Biochemical mechanisms involved in selective fungitoxicity of fungicides which inhibit sterol 14[alpha]-demethylation

J.C. Kapteyn

Research output: Thesisinternal PhD, WU


<p>Sterol demethylation inhibitors (DMIs) are antifungal agents which inhibit the biosynthesis of ergosterol by binding to cytochrome-P450-dependent sterol 14ce-demethylase (P450 <sub><font size="-2">14DM</font></sub> ). These compounds significantly differ in both toxicity and selectivity. This thesis presents results of studies on several potential mechanisms of selective fungitoxicity of DMIs.<p>Chapter 2 gives a literature review on the mode of action of DMIs, their specific interaction with P450 <sub><font size="-2">14DM</font></sub> and selective fungitoxicity.<p>Chapter 3 describes the selective fungitoxic actions of two DMIs, prochloraz and an experimental triazole fungicide, to selected plant pathogenic fungi showing significant differences in sensitivity to these compounds. Results indicate that the relatively high toxicity of prochloraz to the fungi tested could not be explained by the slightly higher acccumulation of prochloraz. As for prochloraz, there was no general correlation between sensitivity of the fungi tested and fungicide accumulation. The same situation held true for the triazole compound. However, the low-DMI-resistant isolate of <em>P. italicum</em> , E <sub><font size="-2">300-3</font></sub> , did exhibit decreased accumulation of both DMIs, which may be responsible for the low level of acquired DMI-resistance in this isolate. Since the high-resistant isolate H <sub><font size="-2">17</font></sub> did not show a further decrease in accumulation of both DMIs, additional mechanisms for resistance in this particular isolate may be involved. Results also show that under the experimental conditions used most fungi tested did not metabolise both DMIs. This implicates that fungal metabolism also does not play a major role in selective fungitoxicity of DMIs. Metabolism also does not generally explain the differences in toxicities of prochloraz and the triazole compound to each individual species. In this respect, <em>Rhizoctonia solani</em> behaved exceptionally, since this fungus metabolised prochloraz to a non- fungitoxic compound. This correlates with its low prochloraz sensitivity.<p>In Chapter 4 the role of plasma membrane potential in mediating the observed reduced accumulation of two other DMIs, fenarimol and imazalil, by resistant isolates of <em>P. italicum</em> was studied. As found for prochloraz and the experimental triazole compound (Chapter 3), the results confirm that decreased accumulation of these DMIs is responsible for a low level of resistance only and that additional mechanisms may operate in isolates with a medium and high degree of resistance. With all isolates of <em>P.</em><em>italicum</em> fenarimol accumulation was mediated by an energy- dependent efflux. This was not obviously the case for imazalil. Since no correlation between the accumulation of DMIs and tetraphenyl phosphonium bromide, a probe for measuring plasma membrane potential, could be found, it was concluded that the plasma membrane potential is not involved in accumulation of fenarimol and imazalil by <em>P. italicum</em> . Hence, the mechanism involved in the reduced fungicide accumulation remains unsolved.<p>Chapter 5 describes a procedure to isolate microsomal cytochrome P450 isozymes from <em>Ustilago maydis</em> . As demonstrated by difference spectroscopy (type II binding spectra) the the DMIs investigated bind with their heterocyclic nitrogen atom to the oxidized haem iron atom in the protoporphyrinic moiety of the isozymes. However, the DMI concentrations which cause half saturation of type II binding spectra (IC <sub><font size="-2">50</font></sub> -type II) did not correlate with the fungicidal activities of the azoles. In the carbon monoxide(CO)-displacement tests binding of CO to ferrous cytochrome-P450 was only slightly inhibited to different degrees by the DMIs tested. The inhibition of CO binding did not correlate with fungitoxicity of these DMIs. It was, therefore, concluded that the spectrophotometric studies are not useful for evaluating toxicity of DMIs to <em>U. maydis</em> .<p>In Chapter 6 the interaction of various DMIs and experimental compounds with P450 <sub><font size="-2">14DM</font></sub> from P. italicum was studied by difference spectroscopy using microsomal P450 isozymes, and assays with cell-free extracts capable of synthesizing ergosterol from [ <sup><font size="-2">14</font></SUP>C] mevalonate. Similar to the results obtained with microsomal preparations of <em>U. maydis</em> (Chapter 5), neither type II binding spectra nor CO-displacement tests were useful to assess fungitoxicity of the test compounds. In contrast, the cell-free sterol 14α-demethylase assay gave valuable information. I <sub><font size="-2">50</font></sub> values (concentrations of compounds which inhibit cell-free sterol synthesis by 50%) varied from 4.3x10 <sup><font size="-2">-9</font></SUP>to 4.4x10 <sup><font size="-2">-5</font></SUP>M. The assay ranked the compounds tested in order of fungitoxicity. Results presented in Chapter 6 support the general view that the inhibitory potency of azole fungicides against P450 <sub><font size="-2">14DM</font></sub> activity is influenced by the N, substituent of the azole moiety, since structural changes in the N <sub><font size="-2">1</font></sub> substituent of prochloraz clearly affected the inhibition of cell- free P450 <sub><font size="-2">14DM</font></sub> activity. However, the intrinsic inhibitory potency of azoles is not exclusively affected by their N <sub><font size="-2">1</font></sub> substituent. It was demonstrated that the nature of the azole moiety is also important.<p>Chapter 7 presents the development of a sterol 14α-demethylase assay with cell-free extracts of the filamentous plant pathogen <em>Botrytis cinerea</em> . Extracts were obtained by mechanical disruption of young germlings in a Bead-Beater apparatus. The C4-desmethyl sterol fraction accounted for 39% of the non-saponifiable lipids formed and consisted of three distinct compounds. Ergosterol was the major one. The cell-free system had a pH optimum for synthesis of C <sub><font size="-2">4</font></sub> -desmethyl sterols at pH 7.3-7.4. Cell-free synthesis of C <sub><font size="-2">4</font></sub> -desmethyl sterols was inhibited by the imidazole fungicide imazalil (IC <sub><font size="-2">50</font></sub> 9.lx10 <sup><font size="-2">-9</font></SUP>M).<p>The Botrytis assay was used to screen prochloraz and several prochloraz analogues for their intrinsic inhibitory potency (Chapter 8). Their IC <sub><font size="-2">50</font></sub> values ranged from 2.6x10 <sup><font size="-2">-9</font></SUP>to 4.4x10 <sup><font size="-2">-7</font></SUP>M. The compounds were also tested in cell-free assays of <em>Saccharomyces cerevisiae</em> . The test compounds were less potent in the yeast assay than in the one of <em>Botrytis</em> . Therefore, it was concluded that the <em>Botrytis</em> assay is more suitable for screening compounds biochemically for their potency to inhibit P450 <sub><font size="-2">14DM</font></sub> activity than the yeast assay. Results confirm that the inhibitory activity of prochloraz analogues on P450 <sub><font size="-2">14DM</font></sub> activity depends on the nature of their N <sub><font size="-2">1</font></sub> substituent and their azole moiety.<p>Unexpectedly, addition of an amino group at C <sub><font size="-2">2</font></sub> in the imidazole moiety of prochloraz caused a change in its mode of action in sterol synthesis of <em>P. italicum</em> and <em>B.</em><em>cinerea</em> (Chapter 8). This was supported by the fact that laboratory-generated triadimenol-resistant strains of <em>B. cinerea</em> exhibited cross resistance to triadimenol and prochloraz, but not to its amino-substituted analogue. Like most compounds, this analogue was less potent in the assays from <em>P. italicum</em> and <em>S. cerevisiae</em> than in the one from <em>B. cinerea.</em> In yeast, this compound did not inhibit sterol biosynthesis at another step in the pathway, but only P450 <sub><font size="-2">14DM</font></sub> activity. These findings indicate that biochemical screening tests should preferentially be carried out with cell-free assays from the target pathogen of interest.<p>It is concluded that the selective fungitoxicity of DMIs is in part determined by their potency to inhibit P450 <sub><font size="-2">14DM</font></sub> activity. Further studies should elucidate which other mechanisms are involved in selective fungitoxicity.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Dekker, J., Promotor, External person
  • de Waard, M.A., Promotor
Award date20 Apr 1993
Place of PublicationS.l.
Print ISBNs9789054851035
Publication statusPublished - 1993


  • plant protection
  • fungicides
  • fungi
  • mycology
  • pesticides
  • pesticidal action
  • pesticidal properties
  • toxicology
  • biochemistry

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