Biology of Sterol-biosynthesis Inhibiting Fungicides

Kück, Karl‐Heinz; Scheinpflug, H.

doi:10.1007/978-3-642-69790-6_3

Cited by 21 publications

(7 citation statements)

References 61 publications

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“…Azoles represent a major class of fungicides that has been extensively used over the past three decades in the control of fungal pathogens of medical and agricultural importance. They are systemic fungicides with both protective and curative activity in disease control 1. Their mode of action is based on inhibition of cytochrome P450 sterol 14α‐demethylase (P450 14DM ), a key enzyme involved in the biosynthesis of ergosterol in fungi.…”

Section: Introductionmentioning

confidence: 99%

Multiple mechanisms account for variation in base‐line sensitivity to azole fungicides in field isolates of Mycosphaerella graminicola

Stergiopoulos¹,

Nistelrooy²,

Kema³

et al. 2003

Pest Management Science

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Molecular mechanisms that account for variation in base-line sensitivity to azole fungicides were examined in a collection of twenty field isolates, collected in France and Germany, of the wheat pathogen Mycosphaerella graminicola (Fuckel) Schroeter. The isolates tested represent the wide baseline sensitivity to the azole fungicide tebuconazole described previously. The isolates were cross-sensitive to other azoles tested, such as cyproconazole and ketoconazole, but not to unrelated chemicals like cycloheximide, kresoxim-methyl or rhodamine 6G. Progenies from a genetic cross between an isolate with an intermediate and a high sensitivity to azoles displayed a continuous range of phenotypes with respect to cyproconazole sensitivity, indicating that variation in azole sensitivity in this haploid organism is polygenic. The basal level of expression of the ATP-binding cassette transporter genes MgAtr1-MgAtr5 from Mgraminicola significantly varied amongst the isolates tested, but no clear increase in the transcript level of a particular MgAtr gene was found in the less sensitive isolates. Cyproconazole strongly induced expression of MgAtr4, but no correlation between expression levels of this gene and azole sensitivity was observed. One isolate with intermediate sensitivity to azoles over-expressed CYP51, encoding cytochrome P450 sterol 14alpha-demethylase from M graminicola. Isolates with a low or high sensitivity to azoles were tested for accumulation of cyproconazole, but no clear correlation between reduced accumulation of the fungicide in mycelium and sensitivity to azoles was observed. Therefore, differences in accumulation cannot account exclusively for the variation in base-line sensitivity of the isolates to azoles. The results indicate that multiple mechanisms account for differences in base-line sensitivity to azoles in field isolates of M graminicola.

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Section: Introductionmentioning

confidence: 99%

Multiple mechanisms account for variation in base‐line sensitivity to azole fungicides in field isolates of Mycosphaerella graminicola

Stergiopoulos¹,

Nistelrooy²,

Kema³

et al. 2003

Pest Management Science

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“…Specific sterol 14α‐demethylation inhibitors (DMIs), such as azoles and other P450 type‐II ligands, are of great importance in agriculture and medicine as plant growth regulators and fungicides [3,13,14]. However, when DMIs display a curative activity against fungal diseases, they also have selective action against enzymes of non‐target organisms [15,16]. As a result, development of new and more selective inhibitors would gain from a better knowledge of the substrate specificity and active‐site structures of organism‐specific enzymes.…”

mentioning

confidence: 99%

Optimized expression and catalytic properties of a wheat obtusifoliol 14α‐demethylase (CYP51) expressed in yeast

Cabello‐Hurtado

Taton

Forthoffer

et al. 1999

European Journal of Biochemistry

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CYP51s form the only family of P450 proteins conserved in evolution from prokaryotes to fungi, plants and mammals. In all eukaryotes, CYP51s catalyse 14a-demethylation of sterols. We have recently isolated two CYP51 cDNAs from sorghum [Bak, S., Kahn, R.A., Olsen, C.E. & Halkier, B.A. (1997) proteins show a high identity (92%) compared with their identity with their fungal and mammalian orthologues (32±39%). Data obtained with plant microsomes have previously suggested that differences in primary sequences reflect differences in sterol pathways and CYP51 substrate specificities between animals, fungi and plants. To investigate more thoroughly the properties of the plant CYP51, the wheat enzyme was expressed in yeast strains overexpressing different P450 reductases as a fusion with either yeast or plant (sorghum) membrane targeting sequences. The endogenous sterol demethylase gene (ERG11) was then disrupted. A sorghum±wheat fusion protein expressed with the Arabidopsis thaliana reductase ATR1 showed the highest level of expression and activity. The expression induced a marked proliferation of microsomal membranes so as to obtain 70 nmol P450´(L culture) 21 , with CYP51 representing 1.5% of microsomal protein. Without disruption of the ERG11 gene, the expression level was fivefold reduced. CYP51 from wheat complemented the ERG11 disruption, as the modified yeasts did not need supplementation with exogenous ergosterol and grew normally under aerobic conditions. The fusion plant enzyme catalysed 14a-demethylation of obtusifoliol very actively (K m,app = 197 mm, k cat = 1.2 min 21) and with very strict substrate specificity. No metabolism of lanosterol and eburicol, the substrates of the fungal and mammalian CYP51s, nor metabolism of herbicides and fatty acids was detected in the recombinant yeast microsomes. Surprisingly lanosterol (K s = 2.2 mm) and eburicol (K s = 2.5 mm) were found to bind the active site of the plant enzyme with affinities higher than that for obtusifoliol (K s = 289 mm), giving typical type-I spectra. The amplitudes of these spectra, however, suggested that lanosterol and eburicol were less favourably positioned to be metabolized than obtusifoliol. The recombinant enzyme was also used to test the relative binding constants of two azole compounds, LAB170250F and g-ketotriazole, which were previously reported to be potent inhibitors of the plant enzyme. The K s of plant CYP51 for LAB170250F (0.29 mm) and g-ketotriazole (0.40 mm) calculated from the type-II sp 2 nitrogenbinding spectra were in better agreement with their reported effects as plant CYP51 inhibitors than values previously determined with plant microsomes. This optimized expression system thus provides an excellent tool for detailed enzymological and mechanistic studies, and for improving the selectivity of inhibitory molecules.

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“…Triazoles, such as propiconazole, may exhibit plant growth regulator properties in addition to antifungal activity. Triazoles were shown to interfere with and inhibit gibberellin biosynthesis and propiconazole was found to increase tree tolerance against frost, salt stress, drought, and to increase chlorophyll content in plants (Fletcher, 1985;Buchel, 1986;Kuck and Scheinpflug, 1986;Davis et al, 1988).…”

Section: Discussionmentioning

confidence: 99%

Persistence of Propiconazole in Peach Roots and Efficacy of Trunk Infusions forArmillariaRoot Rot Control

Amiri

Schnabel

2012

International Journal of Fruit Science

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Trunk infusion of peach with propiconazole has been suggested as a potential strategy to protect roots from colonization by Armillaria species. In the present study, we investigated the persistence of propiconazole in peach roots following fall infusions and its potential for Armillaria root rot control in a commercial peach orchard. Four 12-year-old trees were infused with either 2 liters of a propiconazole solution at 0.4 mg ml -1 or water in September 2007. Bark tissue collected from primary roots of propiconazole-infused trees 48 hr and 6 and 12 months after infusion and analyzed using gas chromatography-mass spectroscopy contained 6.4, 1.4, and 0.9 µg propiconazole per gram fresh bark, respectively. Asymptomatic trees adjacent to trees that had died from Armillaria root rot (protective treatment) and symptomatic but alive trees (curative treatment) were infused with 1 liter of a propiconazole solution (0.4 mg ml -1 ) in spring and fall of 2008 and 2009 at a commercial peach orchard planted in 2005. Trees were rated for Armillaria root rot severity 6, 12, 24, and 36 months post-infusion. In the protective treatment, the survival rate in both non-infused and infused trees was 80% 36 months post-infusion. However, more (60%) of the propiconazole-infused trees remained asymptomatic compared to the control trees (30%). A significant difference was observed between the non-infused and propiconazole-infused trees following curative treatment. None of the control trees were alive after 36 months compared to 40% of propiconazole-infused trees. 438A. Amiri and G. Schnabel We conclude that fall infusion of peripheral trees of infection centers with propiconazole can slow the expansion of Armillaria root rot infection centers in commercial peach orchards.

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Biology of Sterol-biosynthesis Inhibiting Fungicides

Cited by 21 publications

References 61 publications

Multiple mechanisms account for variation in base‐line sensitivity to azole fungicides in field isolates of Mycosphaerella graminicola

Multiple mechanisms account for variation in base‐line sensitivity to azole fungicides in field isolates of Mycosphaerella graminicola

Optimized expression and catalytic properties of a wheat obtusifoliol 14α‐demethylase (CYP51) expressed in yeast

Persistence of Propiconazole in Peach Roots and Efficacy of Trunk Infusions forArmillariaRoot Rot Control

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