Mechanism of resistance in Penicillium italicum to fungicides which inhibit sterol 14alpha-demethylation

The fungitoxic action of azole fungicides is based on inhibition of cytochrome P450-dependent sterol 14α-demethylase (cytochrome P450 14DM ) activity. This thesis presents results of studies on various potential mechanisms of resistance to these sterol 14α-demethylation inhibitors (DMIs) in DMI-resistant isolates of the fungal plant pathogen Penicillium italicum. In chapters 2, 4, 6, 7 and 8 results of studies on four potential mechanisms of resistance are described. In chapters 3 and 5 two important developments in experimental procedures which were essential to test one of the potential mechanisms of resistance, decreased affinity of the target enzyme, are presented.In chapter 2, sterol composition of wild-type isolate W 5 and isolates E 300-3 (low resistance), H 17 (medium resistance), I 33 and J 4 (high resistance) is described. Results showed that in all isolates ergosterol was the major sterol. Inhibition of ergosterol biosynthesis in W 5 was observed at a low concentration of imazalil (0.01 μg m1 -1), while in the resistant isolates much higher concentrations were necessary to achieve the inhibitory effect. Inhibition of ergosterol biosynthesis was accompanied by accumulation of 24-methylenedihydrolanosterol. These results proved that the same target site for imazalil is present in all isolates and that the mechanism of resistance in this fungus is not related to absence of the target enzyme or the presence of a nonfunctional one.Chapter 3 describes an important achievement in studying in vitro enzymes involved in sterol biosynthesis in filamentous plant pathogenic fungi. A novel method to obtain a cell-free extract of P. italicum which was active in synthesizing ergosterol from [ 14C]mevalonate is presented and may serve as a model to study similar processes in other filamentous plant pathogens. The in vitro synthesis of ergosterol in cell-free extracts of P. italicum accounted for about 26% of total non- saponifiable lipids synthesized. The inhibitory effect of various test compounds (DMI fungicides and less-toxic imazalil analogues) on in vitro ergosterol biosynthesis was investigated. IC 50 values (concentrations which inhibit incorporation of radioactivity into ergosterol by 50%) of the highly toxic DMI fungicides imazalil, itraconazole, ketoconazole, penconazole and propiconazole ranged from 6.5 ± 0.5 x 10 -9to 1.7 + 0.7 x 10 -11M. This indicates that DMI fungicides are very potent inhibitors of sterol 14α- demethylase activity in cell-free extracts of the fungus. Less-toxic imazalil analogues had much higher IC 50 values, suggesting that these compounds have a significantly lower potency to inhibit sterol 14α-demethylase activity. The test method used to study ergosterol biosynthesis in the wild-type isolate W 5 could also be applied to DMI-resistant isolates E 300-3 , HP and I 33 , It was noted that the quality of cell-free extracts of wild-type and resistant isolates differed slightly. Nevertheless, IC 50 values of imazalil for inhibition of in vitro ergosterol biosynthesis were not significantly different in wild-type and all DMI-resistant isolates (Chapter 4). This indicates that sterol 14α-demethylase of sensitive and resistant isolates has a similar sensitivity to imazalil. Therefore, reduced affinity of the target enzyme to DMIs in resistant isolates will not play a major role as a mechanism of resistance.In Chapter 5, a method to isolate cytochrome P450 isozymes from wildtype isolate W 5 is described. Interaction between the heterocyclic nitrogen atom of the test compounds (DMI-fungicides and less-toxic imazalil analogues) and the oxidized heme iron atom of the isozymes could be demonstrated by difference spectrophotometry (type II spectra). However, the magnitude of the type II spectra did not correlate with toxicity of the test compounds. A difference in the ability of carbon monoxide (CO) to displace the test compound from reduced cytochrome P450 isozymes was observed. It appeared that the less-toxic imazalil analogues were immediately displaced while the displacement of various DMI fungicides gradually occurred in time. This suggests that the binding affinity of the test compounds to cytochrome P450 isozymes did correlate to some degree with fungitoxicity of the test compounds. However, inconsistent results were observed for DMI fungicides with a large N 1-substituent, like itraconazole and ketoconazole.Cytochrome P450 isozymes were also isolated from DMI-resistant isolates E 300-3 , H 17 , I 33 and J 4 (Chapter 6). However, the procedure for isolate H 17 , I 33 and J 4 had to be slightly modified in order to isolate proper P450 isozymes. The necessary modification of the isolation procedure suggests that the resistant isolates H 17 , I 33 and J 4 differ in some way, possibly in cell wall composition, from that of the wild-type isolate. Maximum and minimum absorbance in type II spectra of P450 isozymes of isolates H 17 , I 33 and J 4 shifted to higher wavelengths as compared with those of the wild-type isolate W 5 and low-resistant isolate E 300-3 , Maximum absorbance in CO spectra of P450 isozymes of isolates H 17 , I 33 and J 4 also shifted to higher wavelengths. Imazalil, itraconazole and ketoconazole were more readily displaced by CO from reduced P450 isozymes of isolates H 17 , I 33 and J 4 than from those of wild-type isolate W 5 and isolate E300-3 (Chapter 6). These results suggest that P450 isozymes of isolates H 17 , I 33 and J 4 had a relatively lower affinity to DMI fungicides. However, due to various factors which hamper a proper interpretation of the results, conclusions derived from spectrophotometric assays may not be reliable enough to compare affinity of cytochrome P450 14DM to DMIs in different isolates. Hence, these results do not provide sound evidence to reject the conclusion that sterol 14α-demethylase of wild-type and DMI-resistant isolates have a similar sensitivity to DMIs (Chapter 4).In Chapter 7, results of experiments on metabolism of imazalil in wildtype and DMI-resistant isolates are presented. It appeared that all isolates metabolized imazalil to its putative 2,3-dihydroxypropyloxy analogue (R42243), which was much less fungitoxic. DMI-resistant isolates showed cross resistance to miconazole which has a similar structure as imazalil except for the propenyloxy side chain. Therefore, it was concluded that metabolism of imazalil in P. italicum is not involved as a mechanism of resistance.Fungicide accumulation studies indicated that the low-resistant isolate E300-3 accumulated a significantly lower level of imazalil and fenarimol than the wild-type isolate W 5 (Chapter 8). The low level of accumulation of the fungicides may be responsible for the relatively low degree of resistance to these fungicides in this isolate. Reduced accumulation of fenarimol may be caused by an increased energy-dependent efflux of the fungicide. However, this mechanism is less obvious for imazalil. Accumulation of both fungicides is probably not mediated by the plasma membrane potential. Medium- and high-resistant isolates H 17 , I 33 and J 4 accumulated similar levels of imazalil and fenarimol as the low-resistant isolate E 300-3 . This suggests that the mechanism of resistance in medium- and high-resistant isolates is not caused by differential accumulation of the DMI fungicides as compared to the lowresistant isolate E 300-3 . However, it might be that differential accumulation of the DMIs between low- and high-resistant isolates is present, but masked by a relatively high background adsorption of the fungicides to mycelium and therefore, not detected with the test method used.From the results of the studies summarized above, it is concluded that the mechanism of resistance in P. italicum to DMIs relates to factors which prevent the fungicides from reaching the target site. Further studies should elucidate which mechanism is relevant in this respect.