azole resistance has been observed through mutation of the 14α-methylase active site and/or the development of alternate ergosterol biosynthesis pathways in Septoria tritici, Saccharomyces cerevisiae and other filamentous fungi. However, the propensity and mechanism of fungicide resistance in Australian L. maculans isolates has not yet been investigated.

KEYWORDS Brassica napus, blackleg fungus, ergosterol biosynthesis, 14α−methylase, mycorrhiza

BACKGROUND INFORMATION

Current methods used to reduce blackleg of Brassica napus (canola) caused by the fungus Leptosphaeria maculans include sowing of cultivars possessing disease resistance, crop rotation regimes, crop residue management and fungicide application (Salisbury et al.1995). As the canola industry continues to expand in Australia, pressure to minimise damage caused by this fungal pathogen is intensifying and the need to enhance these disease control methods is becoming crucial.

The azole fungicide flutriafol (Impact in-furrow) is a highly effective chemical control agent used against blackleg, capable of increasing yields many fold (Ballinger et al. 1988) and currently the fungicide of choice in Australia. However, significant losses following flutriafol application have been observed (Barbetti and Khangura, 1997) and relatively little is known about its mechanism of action in L. maculans.

In fungi, flutriafol appears to act by binding to an enzyme (a 14α-methylase belonging to the Cytochrome P450 superfamily) that is involved in the ergosterol biosynthesis pathway unique to fungi (Joseph-Horne et al. 1995). Ergosterol is critical for maintaining appropriate membrane fluidity, influencing activity of membrane-bound enzymes, regulating membrane permeability and regulating cell growth rates (Lees et al. 1995). A block in its biosynthetic pathway causes the production of substitute sterols that form inadequate membranes, resulting in decreased membrane fluidity, retarded growth and an inability to form invasive mycelia (Joseph-Horne et al. 1995). Azole resistance has been observed through mutation of the 14α-methylase active site and/or the development of alternate ergosterol biosynthesis pathways in Septoria tritici (Joseph-Horne et al. 1996) and Saccharomyces cerevisiae (Lees et al. 1995), microorganisms that share many characteristics of L. maculans. However, little is known about the propensity and mechanism of azole resistance in Australian L. maculans isolates.

The effect of flutriafol on soil microorganisms associated with canola is unknown. Roots of most terrestrial plants are symbiotically associated with fungi, that is, they are mycorrhizal. Growth of the plant is more rapid in these relationships as the presence of the fungi vastly increases the root surface area and expedites nutrient uptake from the soil. In exchange, the plant provides organic compounds for the fungi via root secretions (Brock and Madigan, 1991). Although preliminary studies of the mycorrhizal associations of canola have been performed (Salisbury, personal communication) a complete understanding is not available and little is known about the effect of flutriafol on any beneficial fungal species present.

The inconsistent efficacy of flutriafol as a control agent of L. maculans in Australia and the lack of understanding regarding its effect at molecular and ecological levels suggest that greater knowledge of the fungicide-fungi-crop interaction is required before use of the chemical becomes more routine. These, and topics that become pertinent as research progresses will form the content of my Ph.D. study.

References

1. Ballinger D.J., Salisbury P.A., Kollmorgen J.F., Potter T.D. and Coventry D.R. (1988) Evaluation of rates of flutriafol for control of blackleg of rapeseed. Australian Journal of Experimental Agriculture 28 : 517 - 519

2. Brock T.D. and Madigan M.T. (1991) In : Biology of microorganisms. Sixth Edition. Prentice Hall. pp. 661 - 662

3. Joseph-Horne T., Holloman D., Loeffler R.S.T. and Kelly S.L. (1995) Altered P450 activity associated with direct selection for fungal azole resistance. FEBS Letters. 374 : 174 - 178

4. Joseph-Horne T., Holloman D., Manning N. and Kelly S.L. (1996) Investigation of the sterol composition and azole resistance in field isolates of Septoria tritici. Applied and Environmental Microbiology 62(1) : 184 - 190

5. Lees N.D., Skaggs B., Kirsch D.R. and Bard M. (1995) Cloning of the Late Genes in the Ergosterol Biosynthetic Pathway of Saccharomyces cerevisiae – A Review. Lipids 30(3) : 221 - 226

6. Salisbury P.A., Ballinger D.J., Wratten N., Plummer K.M. and Howlett B.J. (1995) Blackleg disease on oilseed Brassica in Australia: a review. Australian Journal of Experimental Agriculture

7. 35 : 665 - 672

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