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Ultrastructure and sterol composition of laboratory strains of Ustilago avenae resistant to triazole fungicides
PESTICIDE
BIOCHEMISTRY
AND
PHYSIOLOGY
26, 209-219 (1986)
Ultrastructure and Sterol Composition of Laboratory Strains of Ustilago avenae Resistant to Triazole Fungicides SIGRUN *Znstitut fiir Biologie Geschtiftsbereich
HIPPE*
AND
WOLFRAM
KijLLERt~l
ZZZ (Pj7anzenphysiologie), RWTH Aachen, D-5100 Pjlanzenschutz, Biologische Forschung, Bayerwerk, Federal Republic of Germany
Aachen, D-5090
tBayer Leverkusen.
and
AC,
Received November 21. 1985; accepted March 4, 1986 The tine structure and sterol composition of wild-type and triazole-resistant laboratory strains of Ustilago avenue was investigated by electron microscopic and biochemical methods. The growth rate of the mutants was only slightly affected by a fungicide (triadimefon) concentration of about 0.1 mg/ml, whereas the wild-type cells were completely inhibited. Biochemically the sterol composition of wild-type and triazole-resistant strains did not differ. In freeze-fracture electron microscopy no ultrastructural differences were observed between the different untreated strains (wild and resistant). Filipin labeling allowed the localization of ergosterol in the plasmalemma (PF and EF). Generally, wild-type samples and mutants exhibited a clear pattern of Elipin-sterol (FS-) complexes. These results are in accord with the biochemical experiments. Neither a modification of the sterol composition nor an altered localization of sterols seemed to be the prime cause of resistance in C.J. avenue mutants. Alternative explanations for the resistance mechanism are discussed. 0 1986 Academic Press, Inc.
opment of resistance under field conditions when compared with other site-specific antifungal compounds (5). In spite of these promising prospects, research on the biochemical mechanism of resistance remains important since the decreased fitness may or may not be a consequence of the molecular mechanism of resistance. The possibility of a recovery of resistant fungal mutants to full pathogenicity has been discussed (6). This potential recovery would change the likelihood of resistance development under practical conditions. At present several mechanisms of resistance have been described for this class of fungicides. The resistant mutants may present an altered membrane transport system leading to an induced and energy dependent efflux of the inhibitor (3, 4). A deficiency in C-14 methylation of sterols leading to an altered sterol metabolism has been discussed especially for Ustilago species (7-9). A decreased or stereochemically unfavorable conversion to triadimenol has been suggested as a potential mechanism of resistance for triadimefon (10).
INTRODUCTION
Fungicides belonging to the class of triazole derivatives constitute a large and widely used group of systemic compounds developed for the control of plant diseases (1). Triazole fungicides like triadimefon, triadimenol, or bitertanol have been identified as inhibitors of sterol biosynthesis blocking the C-14 demethylation step that occurs during the conversion of lanosterol to ergosterol (2). Although mutants resistant to this group of fungicides have been isolated in vitro with a mutation frequency close to that obtained with other site specific fungicides, a considerable development of resistance has not been observed under practical conditions (3,4). This interesting difference may be explained by a decrease of fitness and pathogenicity, a phenomenon which has been described for pathogens resistant to ergosterol biosynthesis inhibitors (3, 4). For these reasons triazole fungicides have been classified in the low risk category with respect to devel* Present address: Cornell University, New York State Agricultural Experiment Station, Department of Plant Pathology, Geneva, New York 14456. 209
00483575/86 $3.00 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
210
HIPPE
AND
In this report we describe the ultrastructure and sterol composition of Ustilago avenae mutants resistant to ergosterol biosynthesis inhibitors. This fungal test system has been used extensively for studies on the mode of action of triadimefon, triadimenol, and bitertanol ( 11- 16).
KOLLER
n
10
5en
/
t
5
1.
m
METHODS
Fungi. Resistant strains of Ustilago avenae were obtained by selection of uvirradiated sporidia on medium containing triadimefon at a concentration of 0.1 mg/ml. Resistant strains of fungi were maintained on malt agar slants containing triadimefon. Culturing conditions. The fungi were grown aerobically in loo-ml Erlenmeyer flasks containing 20 ml growth medium (15) on a reciprocal shaker at 20°C. Growth was determined on the basis of turbidity measurements at 660 nm. Electron microscopy. Sporidia of U. avenae were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, and simultaneously treated with filipin. Filipin (a gift from Dr. H. G. Endries, Upjohn) was dissolved in 100% ethanol and added to the fixative giving a final concentration of 50 pg filipin/ml fixative and 1% ethanol. Fixation and treatment with filipin was performed for 6 hr after which the cells were rinsed several times with 0.1 M phosphate buffer and rapidly cryofixed by a simple propane-double jet technique using copper-copper sandwiches (17). The freeze-etching procedure was performed in a Leybold-Heraeus BIOETCH 2005 Freeze Etcher under improved vacuum conditions of 5 x lop6 Pa (17). The replicas were cleaned carefully with 40% chromic acid overnight, rinsed several times in bidistilled water, and mounted on uncoated copper grids (150 mesh). According to routine methods a Zeiss EM 10 electron microscope operated at 60 kV was used for the analysis of the replicas. Sterol analysis. Sterols were extracted from freeze-dried sporidia and analyzed by
/ LI
ii
0
/rn
0 TIME
FIG.
1. Growth
resistant in liquid mefon
(rl, r2, culture. (0.1
of sporidia r8.
50
100
(hours)
of
r13) mutants n , control; 0,
\ci/d-type (sen) und of Ustilago avenae treated with triadi-
mglmn.
GC-MS as described elsewhere (18). Relative amounts were calculated by peak integration using a HP 5880 gas chromatograph. Toxicity assays. The activity of chemicals was tested by a radial diffusion assay. Filter paper disks (diameter 1 cm) were dipped in an acetonic solution of the test chemicals at various concentrations. The wet disks were air dried for 30 min and placed on the surface of a medium (15) containing agar and sporidia of U. avenue
TRIAZOLE
FUNGICIDE,
RESISTANT TABLE
Effects
of Toxicants
STRAINS
OF Ustilago
211
avenae
1
on the Growth
of Ustilago
Minimum Inhibitor
avenue
Concentration”
Toxicant
senb
rlc
r2C
r8’
r13’
Triadimenol Bitertanol Diclobutrazol Propiconazole Fenpropimorph Nvstatin
0.7 1.1 0.4 1.4 10.3 4.3
3.50 150 7 32 16 4
335 30 7 13 5 3
270 30 11 41 10 4
230 20 14 40 10 4
0 Lowest concentration (p&r/ml) of the fungicide on the saturated filter pads to obtain an inhibition b Wild-type strain. c Mutants selected on triadimefon.
(106/ml). Fungal growth was assessed after 2 days of incubation at 22°C by measuring the diameter of the inhibition zone. The minimum inhibitory concentrations was the lowest concentration producing an inhibition zone. The values were derived from dosage-response curves. RESULTS
Growth Rate Mutants of U. avenae resistant to triadimefon were readily obtained under laboratory conditions. The growth rate of all four mutants selected for this study was similar to the growth of the wild-type strain (Fig. 1). The replication of the wild-type sporidia, however, was completely inhibited by 0.1 mg/ml triadimefon whereas growth of the mutants appeared to be only slightly affected at this concentration (Fig. 1). Cross Resistance The sensitivity of triadimefon-resistant mutants to different representatives of sterol C-14 demethylation inhibitors (2) is indicated in Table 1. Cross-resistance appeared to be present in all cases. The cross-resistance between triadimefon and triadimenol clearly shows that an unfavorable activation of triadimefon can be ruled out as a mechanism of resistance (10). The mutants retained their normal sensitivity to fenpropimorph, an inhibitor of sterol C-14 (15) -double bond reduction (2), and nys-
zone.
tatin, like filipin a polyene macrolide antibiotic interacting with sterols in biological membranes (19). Fine Structure of Wild-Type and Fungicide-Resistant Sporidia A comparison of the fine structure of wild-type sporidia and triazole-resistant mutants using “rl”, as a representative strain, is shown in Figs. 2 and 3. No ultrastructural differences are observed between wild-type cells (Fig. 2) and mutants (Fig. 3). The following fine structure details are illustrated in Figs. 2 and 3: the cell wall (CW),2 the two fracture planes of the plasmalemma (PF, plasmatic fracture face and EF, extraplasmatic fracture face); the nucleus (N), mitochondria (M), vacuoles (V), vesicles (VE), and endoplasmatic reticulum (ER) are embedded in a homogeneously structured cytoplasma (CY) showing no signs of ice crystal formation. Sterols The sterols identified in resistant mutants of U. avenae were similar to the wildtype strain, both qualitatively and quantita-
2 Abbreviations used: CW, peripheric cell wall; PF, plasmatic fracture face, plasmalemma; EF, extraplasmatic fracture face, plasmalemma; N, nucleus; M, mitochondrion; V, vacuole; VE, vesicle; ER, endoplasmatic reticulum: CY, cytoplasm; FS filipin-sterol; IMP. intramembrane particles.
HIPPE
AND
KOLLER
FIGS. 2 AND 3. Fine stractare of freeze-etched wild-type sporidia (Fig. 2) und triawle-re,cistant specimens (Fig. 3) of Ustilago avenae. The followirlg altrastractural details are raisible: cell bc,all (CW), plasmatic fracture face (PF), and extraplasmatic fracture face (EF) of the pla.smalemma. mtclew (N), mitochondrion (M). vacuole (V). vesicles (VE). endoplasmatic reticalum (ER), cytoplasm (CY). The arrow’s at the bottom left of the figures indicate the direction of shadowing. FIG. 2. Wild-type sporidium of Ustilago avenae withoat addition of.filipin. Bar = 0.5 pm.
TRIAZOLE
FUNGICIDE,
RESISTANT
STRAINS
OF
Ustilago
avenae
213
FIG. 3. Triazole-resistant sporidia of Ustilago avenae. strain rl, in the absence offilipin. The two sporidia are longitudinally fractured. In comparison with Fig. 2, there are no differences in the ultrastructure between mutants and Triazole-sensitive, wild-type cells. Bar = 1 km. FIG. 4. Ultrastructure of triazote-resistant sporidia (rl strain) of Ustilago avenae after filipin labeling. The plasmalemma fracture faces (PF and EF) reveal distinct FS-complexes in form of pits on PF (b) and protrusions on EF (b). On the subcellular membrane system (nucleus, N; mitochondria, M; vacuoles, V; vesicles, VE) no FS-complexes are detectable at the time of investigation. Bar = 1 pm. The arrow at the bottom left of the$gure indicates the direction of shadowing.
214
HIPPE AND KOLLER
Distribution Sterol (% Total sterols) 24-Methylenedihydrolanosterol Obtusiofoliol 14-Methylfecosterol Ergosta-5,7-dienol Ergosterol Relative amountsd of total sterols
TABLE 2 of Sterols in Ustilago Species
Ustilago maydis5
Ustilago avenue
Wild type
erg 40
senb
rlc
r2’
r8C
r13c
22.9 0.4 5.9 70.8
65.2 21.1 13.7 -
100
2.3 97.7
loo
100
15.7 84.3
100
86
85
112
99
100
273
a Values calculated from (7). b Wild-type strain. c Mutants selected on triadimefon. d Based on dry weight (U. maydis) or cell number (U. avenae).
tively (Table 2). Ergosterol was characterized as the exclusive sterol in the sensitive wild-type strain and in resistant strains r2 and r8. Small amounts of 24-methylene dihydrolanosterol were detected in strains rl and r13 as a second sterol component. This precursor of ergosterol has been detected in wild-type U. maydis (7) and seems not to be related with the mechanism of resistance. The sterol composition of CJ. avenue mutants described in this report, however, is strikingly different from sterols identified in a U. maydis mutant (erg 40) resistant to ergosterol biosynthesis inhibitors (7). Ergosterol was completely absent in this mutant and C-14 methylated sterols strongly accumulated. Moreover, the total sterol content of this mutant was found to be significantly higher than that of the wild-type strain (Table 2). The presence of ergosterol and an absence of a large accumulation of C-14 methylated sterols were observed in mutants of U. avenue. Filipin Labeling Filipin labeling has been applied to wildtype and triazole-resistant strains of U. avenue to examine the planar distribution of ergosterol in the membranes. Generally, wild-type samples and mutants exhibit a clear pattern of filipin-sterol (FS)-com-
plexes under described conditions (Figs. 4, 7-10). FS-complexes can be visualized as small pits on PF formed in the lipid matrix in between the intramembrane particles (IMP) and corresponding protuberances on EF. During filipin labeling glutaraldehyde fixative is simultaneously applied both on the wild-type specimens and on the mutants. Thus, the membranes are stabilized, cell destruction by the polyene antibiotic is avoided, and a distinct formation of FScomplexes becomes possible. Organelle membranes such as those of the nuclei, the mitochondria, the vacuoles, and the vesicles as well as those of the endoplasmatic reticulum do not show any indication of filipin-sterol interaction (Fig. 4). The plasmalemma fracture planes appear smooth (Figs. 5 and 6) and after filipin treatment heterogeneously studded with FS-complexes (Figs. 4, 7-lo), thus indicating that the plasmalemma is the primary, sterol containing membrane in wild-type and triazole-resistant cells. In Figs. 5 and 6 the intramembrane structure of the plasmalemma, PF and EF, of wild-type samples prior to filipin application is shown. In the presence of filipin the lipid matrix forms pits on PF (Fig. 7) and protrusions on EF (Fig. 8) in dispersive distribution. Filipin as a cytochemical probe for ergosterol local-
FIGS. 5 AND 6. Plasmatic fracture face (PF, Fig. 5) and extraplasmatic fracture face (EF. Fig. 6) of the plasmalemma of wild-type sporidia of Ustilago avenae prior toj?lipin labeling. PF shows a high density of intramembrane particles (IMPS) whereas on EF far fewer IMPS are visible. The particles are embedded in a smooth and homogeneously structared lipid matrix. In comparison to the resistant strains there are no differences in the intramembrane ultrastructare of the plasmalemma. Bars = 0.1 km. The arrows at the bottom left at the figures indicate the direction of shadowing. FIGS. 7 AND 8. Sections of the plasmatic fracture face (PF, Fig. 7) and extraplasmatic fracture face (EF, Fig. 7) of the plasmalemma of wild-type sporidia of Ustilago avenae after filipin treatment. FS-complexes 0) can be visualized as distinct pits on PF (Fig. 7) and corresponding protrusions of EF (Fig. 8) formed in the lipid matrix in between the intramembrane particles in dispersive distribution. Bars = 0.1 pm. The arrows at the bottom of the figures indicate the direction of shadowing. 215
216
HIPPE
AND
FIGS. 9 AND 10. Sections of the plasmatic and zole-resistant sporidia (Figs. 9 and 10; strain r8) plexes (3) are randomly distributed on PF (Fig. 9) the bottom of the figures indicate the direction of
ization has also been used in triazole-resistant specimens. Figures 9 and 10 show two examples for the formation of FS-complexes on PF and EF of the r8 mutant. DISCUSSION
Fungal strains resistant to fungicides with a specific mode of action can be easily obtained in laboratory experiments (5). Therefore, it is not surprising that in vitro resistance to inhibitors of the C-14 demethylation step in lanosterol conversion has been described (3, 4). Failures of disease control due to the development of resistance under practical conditions, however, are not only dependent on the existence of resistant strains of the pathogen but depend on various factors including the fitness of resistant mutants, the type of disease, the mode of resistance development or the biochemical mechanism of resistance, (5, 20, 21). Thus, compared with other groups of
K6LLER
extraplasmatic fracture faces (PF and EF) of triaof Ustilago avenae after filipin labeling. FS-wrnand on EF (Fig. IO). Bars = 0.2 pm. The arrows at shadowing.
site-specific fungicides (22, 23), severe failures of disease control have not been reported for the widely used group of triazole fungicides (5). This striking difference might be related to the biochemical mechanism of resistance. Laboratory resistance to C-14 demethylation inhibitors has been reported for many fungi including Cladosporium cucumerinum, Aspergillus nidulans, Penicillium expansum, Botrytis cinerea, Ustilago maydis, Ustilago avenue, or Candida albicans (3, 4, 9, 24, 25). Only limited infor-
mation, however, is available on the ultrastructure and sterol composition of resistant strains. The results presented in this report indicate no significant difference between triazole fungicide-resistant U. avenae mutants and the wild-type strain regarding growth rate, ultrastructure, and sterol composition. Chemical analysis revealed the plasma
TRIAZOLE
FUNGICIDE,
RESISTANT
membrane as the predominant site for ergosterol in fungi [e.g., (26)]. The present freeze-fracture procedure allows the localization of ergosterol by means of the polyene antibiotic tilipin as a cytochemical probe. The membrane lesions in form of pits of PF and protrusions on EF arise from structural rearrangement of membrane lipids due to the formation of multimolecular complexes of tilipin and ergosterol (27-36). In spite of the feasibility of tilipin as a cytochemical probe care has to be taken in the interpretation of results. The effectiveness of the labeling, i.e., the filipin binding to the target site (sterol), is influenced by the preparation conditions (fixed or unfixed specimens), by the concentration of the filipin dose, by the labeling time, and, of course, by the penetration rate (37-39). Further critical factors determining the number and planar distribution of FS-complexes are the sterol content of the membranes and the mode of growth of the organisms (40,41). These difficulties might be responsible for differences between literature data about filipin binding on subcellular structures. On the one hand it has been reported that filipin does not bind on membranes of organelles (42-44); however, FS-complexes have been detected on intracellular membrane systems both on fungal and on animal specimens (4.5-50). The results presented for U. avenae in this report clearly indicate the plasma membrane as the predominant site of ergosterol. Furthermore, no differences are observed between wild-type sporidia and mutants resistant to triazole fungitides. Neither a modification of the sterol composition nor an altered localization of sterols seems to be the prime cause of resistance in U. avenae mutants. Thus, the mechanism of resistance is not related with the site of action of the C-14 demethylation inhibitors, a mechanism of resistance, which has been described for mutants of U. rnaydis (7) and U. avenae (8). These mutants, deficient in C-14 demethylation of sterols, showed a considerably slower
STRAINS OF Vstilago
avenae
217
growth rate and a heterogeneous and abnormal morphology. The U. avenae described in this report are clearly different. Although two different types of resistant Ustilago mutants seem to be possible (7-9, 24), only the mutants with an unmodified sterol composition seem to be a valuable model system for filamentous fungi. Attempts to select mutants of filamentous fungi with a deficient C-14 demethylation system have been unsuccessful (51), indicating that this defect might be lethal in this class of fungi. A mechanism of resistance based on an unaltered sterol metabolism might be explained by a modified target site, a reduced uptake as observed with fenarimol (3, 4), or a rapid detoxification of the inhibitors. These possibilities will be investigated in further experiments. ACKNOWLEDGMENTS The authors thank Professor Dr. H. J. Reisener (RWTH Aachen) and Professor Dr. H. Scheinpflug (Bayer AG) for their interest in this work. Thanks are also due to Mrs. M. Hermanns and Mr. R. Schiller for their skilled technical assistance. REFERENCES 1. E J. Schwinn, Ergosterol biosynthesis inhibitors. An overview of their history and contribution to medicine and agriculture. Pestic. Sci. 15, 40 (1983). 2. H. D. Sisler and N. N. Ragsdale, Biochemical and cellular aspects of the antifungal action of ergosterol biosynthesis inhibitors; in, “Mode of Action of Antifungal Agents” (A. P. J. Trinci and J. F. Ryley, Eds.), pp. 257, Cambridge Univ. Press, Cambridge, 1984. 3. M. A. de Waard and A. Fuchs, Resistance to ergosterol-biosynthesis inhibitors. II. Genetics and physiological aspects, in “Fungicide resistance in Crop Protection” (J. Dekker and S. G. Georgopoulos, Eds.), pp. 87, Pudoc, Wageningen, 1982. 4. M. A. de Waard and A. Fuchs, Resistance to ergosterol biosynthesis inhibiting fungicides, in “Systemische Fungizide und antifungale Verbindungen” (H. Lyr and C. Polters, Eds.). pp. 429. Akademie-Verlag. Berlin, 1983. 5. J. Dekker, Development of resistance to antifungal agents, in “Mode of Action of Antifungal Agents” (A. P. J. Trinci and J. F. Ryley, Eds.). pp. 89, Cambridge Univ. Press, Cambridge, 1984. 6. M. A. de Waard, H. Groeneweg, and J. G. M.
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RESISTANT
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46.
47.
48.
49.
50.
51.
STRAINS OF Usrilago
219
avenae
lipin treatment, J. Gen. Microbial. 131, 309 (1985). P. M. Elias, D. S. Friend, and J. Goerke, Freezefracture localization of cholesterol in cell and liposome membranes with saponins and filipin, .I. Ceil Biol. 79, 232 (1978). G. R. Gale, Cytology of Candida albicans as influenced by drugs acting on the cytoplasmic membrane, J. Bacreriol. 86, 151 (1963). S. C. Kinsky, G. R. Gronau, and M. M. Weber, Interaction of polyene antibiotics with subcellular membrane systems. Mol. Pharmacol. 1, 190 (1965). J. R. Sommer, P. C. Dolber, and I. Taylor, Filipincholesterol complexes in the sarcoplasmic reticulum of frog skeletal muscle, J. Ultrastruct. Res. 72, 272 (1980). H. Fujita, K. Ishimura, and H. Matsuda, Freezefracture images on filipin-sterol complexes in the thyroid follicle epithelial cell of mice with special regard to absence of cholesterol at the site of micropinocytosis, Hisiochemistly 73, 57 (1981). D. E. Harder and K. Mendgen, Filipin-sterol complexes in bean rust- and oat crown rustfungahplant interactions: Freeze-etch electron microscopy, Protoplasma 112, 46 (1982). L. Orci, R. G. Miller, R. Montesano, A. Perrelet, and M. Amherdt, Opposite polarity of filipininduced deformations in the membrane of condensing vacuoles and zymogen granules, Science 210, 1019 (1980). L. Orci, R. Montesano, P. Meda, F. MalaisseLagae, D. Brown, A. Perrelet. and P. Vassalli, Heterogeneous distribution of filipin-cholesterol complexes across the cisternae of the golgi apparatus, Proc. Natl. Acad. Sci. USA 78, 293 (1981). J. Ahoy, F. B. Merk, V. Goyal, and A. Ucci, Heterogeneous distribution of filipin-sterol complexes in nuclear membranes, Biochim. Biophys. Acta 649, 239 (1981). B. N. Ziogas, H. D. Sisler, and W. R. Lusby. Sterol content and other characteristics of pimaricin-resistant mutants of Aspergillus nidulans,
(1983).
Pesfic.
Biochem.
Physiol.
20,
320
BIOCHEMISTRY
AND
PHYSIOLOGY
26, 209-219 (1986)
Ultrastructure and Sterol Composition of Laboratory Strains of Ustilago avenae Resistant to Triazole Fungicides SIGRUN *Znstitut fiir Biologie Geschtiftsbereich
HIPPE*
AND
WOLFRAM
KijLLERt~l
ZZZ (Pj7anzenphysiologie), RWTH Aachen, D-5100 Pjlanzenschutz, Biologische Forschung, Bayerwerk, Federal Republic of Germany
Aachen, D-5090
tBayer Leverkusen.
and
AC,
Received November 21. 1985; accepted March 4, 1986 The tine structure and sterol composition of wild-type and triazole-resistant laboratory strains of Ustilago avenue was investigated by electron microscopic and biochemical methods. The growth rate of the mutants was only slightly affected by a fungicide (triadimefon) concentration of about 0.1 mg/ml, whereas the wild-type cells were completely inhibited. Biochemically the sterol composition of wild-type and triazole-resistant strains did not differ. In freeze-fracture electron microscopy no ultrastructural differences were observed between the different untreated strains (wild and resistant). Filipin labeling allowed the localization of ergosterol in the plasmalemma (PF and EF). Generally, wild-type samples and mutants exhibited a clear pattern of Elipin-sterol (FS-) complexes. These results are in accord with the biochemical experiments. Neither a modification of the sterol composition nor an altered localization of sterols seemed to be the prime cause of resistance in C.J. avenue mutants. Alternative explanations for the resistance mechanism are discussed. 0 1986 Academic Press, Inc.
opment of resistance under field conditions when compared with other site-specific antifungal compounds (5). In spite of these promising prospects, research on the biochemical mechanism of resistance remains important since the decreased fitness may or may not be a consequence of the molecular mechanism of resistance. The possibility of a recovery of resistant fungal mutants to full pathogenicity has been discussed (6). This potential recovery would change the likelihood of resistance development under practical conditions. At present several mechanisms of resistance have been described for this class of fungicides. The resistant mutants may present an altered membrane transport system leading to an induced and energy dependent efflux of the inhibitor (3, 4). A deficiency in C-14 methylation of sterols leading to an altered sterol metabolism has been discussed especially for Ustilago species (7-9). A decreased or stereochemically unfavorable conversion to triadimenol has been suggested as a potential mechanism of resistance for triadimefon (10).
INTRODUCTION
Fungicides belonging to the class of triazole derivatives constitute a large and widely used group of systemic compounds developed for the control of plant diseases (1). Triazole fungicides like triadimefon, triadimenol, or bitertanol have been identified as inhibitors of sterol biosynthesis blocking the C-14 demethylation step that occurs during the conversion of lanosterol to ergosterol (2). Although mutants resistant to this group of fungicides have been isolated in vitro with a mutation frequency close to that obtained with other site specific fungicides, a considerable development of resistance has not been observed under practical conditions (3,4). This interesting difference may be explained by a decrease of fitness and pathogenicity, a phenomenon which has been described for pathogens resistant to ergosterol biosynthesis inhibitors (3, 4). For these reasons triazole fungicides have been classified in the low risk category with respect to devel* Present address: Cornell University, New York State Agricultural Experiment Station, Department of Plant Pathology, Geneva, New York 14456. 209
00483575/86 $3.00 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
210
HIPPE
AND
In this report we describe the ultrastructure and sterol composition of Ustilago avenae mutants resistant to ergosterol biosynthesis inhibitors. This fungal test system has been used extensively for studies on the mode of action of triadimefon, triadimenol, and bitertanol ( 11- 16).
KOLLER
n
10
5en
/
t
5
1.
m
METHODS
Fungi. Resistant strains of Ustilago avenae were obtained by selection of uvirradiated sporidia on medium containing triadimefon at a concentration of 0.1 mg/ml. Resistant strains of fungi were maintained on malt agar slants containing triadimefon. Culturing conditions. The fungi were grown aerobically in loo-ml Erlenmeyer flasks containing 20 ml growth medium (15) on a reciprocal shaker at 20°C. Growth was determined on the basis of turbidity measurements at 660 nm. Electron microscopy. Sporidia of U. avenae were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, and simultaneously treated with filipin. Filipin (a gift from Dr. H. G. Endries, Upjohn) was dissolved in 100% ethanol and added to the fixative giving a final concentration of 50 pg filipin/ml fixative and 1% ethanol. Fixation and treatment with filipin was performed for 6 hr after which the cells were rinsed several times with 0.1 M phosphate buffer and rapidly cryofixed by a simple propane-double jet technique using copper-copper sandwiches (17). The freeze-etching procedure was performed in a Leybold-Heraeus BIOETCH 2005 Freeze Etcher under improved vacuum conditions of 5 x lop6 Pa (17). The replicas were cleaned carefully with 40% chromic acid overnight, rinsed several times in bidistilled water, and mounted on uncoated copper grids (150 mesh). According to routine methods a Zeiss EM 10 electron microscope operated at 60 kV was used for the analysis of the replicas. Sterol analysis. Sterols were extracted from freeze-dried sporidia and analyzed by
/ LI
ii
0
/rn
0 TIME
FIG.
1. Growth
resistant in liquid mefon
(rl, r2, culture. (0.1
of sporidia r8.
50
100
(hours)
of
r13) mutants n , control; 0,
\ci/d-type (sen) und of Ustilago avenae treated with triadi-
mglmn.
GC-MS as described elsewhere (18). Relative amounts were calculated by peak integration using a HP 5880 gas chromatograph. Toxicity assays. The activity of chemicals was tested by a radial diffusion assay. Filter paper disks (diameter 1 cm) were dipped in an acetonic solution of the test chemicals at various concentrations. The wet disks were air dried for 30 min and placed on the surface of a medium (15) containing agar and sporidia of U. avenue
TRIAZOLE
FUNGICIDE,
RESISTANT TABLE
Effects
of Toxicants
STRAINS
OF Ustilago
211
avenae
1
on the Growth
of Ustilago
Minimum Inhibitor
avenue
Concentration”
Toxicant
senb
rlc
r2C
r8’
r13’
Triadimenol Bitertanol Diclobutrazol Propiconazole Fenpropimorph Nvstatin
0.7 1.1 0.4 1.4 10.3 4.3
3.50 150 7 32 16 4
335 30 7 13 5 3
270 30 11 41 10 4
230 20 14 40 10 4
0 Lowest concentration (p&r/ml) of the fungicide on the saturated filter pads to obtain an inhibition b Wild-type strain. c Mutants selected on triadimefon.
(106/ml). Fungal growth was assessed after 2 days of incubation at 22°C by measuring the diameter of the inhibition zone. The minimum inhibitory concentrations was the lowest concentration producing an inhibition zone. The values were derived from dosage-response curves. RESULTS
Growth Rate Mutants of U. avenae resistant to triadimefon were readily obtained under laboratory conditions. The growth rate of all four mutants selected for this study was similar to the growth of the wild-type strain (Fig. 1). The replication of the wild-type sporidia, however, was completely inhibited by 0.1 mg/ml triadimefon whereas growth of the mutants appeared to be only slightly affected at this concentration (Fig. 1). Cross Resistance The sensitivity of triadimefon-resistant mutants to different representatives of sterol C-14 demethylation inhibitors (2) is indicated in Table 1. Cross-resistance appeared to be present in all cases. The cross-resistance between triadimefon and triadimenol clearly shows that an unfavorable activation of triadimefon can be ruled out as a mechanism of resistance (10). The mutants retained their normal sensitivity to fenpropimorph, an inhibitor of sterol C-14 (15) -double bond reduction (2), and nys-
zone.
tatin, like filipin a polyene macrolide antibiotic interacting with sterols in biological membranes (19). Fine Structure of Wild-Type and Fungicide-Resistant Sporidia A comparison of the fine structure of wild-type sporidia and triazole-resistant mutants using “rl”, as a representative strain, is shown in Figs. 2 and 3. No ultrastructural differences are observed between wild-type cells (Fig. 2) and mutants (Fig. 3). The following fine structure details are illustrated in Figs. 2 and 3: the cell wall (CW),2 the two fracture planes of the plasmalemma (PF, plasmatic fracture face and EF, extraplasmatic fracture face); the nucleus (N), mitochondria (M), vacuoles (V), vesicles (VE), and endoplasmatic reticulum (ER) are embedded in a homogeneously structured cytoplasma (CY) showing no signs of ice crystal formation. Sterols The sterols identified in resistant mutants of U. avenae were similar to the wildtype strain, both qualitatively and quantita-
2 Abbreviations used: CW, peripheric cell wall; PF, plasmatic fracture face, plasmalemma; EF, extraplasmatic fracture face, plasmalemma; N, nucleus; M, mitochondrion; V, vacuole; VE, vesicle; ER, endoplasmatic reticulum: CY, cytoplasm; FS filipin-sterol; IMP. intramembrane particles.
HIPPE
AND
KOLLER
FIGS. 2 AND 3. Fine stractare of freeze-etched wild-type sporidia (Fig. 2) und triawle-re,cistant specimens (Fig. 3) of Ustilago avenae. The followirlg altrastractural details are raisible: cell bc,all (CW), plasmatic fracture face (PF), and extraplasmatic fracture face (EF) of the pla.smalemma. mtclew (N), mitochondrion (M). vacuole (V). vesicles (VE). endoplasmatic reticalum (ER), cytoplasm (CY). The arrow’s at the bottom left of the figures indicate the direction of shadowing. FIG. 2. Wild-type sporidium of Ustilago avenae withoat addition of.filipin. Bar = 0.5 pm.
TRIAZOLE
FUNGICIDE,
RESISTANT
STRAINS
OF
Ustilago
avenae
213
FIG. 3. Triazole-resistant sporidia of Ustilago avenae. strain rl, in the absence offilipin. The two sporidia are longitudinally fractured. In comparison with Fig. 2, there are no differences in the ultrastructure between mutants and Triazole-sensitive, wild-type cells. Bar = 1 km. FIG. 4. Ultrastructure of triazote-resistant sporidia (rl strain) of Ustilago avenae after filipin labeling. The plasmalemma fracture faces (PF and EF) reveal distinct FS-complexes in form of pits on PF (b) and protrusions on EF (b). On the subcellular membrane system (nucleus, N; mitochondria, M; vacuoles, V; vesicles, VE) no FS-complexes are detectable at the time of investigation. Bar = 1 pm. The arrow at the bottom left of the$gure indicates the direction of shadowing.
214
HIPPE AND KOLLER
Distribution Sterol (% Total sterols) 24-Methylenedihydrolanosterol Obtusiofoliol 14-Methylfecosterol Ergosta-5,7-dienol Ergosterol Relative amountsd of total sterols
TABLE 2 of Sterols in Ustilago Species
Ustilago maydis5
Ustilago avenue
Wild type
erg 40
senb
rlc
r2’
r8C
r13c
22.9 0.4 5.9 70.8
65.2 21.1 13.7 -
100
2.3 97.7
loo
100
15.7 84.3
100
86
85
112
99
100
273
a Values calculated from (7). b Wild-type strain. c Mutants selected on triadimefon. d Based on dry weight (U. maydis) or cell number (U. avenae).
tively (Table 2). Ergosterol was characterized as the exclusive sterol in the sensitive wild-type strain and in resistant strains r2 and r8. Small amounts of 24-methylene dihydrolanosterol were detected in strains rl and r13 as a second sterol component. This precursor of ergosterol has been detected in wild-type U. maydis (7) and seems not to be related with the mechanism of resistance. The sterol composition of CJ. avenue mutants described in this report, however, is strikingly different from sterols identified in a U. maydis mutant (erg 40) resistant to ergosterol biosynthesis inhibitors (7). Ergosterol was completely absent in this mutant and C-14 methylated sterols strongly accumulated. Moreover, the total sterol content of this mutant was found to be significantly higher than that of the wild-type strain (Table 2). The presence of ergosterol and an absence of a large accumulation of C-14 methylated sterols were observed in mutants of U. avenue. Filipin Labeling Filipin labeling has been applied to wildtype and triazole-resistant strains of U. avenue to examine the planar distribution of ergosterol in the membranes. Generally, wild-type samples and mutants exhibit a clear pattern of filipin-sterol (FS)-com-
plexes under described conditions (Figs. 4, 7-10). FS-complexes can be visualized as small pits on PF formed in the lipid matrix in between the intramembrane particles (IMP) and corresponding protuberances on EF. During filipin labeling glutaraldehyde fixative is simultaneously applied both on the wild-type specimens and on the mutants. Thus, the membranes are stabilized, cell destruction by the polyene antibiotic is avoided, and a distinct formation of FScomplexes becomes possible. Organelle membranes such as those of the nuclei, the mitochondria, the vacuoles, and the vesicles as well as those of the endoplasmatic reticulum do not show any indication of filipin-sterol interaction (Fig. 4). The plasmalemma fracture planes appear smooth (Figs. 5 and 6) and after filipin treatment heterogeneously studded with FS-complexes (Figs. 4, 7-lo), thus indicating that the plasmalemma is the primary, sterol containing membrane in wild-type and triazole-resistant cells. In Figs. 5 and 6 the intramembrane structure of the plasmalemma, PF and EF, of wild-type samples prior to filipin application is shown. In the presence of filipin the lipid matrix forms pits on PF (Fig. 7) and protrusions on EF (Fig. 8) in dispersive distribution. Filipin as a cytochemical probe for ergosterol local-
FIGS. 5 AND 6. Plasmatic fracture face (PF, Fig. 5) and extraplasmatic fracture face (EF. Fig. 6) of the plasmalemma of wild-type sporidia of Ustilago avenae prior toj?lipin labeling. PF shows a high density of intramembrane particles (IMPS) whereas on EF far fewer IMPS are visible. The particles are embedded in a smooth and homogeneously structared lipid matrix. In comparison to the resistant strains there are no differences in the intramembrane ultrastructare of the plasmalemma. Bars = 0.1 km. The arrows at the bottom left at the figures indicate the direction of shadowing. FIGS. 7 AND 8. Sections of the plasmatic fracture face (PF, Fig. 7) and extraplasmatic fracture face (EF, Fig. 7) of the plasmalemma of wild-type sporidia of Ustilago avenae after filipin treatment. FS-complexes 0) can be visualized as distinct pits on PF (Fig. 7) and corresponding protrusions of EF (Fig. 8) formed in the lipid matrix in between the intramembrane particles in dispersive distribution. Bars = 0.1 pm. The arrows at the bottom of the figures indicate the direction of shadowing. 215
216
HIPPE
AND
FIGS. 9 AND 10. Sections of the plasmatic and zole-resistant sporidia (Figs. 9 and 10; strain r8) plexes (3) are randomly distributed on PF (Fig. 9) the bottom of the figures indicate the direction of
ization has also been used in triazole-resistant specimens. Figures 9 and 10 show two examples for the formation of FS-complexes on PF and EF of the r8 mutant. DISCUSSION
Fungal strains resistant to fungicides with a specific mode of action can be easily obtained in laboratory experiments (5). Therefore, it is not surprising that in vitro resistance to inhibitors of the C-14 demethylation step in lanosterol conversion has been described (3, 4). Failures of disease control due to the development of resistance under practical conditions, however, are not only dependent on the existence of resistant strains of the pathogen but depend on various factors including the fitness of resistant mutants, the type of disease, the mode of resistance development or the biochemical mechanism of resistance, (5, 20, 21). Thus, compared with other groups of
K6LLER
extraplasmatic fracture faces (PF and EF) of triaof Ustilago avenae after filipin labeling. FS-wrnand on EF (Fig. IO). Bars = 0.2 pm. The arrows at shadowing.
site-specific fungicides (22, 23), severe failures of disease control have not been reported for the widely used group of triazole fungicides (5). This striking difference might be related to the biochemical mechanism of resistance. Laboratory resistance to C-14 demethylation inhibitors has been reported for many fungi including Cladosporium cucumerinum, Aspergillus nidulans, Penicillium expansum, Botrytis cinerea, Ustilago maydis, Ustilago avenue, or Candida albicans (3, 4, 9, 24, 25). Only limited infor-
mation, however, is available on the ultrastructure and sterol composition of resistant strains. The results presented in this report indicate no significant difference between triazole fungicide-resistant U. avenae mutants and the wild-type strain regarding growth rate, ultrastructure, and sterol composition. Chemical analysis revealed the plasma
TRIAZOLE
FUNGICIDE,
RESISTANT
membrane as the predominant site for ergosterol in fungi [e.g., (26)]. The present freeze-fracture procedure allows the localization of ergosterol by means of the polyene antibiotic tilipin as a cytochemical probe. The membrane lesions in form of pits of PF and protrusions on EF arise from structural rearrangement of membrane lipids due to the formation of multimolecular complexes of tilipin and ergosterol (27-36). In spite of the feasibility of tilipin as a cytochemical probe care has to be taken in the interpretation of results. The effectiveness of the labeling, i.e., the filipin binding to the target site (sterol), is influenced by the preparation conditions (fixed or unfixed specimens), by the concentration of the filipin dose, by the labeling time, and, of course, by the penetration rate (37-39). Further critical factors determining the number and planar distribution of FS-complexes are the sterol content of the membranes and the mode of growth of the organisms (40,41). These difficulties might be responsible for differences between literature data about filipin binding on subcellular structures. On the one hand it has been reported that filipin does not bind on membranes of organelles (42-44); however, FS-complexes have been detected on intracellular membrane systems both on fungal and on animal specimens (4.5-50). The results presented for U. avenae in this report clearly indicate the plasma membrane as the predominant site of ergosterol. Furthermore, no differences are observed between wild-type sporidia and mutants resistant to triazole fungitides. Neither a modification of the sterol composition nor an altered localization of sterols seems to be the prime cause of resistance in U. avenae mutants. Thus, the mechanism of resistance is not related with the site of action of the C-14 demethylation inhibitors, a mechanism of resistance, which has been described for mutants of U. rnaydis (7) and U. avenae (8). These mutants, deficient in C-14 demethylation of sterols, showed a considerably slower
STRAINS OF Vstilago
avenae
217
growth rate and a heterogeneous and abnormal morphology. The U. avenae described in this report are clearly different. Although two different types of resistant Ustilago mutants seem to be possible (7-9, 24), only the mutants with an unmodified sterol composition seem to be a valuable model system for filamentous fungi. Attempts to select mutants of filamentous fungi with a deficient C-14 demethylation system have been unsuccessful (51), indicating that this defect might be lethal in this class of fungi. A mechanism of resistance based on an unaltered sterol metabolism might be explained by a modified target site, a reduced uptake as observed with fenarimol (3, 4), or a rapid detoxification of the inhibitors. These possibilities will be investigated in further experiments. ACKNOWLEDGMENTS The authors thank Professor Dr. H. J. Reisener (RWTH Aachen) and Professor Dr. H. Scheinpflug (Bayer AG) for their interest in this work. Thanks are also due to Mrs. M. Hermanns and Mr. R. Schiller for their skilled technical assistance. REFERENCES 1. E J. Schwinn, Ergosterol biosynthesis inhibitors. An overview of their history and contribution to medicine and agriculture. Pestic. Sci. 15, 40 (1983). 2. H. D. Sisler and N. N. Ragsdale, Biochemical and cellular aspects of the antifungal action of ergosterol biosynthesis inhibitors; in, “Mode of Action of Antifungal Agents” (A. P. J. Trinci and J. F. Ryley, Eds.), pp. 257, Cambridge Univ. Press, Cambridge, 1984. 3. M. A. de Waard and A. Fuchs, Resistance to ergosterol-biosynthesis inhibitors. II. Genetics and physiological aspects, in “Fungicide resistance in Crop Protection” (J. Dekker and S. G. Georgopoulos, Eds.), pp. 87, Pudoc, Wageningen, 1982. 4. M. A. de Waard and A. Fuchs, Resistance to ergosterol biosynthesis inhibiting fungicides, in “Systemische Fungizide und antifungale Verbindungen” (H. Lyr and C. Polters, Eds.). pp. 429. Akademie-Verlag. Berlin, 1983. 5. J. Dekker, Development of resistance to antifungal agents, in “Mode of Action of Antifungal Agents” (A. P. J. Trinci and J. F. Ryley, Eds.). pp. 89, Cambridge Univ. Press, Cambridge, 1984. 6. M. A. de Waard, H. Groeneweg, and J. G. M.
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FUNGICIDE,
RESISTANT
membranes of ergosterol-replaced Terrahymena cells, as studied by freeze-fracture electron microscopy, Biochim. Biophys. Acra 550, 269 (1979). 32. Y. Kitajima, T. Sekiya, and Y. Nozawa, Freezefracture ultrastructural alterations induced by filipin, pimaricin, nystatin and amphotericin B in the plasma membranes of Epidermophyron. Saccharomyces and red blood cells. A proposal of models for polyene-ergosterol complex-induced membrane lesions, Biochim. Biophys. Acfa 445, 452 (1976). 33. P. M. Elias, D. S. Friend, and .I. Goerke, Membrane sterol heterogeneity: Freeze-fracture detection with saponins and filipin, J. Histochem. Cytochem.
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