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Mechanism of resistance to fenarimol in Aspergillus nidulans
PESTICIDE
BIOCHEMISTRY
Mechanism Laboratory
AND
PHYSIOLOGY
10,219-229
(1979)
of Resistance
to Fenarimol
M. A. DE
AND J. G. M. VAN NISTELROOY
of Phytopathology,
WAARD
Binnenhaven
9, 6709 PD
in Aspergilhs Wageningen.
nidulans
The Netherlands
Received April 13, 1978; accepted June 21, 1978 Mycelial uptake of [‘4C]fenarimol (10 &ml) by 20 fenarimol-resistant mutants of Aspergiks was compared with uptake by wild-type strain 003. Uptake of the fungicide during the initial 10 min of incubation was significantly lower in all mutant strains than in the wild-type strain indicating that resistance is related with reduced uptake. Upon prolonged incubation a gradual decrease of accumulated radioactivity in the wild-type strain was observed. A few mutants displayed resistance to unrelated chemicals such as p-fluorophenylalanine or o-serine; this phenomenon appeared not to be due to a decreased uptake of the corresponding natural amino acids. Incorporation of [Wladenine and [‘4C]leucine by mycelium of mutant Ml93 was hardly inhibited after 5 hr of incubation with the fungicide, whereas a distinct effect was found with the wild-type strain. At this time also fungitoxicity to the wild-type strain became apparent. Probably, this effect is indirectly caused by inhibition of ergosterol biosynthesis. Mycelium of mutant Ml93 incorporated [‘PC]acetate slightly less effectively than the wild-type strain. After 2 hr of incubation with this radiochemical leakage of [‘4C]acetate metabolites from mycelium of the mutant strain was observed. This indicates that resistance might be correlated with increased excretion of fungal metabolites, which in turn may be related with reduced fitness of fenarimol-resistant mutants. nidulans
INTRODUCTION
Fenarimol [EL-222; cu-(Zchlorophenyl)a-(4-chlorophenyl)-5pyrimidinemethanoll is a new systemic fungicide for control of diseases such as powdery mildews and apple scab (1). It is a broad-spectrum fungicide with antifungal in vitro activity against several Ascomycetes, Basidiomycetes, and Fungi imperfecti (Technical Report on EL222). Also some Oomycetes are sensitive (2). Both fenarimol and the structurally related chemical triarimol [EL-273; a-(2,4dichlorophenyl)-cY-phenyl-5-pyrimidinemethanol] are highly toxic to, among others, Aspergillus nidulans (3). The chemicals primarily inhibit germ tube elongation and mycelial growth, while spore germination is significantly less sensitive. Triarimol is a potent inhibitor of ergosterol biosynthesis in Ustilago maydis (4-6). Fenarimol has a similar effect in A. nidulans (2) and in U. avenue (7). Therefore, fenarimol can be classified in the group of ergosterol biosynthesis-inhibiting fungicides such as triforine (5, 8)) Denmert (S-1358) (9), triadimefon (lo-12), flue-
trimazole (lo), imazalil (13), and nuarimol (7). Development of resistance in fungi to systemic fungicides with a specific mechanism of action is quite common ( 14). Therefore, it is not surprising that in vitro resistance to ergosterol biosynthesisinhibiting fungicides has been observed (3, 8, 15- 17). Up until now no development of resistance to these fungicides under practical conditions has been reported ( 17, 18). Absence of resistance under practical conditions may be due to decreased pathogenicity of resistant strains; this phenomenon, related with diminished spore germination, germ tube elongation, mycelial growth, and sporulation has been described for triarimol- and triforine-resistant mutants of Cladosporium cucumerinum (17, 19). Decreased fitness of fenarimoland imazalil-resistant strains of A. nidufans appeared to be due to the same phenomena (3, 16). Since the practical importance of ergosterol biosynthesis-inhibiting fungicides is 219 0048-3575179/020219-11$02.00/O Copyright @ 1979 by Academic Press. Inc. AU rights of reproduction in any form rrwrved.
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AND
still increasing, it seemed worthwhile to investigate the biochemical mechanism of resistance. According to previous studies with fenarimol and A. nidulans, resistance proved not to be caused by enhanced metabolism of fenarimol (2) nor was it accompanied with a major change in sterol patterns (20). This paper deals with other possible mechanisms of fenarimol resistance . MATERIALS
AND
METHODS
Organism. Aspergillus nidulans biA1 acrA1 requiring biotin, resistant to acriflavin, and with wild-type sensitivity to fenarimol and related chemicals (strain 003) was used in all experiments. Fenarimolresistant strains were obtained by selection of untreated (sp) or uv-irradiated (uv) conidia on fenarimol-containing media (15, 16). The genetic basis for resistance was due to an imaB gene in strain 5146 (sp) and Ml93 (uv) and an imaD gene in strain 5250 (sp). Strain 5146’ is a new subculture of 5146 selected on fenarimol-containing agar. Strain R264 is a recombinant strain carrying imaA and imaB genes and a gene which fortifies the action of the imaA gene (15). The genetic basis of all other strains used is unknown. Chemicals. Fenarimol and [a-14C]fenarimol (sp act 19 @i/mg) were gifts of Eli Lilly Company, Indianapolis, Illinois. The radiochemicals [U-14C]acetate, [8-3H]adenine, L-[U-14C]leucine, L-[U-14C]phenylalanine, and t.-[U-14C]serine were obtained from the Radiochemical Centre, Amersham, England. Culture methods. Conidia of A. nidulans strains were grown on a 2% malt extract agar medium, supplemented with 1 mg biotin/liter. Conidial suspensions with approximately IO9 conidia/ml were added to Erlenmeyer flasks which contained 100 ml glucose-nitrate medium (21) supplemented with biotin, to give a final concentration of 10’ conidia/ml. Flasks were incubated at 37°C on a Gallenkamp orbital shaker at 180 rpm for about 16 hr. Then, the mycelium was harvested by filtration on a Biichner
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filter and washed two times by resuspending 5 g of wet mycelium in 250 ml of glucose-nitrate medium. The final standard mycelial suspension was made by resuspending 1 g of mycelium in 50 ml of fresh medium. Thereafter, incorporation experiments were mostly started after 30-45 min preincubation of the suspension at 37°C in a reciprocal water-bath shaker. Toxicity of fungicides. Fungitoxicity of chemicals was usually determined in mycelial growth tests. Chemicals were added as a solution in methanol or water to molten glucose-nitrate agar medium. The final methanol concentration in the medium, identical in controls and treatments, never exceeded 1%. The medium was poured in 9-cm Petri dishes and inoculated by placing three inverted 5-mm agar disks with young mycelium on the agar. Radial growth was measured in duplicate after 3 days of incubation at 37°C. Toxicity of fungicides in shake cultures was established by adding the chemicals to standard mycelial suspensions in Erlenmeyer flasks. Growth was measured by determining increase in fungal dry weight of the cultures. Three IO-ml samples per treatment were filtered using preweighed filter papers. After washing with distilled water the filter papers were dried overnight at 60°C and reweighed. Uptake and incorporation of radiochemicals by mycelium. Standard mycelial suspensions were incubated in a reciprocal shaker at 37°C. Radiochemicals were added to the suspension at final concentrations of 100 /.L&! acetate (sp act 215 &i/mmol), 10 @V adenine (sp act 5 mCi/mmol), 30 w fenarimol (sp act ca. 67 &i/mmol), 100 m L-leucine (sp act 116 ,&i/mmol), 100 m L-phenylalanine (sp act 230 &i/mmol), and 100 /..N L-serine (sp act 447 &ilmmol). After various incubation times 5-ml samples of mycelial suspensions were filtered over Whatman GF/A glass filter paper using a Millipore sampling manifold filter apparatus. The mycelial residues were washed five times within 30 set with 5 ml
RESISTANCE
TO
FENARIMOL
glucose-nitrate medium. Radioactivity was determined by transferring the filters with mycelial residue into vials containing 10 ml scintillation- liquid (3 g PPO in a mixture of 750 ml xylene and 250 ml Triton X-100; Lumac B.V., Amsterdam, The Netherlands) and by counting in a Nuclear Chicago Mark I liquid scintillation spectrometer. Linear regression analysis and calculation of correlation coefficients (Y) of the relation between uptake and incorporation of radiochemicals versus incubation time were carried out with a Diehl algotronic calculator. RESULTS
Toxicity
of Fenarimol
The ED5,, value of fenarimol for radial mycelial growth of the wild-type strain 003 of A. nidulans was found to be about 3.8 pg/ml (Fig. 1). ED,,, values for radial growth of the resistant strains tested were maximally ca. fivefold as high as that of the wild-type strain, indicating that the degree of resistance was relatively low. Differences between minimal inhibitory concentrations, on the other hand, were much larger since 30 &ml inhibited growth of the wild-type strain completely, while some resistant mutants even grew at 500 &ml. Fenarimol added to standard mycelial suspensions hardly inhibited growth during the initial 3.5 hr of incubation irrespective
IN
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NIDULANS
of the concentration used (Fig. 2). Thereafter, growth was inhibited by a concentration of at least 10 &ml. This concentration was used in most of the following experiments. Cross-resistance
In previous studies (2, 3, 5, 8, 15, 16) fenarimol-resistant strains of A. nidulans often displayed cross-resistance to other inhibitors of sterol biosynthesis such as imazalil and triadimefon. Cross-resistance to a number of unrelated chemicals was investigated in this study in radial growth tests. Four out of 12 resistant mutants tested showed a pronounced change in sensitivity to chemicals such as 8-azaguanine (J146), p-fluorophenylalanine (M 193 and J250), n-serine (M193), thiourea (Ml93 and 5146) and 3-aminotyrosine (M193) (Table I). Uptake
of Amino
Acids
Resistance to antimetabolites like p-fluorophenylalanine and n-serine is often ascribed to a deficiency in amino acid transport (22-24). Strain J250 and Ml93 with resistance to p-fluorophenylalanine and n-serine, respectively, might therefore be unable to take up L-phenylalanine and L-serine. This was tested by measuring the
100 -g ’
8
z 5 ,” 2
90 80 70 60 50 LO
g 30 z r, 20 e 10
0)
0 1 FIG.
10 concentration 1. Dosage
radial growth fenarimol-resistant
100 (jdg/ml
500 1
-response curves of fenarimol for of the wild-type strain 003 and mutants of Aspergillus nidulans.
0
4 incubation
time
8 (h 1
FIG. 2. Toxicity offenarimol for mycelial growth of the wild-type strain 003 of Aspergillus nidulans in liquid cultures. Treatments: control (0). 5 (0). 10 fO), 20 (m), and 40 f x) pglml fenarimol.
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Cross-Resistance
WAARD
of Fenarimol-Resistant
Compound
I-Azaguanine p-Fluorophenylalanine n-Serine Thiourea
AND
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NISTELROOY
TABLE
1
Mutants
of Aspergillus
nidulans
to Antimetabolites
003
Ml93
5146
5250
R264
10 24 112 70
EDso G.&N 10 12 MOO 180
4 16 120 240
12 2100 85 -
9 30 70 -
82 60 41
107 52 36
Growth as percentage of control 3-Aminotyrosine (3 mg/ml) L-Ethionine (5 mgknl) Thiourea (100 &ml)
88 47 47
uptake of L-[ 14C]phenylalanine and L-[ ‘4Clserine in standard mycelial suspensions. Uptake of both amino acids by the mutant strains proved to be similar to that of the wild-type strain during 1 hr of incubation (Fig. 3). Utilization of the amino acids was also tested by growing the wild-type and mutant strains on synthetic agar media with L-phenylalanine and L-serine as sole nitrogen source (22). Results showed that radial growth of all strains tested did not differ much (Table 2), indicating again normal ability to take up the amino acids.
F 5 1601 1LO 120 100 80 60 LO 20 0 0
10
20 30 incubation
LO 50 60 time ( min 1
FIG. 3. Uptake and incorporation of L-[Tlserine by mycelium of Aspergillus nidulans wild-type strain 003 (O), and D-serine-tolerant mutant Ml93 (0); ditto of [‘4C]phenylalanine by the wild-type strain (0) and p-jiuorophenylalanine-tolerant mutant 5250 (U). Dry weights of IO-ml mycelial suspension (0) 27.7. (0) 33.2, (0) 30.0, and (m) 31 .I mg.
33 66 86
94 67 83
Effect of Fenarimol on Uptake and Incorporation of Radiochemicals The fungicide miconazole inhibits uptake of adenine, guanine, and hypoxanthine by cells of Candida al&cans (25). The fungicide is chemically related to imazalil. Both fenarimol and imazalil inhibit ergosterol biosynthesis in fungi (2,7). Therefore, it was of interest to examine whether fenarimol affected the uptake of the radiochemicals [ ‘4C]acetate, [ 3H]adenine, and [‘4C]leucine by mycelium of the wildtype strain and of mutant M193. Uptake and incorporation of [‘T]acetate was investigated by addition of the radiochemical to mycelial suspensions with 10 Mm1 fenarimol. Every 5 min radioactivity in washed mycelium of 5-ml samples was measured. In both strains uptake and incorporation proved to be almost linear with time during the first hr of incubation and was unaffected by the presence of fenarimol (Fig. 4). The observed relatively low uptake and incorporation of [%]acetate by mutant strain Ml93 (Fig. 4) might also be due to increased excretion of [ Ylacetate metabolites. Therefore, in a following experiment it was examined whether after 2 hr of preincubation of mycelium with [%]acetate when maximal uptake and incorporation of the radiochemical was found, addition of 10 pug/ml fenarimol induced a leakage of [%]acetate and [ 14C]acetate metabolites. Figure 5
RESISTANCE
TABLE
TO
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IN
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NIDULANS
2
Effect of the Nitrogen Source in Synthetic Nutrient Medium on Radial Growth ofAspergillus nidulans 003 (wild-type strain), Ml93 (D-serine tolerant), and J250 (p-fluorophenylalanine tolerant) Radial growth Strain
NaNO,
003 Ml93
25.0 21.3 24.0
J250 ” Relatively
(mm)
L-Phenylalanine
+ 0 + 1.2 + 0
19.7 + 0.6 24.0 A 1.0’ 22.0 ‘- 1.0
thin mycelium
L-Serine
003-control 003-fenarmol
lo1 (01
r 012 032
19.7 e 0.6
M193-control M193-fenarlmol
IO) (=I
-0Ll -051
18.0 k 1.0 f 2.1
22.3
8
I
0
mat.
10
I
20
I
1
I
I
30
LO
50
60
incubation
shows that radioactivity in mycelium of the wild-type strain remained essentially constant during 1 hr of incubation, while in strain Ml93 a slight decrease of radioactivity was noticed. The latter results might indicate that low amounts of fungal constituents were excreted by mycelium of strain M193. No differences were observed between controls and fenarimol treatments. Uptake and incorporation of [W]adenine and [‘CJleucine were investigated as described before for [‘4C]acetate and measured after 1, 3, and 5 hr of preincubation 5 2201
timt,
( mm 1
5. Effect offenarimol(l0 Hplml) on content of [Tlacetate and [Wlacetate metabohtes in myrelium of Aspergillus nidulans strain 003 (wild type) and Ml93 (fenarimol resistant). Fenarimol added at zero time after2 hr preincubation with [‘Tlacetate. Dry weights of IO-ml suspension strain 003: 28.4 and Ml93: 25.6 w. FIG.
with 10 CLg/ml fenarimol. Pronounced differences with both chemicals were only observed with mycelium of the wild-type strain 003, preincubated with fenarimol for 5 hr; under similar conditions such differences were not found with mycelium of strain Ml93 (Figs. 6 and 7). Uptake of [14C]Fenarimol
Uptake of [ “‘Qfenarimol was determined by measuring radioactivity in mycelium of 5-ml samples after filtration over glass filter paper and five consecutive washings with
003-fencmmol M 193 -control M193-fenanmol
0
10
20 30 incubation
(*I 099 (01 0 99 (*I 098
LO 50 60 time (mm )
FIG. 4. Effect offenarimol (IO pglml) on uptake and incorporation of [‘Tlacetate by mycelium of Aspergillus nidulans strain 003 (wild-type) and Ml93 (fenarimol-resistant). [‘*C]Acetate added at zero time after I hr preincubation with fenarimol. Dry weight of JO-ml suspension strain 003: 32.4 and MJ93: 34.1 mg.
FIG. 6. Effect offenarimol (JO pglml) on uptake and incorporation of [3H]adenine in mycelium of Aspergillus nidulans strain 003 (wild type) and MJ93 (fenarimol resistant). [3H]adenine added at zero time after I (0.a). 3 (0,m). and 5 (V.V) hr ofpreincubation with fenarimol. Controls (O,O, V). fenarimol treatments (0, n , V). Dry weights of IO-ml suspension’strain 003: 28.7 and M193: 31.6 mg.
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10
20
30
LO 50 60 0 mcubation time
WAARD
10 20 ( min I
30
LO 50
AND
60
FIG. 7. Effect offenarimol (IO CLglml) on uptake and incorporation of L-[W]/eucine in mycelium of Aspergillus nidulans strain 003 (wild type) and Ml93 (fenarimol resistant). L-[W]Leucine added at zero time after I (O,O), 3 (O,m), and 5 (0.v). hr ofpreincubation with fenarimol. Controls (0, q ,V), fenarimol treatments (0, H, V). Dry weights of IO-ml suspension strain 003: 30.3, and M193: 32.6 mg.
synthetic medium as described under Materials and Methods. Results showed that [‘4C]fenarimol was rapidly taken up by mycelium of the wildtype strain 003 as compared with mycelium of the fenarimol-resistant mutants J146’, M193, and R264 (Fig. 8). With the wild-type strain an evident maximum in uptake was found after 5- 10 min of incubation. At that time generally about 13- 16% of the radioactivity added was present in the washed mycelial pellet. Unwashed pellets generally contained about 30-40% of the
o!
0
1
0
3
10
20
30
1
LO
incubation
FIG. 8. Uptake
I
50 time
,“1
60
120 210 (mm)
of [‘4Clfenarimol by mycelium of the strain ooj (0) and fenarimoi-resistant strains 3146’ (O), Ml93 (0). and R264 R) of Aspergillus nidulans. [‘T]Fenarimol (10 pglml: sp act 447,100 dpml mg) added at zero time. Dry weight of IO-ml suspension strain 003: 21.9, J146’: 27.2, M193: 28.1, and R264: 21.7 mg. wild-type
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radioactivity added. After 10 min of incubation radioactivity in mycelium of strain 003 decreased almost linearly with time, until after about I hr a minimum level of about 2% of the radioactivity added was reached. Usually radioactivity was only measured in washed mycelial pellets. In one separate experiment, however, radioactivity w,as also measured in the culture medium and successive rinse waters. With three out of four samples of strain 003 and Ml93 about 70-75% of the added radioactivity could be recovered in the culture medium; the rest of the radioactivity was almost totally found in the first three water rinses (Table 3). In contrast, recovery of radioactivity in samples of strain 003 taken after 10 min of incubation showed a different pattern: only about 60% of the added radioactivity was present in the culture medium while in the last two rinse waters and the mycelial pellet still a significant amount of radioactivity was found. Radioactivity remaining in the mycelial pellet of this particular sample was about half of the amount mentioned above; this is probably due to the longer lasting rinsing procedure in this experiment. With boiled mycelium of 003 no difference in uptake of [‘4C]fenarimol after 10 and 60 min of incubation was found (Table 3). In further experiments uptake of [‘4C]fenarimol was investigated in a large number of fenarimol-resistant mutants ofA. nidulans. First, new mutant strains were selected by plating lo8 viable conidia of the wild-type strain 003 on synthetic medium containing 50, 100, and 500 pug/ml fenarimol. Isolates from developing colonies (Fig. 9) were transferred at least three times to fungicide-free media and tested for resistance in radial growth tests. EDsO values of fenarimol for these isolates were of the same magnitude as values for strains M 193 and R264 (Table 4). Uptake of [Ylfenarimol by these strains was tested by measuring radioactivity in 5-ml mycelium samples after 10 and 60 min of incubation with the fungicide. All strains
RESISTANCE
TO
FENARIMOL
IN
ASPERGILLUS
FIG.
275
NIDULANS
of fenarimol-resistant
9. Development
of Aspergillus
nidulans
on
and 500 conidia
fenarimol. wild-type
Per Petri dish ca. strain 003 M’ere seeded
agar
pg/m/
of the
media
containing
strains 50. 100. 5
): 10’ OII ihe
surflcrcr.
tested in this way showed a significantly lower uptake after 10 min of incubation than the wild-type strain 003 (Table 4). Complete time-course experiments were performed with four of these strains (Fig. 10). Again only the wild-type strain 003 showed a distinct optimum in the uptake of the fungicide. DISCUSSION
am -.*m Ad--d-; +I ti tl 0-0000 v;v;v;v;-;
q 0 +I
fl -
tl d ti -
The main difference found in this study between the wild-type strain and fenarimol-resistant mutants of A. niduhs concerns mycelial the uptake of [ ‘4Clfenarimol. Under the test conditions used radioactivity recovered in unwashed and washed mycelium of the wild-type strain 003 after 10 min of incubation amounted to about 30-40% and about 13- 16% of the quantity of fungicide added, respectively. Since the mycelial volume was estimated to be maximally 2% of the suspension, the fungicide was assumed to accumulate to a certain degree in the mycelium. Uptake may also have been partly due to adsorption on the mycelial cell surface. However, no attempts were made to discriminate between these different processes. Under the same test conditions uptake of [ ‘?]fenarimol was much lower in all resistant strains tested than in the wildtype strain (Figs. 8 and 10; Table 4). Therefore, the mechanism of resistance may be a reduced uptake of fenarimol. Reduced uptake has also been suggested as the mechanism of resistance to the fungicides
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TABLE Uptake
NISTELROOY
4
of [W]Fenarimol (10 uglml; spec act 489,300 dpmlmg) Aspergillus nidulans Carrying Spontaneous Mutations
Strain
EDm radial growth (&ml)
Dry weight (mg/lO ml supension)
3.1 18.0 18.0 22.0 19.0 12.5 18.0 17.0 18.0 12.0 12.5 18.0 17.0 14.5 14.0 14.5 13.5 19.5 12.0 15.0 14.5
30.4 34.8 27.9 28.2 27.9 35.8 28.9 32.3 30.4 30.7 33.8 31.0 33.5 34.5 31.9 35.8 33.3 32.1 33.7 29.7 35.4
003 5146’ Ml93 R264 003.50.20 003.50.23 003.50.24 003.100.11 003.100.18 003.100.19 003.100.20 003.100.21 003.500.1 003.500.3 003.500.4 003.500.10 003.500.14 003.500.16 003.500.22 003.500.23 003.500.24
by Mycelial Suspensions of 17 Isolates for Resistance to Fenarimola
Uptake of [ 14C]fenarimol (dpm)
of
10 min
60 min
Ratio of uptake after 10 and 60 min
4336 567 767 13% 1302 1091 788 1285 1200 1327 1268 2200 1172 687 701 835 1005 1071 906 1309 640
411 587 616 405 438 434 493 522 518 403 388 733 720 310 181 605 242 606 360 602 303
10.5 1.0 1.2 3.4 3.0 2.5 1.6 2.5 2.3 3.3 3.3 3.0 1.6 2.2 3.9 1.4 4.3 1.8 2.5 2.2 2.1
u [W]Fenarimol, 10 &ml, sp act 489,300 dpm/mg. Strains 003, J146’, M193, and R264 were used for comparison. Uptake of [Wlfenarimol indicated as dpm in washed mycelium of 5-ml samples.
gE tzLooo-l - ‘o A LOO0 a tc 3000s %
2 2000-
3000
_
aAx \ o 0
2000
120 2LO lncubotlon
FIG. 10. Uptake
tlme(mfnl
of [‘Tlfenarimol by mycelium of the wild-type strain 003 (0) and fenarimolstrains 003.500.16 ( l ), 003.50.24 (x), 003.500.10 (o), and 003.500.24 (W) of Aspergillus nidulans. [‘*C]Fenarimol (10 uglml; sp act exp. A 489.300 dpmlmg, exp. B 453,100 dpmllmg) added at Zero time. Dry weights of IO-ml suspension exp. A strain 003: 28.5, 003.500.16: 29.0 and 003.50.24: 30.6 mg; ditto exp. B strain 003: 28.9, 003.500.10: 33.1, and 003.500.24: 33.0 mg. resistant
RESISTANCE
TO
FENARIMOL
blasticidin in Pyricularia oryzae (26) and to polyoxin in Alternaria kikuchiana (27). In the latter study transport in the wild-type strain was supposed to be partly active, sensitive to, among others, sodium azide and blasticidin, and partly passive caused by adsorption to cellular debris. In resistant strains only the latter process might operate. The rapid uptake of [Wlfenarimol by mycelium of the wild-type strain 003 is probably mainly caused by a physicochemical partitioning between lipid fractions of mycelium and the culture medium. This hypothesis is supported by the fact that radioactivity in culture medium of boiled mycelium of strain 003 incubated for 10 min with [Wlfenarimol was at least as low as that in fresh mycelium (Table 3); furthermore, sodium azide did not inhibit the initial uptake of fenarimol in the wild-type strain (to be published). A similar physicochemical uptake has been described for other fungicides such as dodine (28-30) and glyodin (31). Reduced active transport has frequently been shown to be involved in resistance of fungi and bacteria to toxic amino acid analogs. Kinghorn and Pateman (22) reported that mutation in A. nidulans in at least four different loci can result in impaired transport of certain amino acids. Impaired transport of natural amino acids such as L-serine and L-phenylalanine resulted in resistance to D-serine and p-fluorophenylalanine and poor growth on media with the natural amino acid as sole nitrogen source. However, fenarimol-resistant mutants of A. nidulans with cross-resistance to D-serine (M193) and p-fluorophenylalanine (5250) showed almost normal uptake patterns of natural amino acids (Fig. 3) and growth on media with the same amino acids as sole nitrogen source (Table 2). This type of p-fluorophenylalanine resistance has also been described for mutants of Pseudomonas aeruginosa (32). Therefore, in these particular strains of A. nidufans resistance to fenarimol seems not related with a deficiency in amino acid transport. This
IN
ASPERGILLUS
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227
view is corroborated by the supposed physicochemical uptake of fenarimol mentioned earlier. Major sterol patterns in mycelia of resistant mutants tested are similar to that of the wild-type strain (20). Hence, a mechanism of resistance as described in yeast for polyene antibiotics [cf. (33)] does not apply. Other mechanisms of resistance related with the decreased uptake of [ WJfenarimol by resistant mutants of A. nidulans might be reduced (passive) permeability of the cell membrane or a decrease in the number, affinity, or activity of intracellular receptors of the compound [cf. (34)]. Furthermore, an apparent decreased uptake may also be due to a fast detoxification and efflux of the fungicide from the mycelium. However, this possibility seems improbable since in culture media of neither the wildtype strain nor fenarimol-resistant mutants of A. nidulans could conversion products of fenarimol be detected (2). Most of the [‘Flfenarimol present in or on mycelium of the wild-type strain after 10 min of incubation can be washed away in successive rinse waters (Table 3), indicating that the fungicide is not strongly bound. In contrast dodine taken up by conidia of several fungi is firmly bound (29). Fenarimol is also released from mycelium of the wild-type strain upon prolonged incubation. The mechanism of this phenomenon is not clear. Release of glyodin or dodine from mycelial cells after accumulation has not been reported, although these fungicides affect cell membrane permeability with the resulting loss of cellular constituents (28, 30). In the case of macroconidia of Fusarium solani f. sp. phaseoli incubated with [ ‘%]dodine, release of [‘“Cllabeled material was caused by metabolic detoxification (35). In our studies, other efflux mechanisms might operate, since no conversion products were found (2). Another possibility might be that fenarimol taken up by mycelium of the wild-type strain disrupts cell membrane permeability and as a consequence will be
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WAARD
AND
leached from the mycelium. In that case, however, leakage should be very selective for fenarimol, since under the same test conditions fungitoxicity only became apparent after 3-4 hr of incubation (Fig. 2). Furthermore, uptake and incorporation of [Ylacetate (Fig. 4), [-ladenine (Fig. 6), and L-[%]leucine (Fig. 7) appeared hardly affected. Therefore, another more plausible explanation might be that fenarimol, possibly complexed with cellular constituents, is passively or actively released into the medium. Van den Bossche et al. (25) reported that miconazole, a fungicide related to imazalil, which like fenarimol affects ergosterol biosynthesis, inhibits the rate of uptake of adenine, guanine, and hypoxanthine by Candida afbicuns cells and hence inhibited incorporation of these purines into nucleic acids. In our experiments fenarimol also inhibited incorporation of [ 3H]adenine and, though to a lesser extent, [‘4C]leucine into mycelium of the wild-type strain. Since these effects only became apparent after 5 hr of preincubation with the fungicide, fenarimol evidently does not inhibit uptake of the radiochemicals directly, but does so indirectly by affecting membrane structure through its interference with ergosterol biosynthesis. Incorporation of [%I]adenine and [%]leucine was much less affected in the fenarimol-resistant mutant Ml93 (Figs. 6 and 7). This might be due to uninhibited ergosterol biosynthesis which in turn results in normally functioning cell membranes . Uptake and incorporation of [ %]acetate by mycelium of the fenarimol-resistant strain Ml93 proved to be slightly less effective than by mycelium of the wild-type strain (Fig. 4). Furthermore, mycelium of strain Ml93 released [14C]acetate metabolites, both in the absence and presence of fenarimol (Fig. 5). This might indicate that resistance is correlated with increased excretion of fungal constituents. With strain Ml93 this also became apparant in uv spectra of culture media, since untreated
VAN
NISTELROOY
mycelium of this mutant secreted significantly more substances absorbing at 260 nm than that of the wild-type strain (to be published). These phenomena might indicate a less effective functioning of the cell membrane, which in turn may be related with the relatively low fitness of fenarimolresistant mutants of A. niduluns (3, 16). ACKNOWLEDGMENTS
The authors are grateful to Dr. J. Dekker and Dr. A. Fuchs for a critical reading of the manuscript. REFERENCES
1. I. F. Brown, H. M. Taylor, and H. R. Hall, EL222, a new systemic fungicide with eradicative activity, Proc. Amer. Phyropathol. Sot. 2, 31 (1975).
M. A. de Waard and N. N. Ragsdale, Fenarimol, a new systemic fungicide, in “Proceedings of the Vth Reinhardsbrunn Symposium on Systemic Fungicides,” 1977. 3. M. A. de Waard and H. D. Sisler, Resistance to fenarimol in Aspergillus niduluns, Meded. Fuc. Landbouwwer. R$isuniv. Gent 41, 571 (1976). 4. N. N. Ragsdale and H. D. Sisler, Inhibition of ergosterol synthesis in Ustilago maydis by the fungicide triarimol, Biochem. Biophys. Res. 2.
Commun.
46, 2048 (1972).
5. J. L. Sherald, N. N. Ragsdale, and H. D. Sisler, Similarities between the systemic fungicides triforine and triarimol, Pesfic. Sci. 4,719 ( 1973). 6. N. N. Ragsdale, Specific effects of triarimol on sterol biosynthesis in Ustilago maydis, Biochim. Biophgs. Acta 380, 81 (1975). 7. H. Buchenauer, Biochemical effects of the systemic fungicides fenarimol (EL-222) and nuarimol (EL-228) in Vsfilago avenae. Z. Pfianzenkr. Pflanzenschutz. 84, 286 (1977). 8. J. L. Sherald and H. D. Sisler, Antifungal mode of action of triforine, Pestic. Biochem. Physiol. 5, 477 (1975).
T. Kato, S. Tanaka, M. Ueda, and Y. Kawase, Effects of the fungicide S-1358 on general metabolism and lipid biosynthesis in Monilia fructigena. Agr. Biol. Chem. 38, 2377 (1974). 10. H. Buchenauer, Hemmung der Ergosterinbiosynthese in Ustilago aveliae durch Triadimefon und Fluotrimazol, Z. Pj7anzenkr. Pflanzenschutz. 83, 363 (1976). 11. P. Leroux and M. Gredt, Comparaisons des modes d’action fongitoxique du triadimefon (Meb 6447). du triarimol et de la triforine, Phyropathol. Z. 86, 276 (1976). 12. H. Buchenauer, Mode of action of triadimefon in 9.
Ustilago avenae, 309 (1977).
Pestic.
Biochem.
Physiol.
7,
RESISTANCE
TO
FENARIMOL
13. H. Buchenauer, Mechanisms of action of the fungicide imazalil in Ustilago avenae, Z. Pflanzenkr. Pflanzenschutz. 84,440 (1977). 14. J. Dekker, Acquired resistance to fungicides, Annu. Rev. Phytopathol. 14, 405 (1976). 15. J. M. van Tuyl, Genetics of fungal resistance to systemic fungicides, Meded. Landbouwhogesch. Wageningen, 77-2, 1 (1977). 16. M. A. de Waard and S. A. Gieskes, Characterization of fenarimol-resistant mutants of Aspergillus nidulans, Neth. J. Plant Pathol. 83S, 177 (1977). 17. A. Fuchs and M. Viets-Verwey, Permanent and transient resistance to triarimol and triforine in some phytopathogenic fungi, Meded. Fat. Landbouwwet. Rijksuniv. Gent 40,669 (1975). 18. A. Fuchs and C. A. Drandarevski, The likelihood of development of resistance to systemic fungicides, which inhibit ergosterol biosynthesis, Neth. J. Plant Pathol. 82, 85 (1976). 19. A. Fuchs, S. P. de Ruig, J. M. van Tuyl, and F. W. de Vries, Resistance to triforine: A nonexistent problem? Neth. J. Plant Pathol. 83S, 189 (1977). 20. N. N. Ragsdale and M. A. de Waard, Sterol patterms in fenarimol-susceptible and tolerant strains of Aspergillus nidulans, Proc. Amer. Phytopathol. Sot. 4, 218 (1977). 21. R. F. Rosenberger and M. Kessel, Synchrony of nuciear replication in individual hyphae of Aspergillus nidulans, J. Bacterial. 94,1464 (1%7). 22. J. R. Kinghorn and J. A. Pateman, Mutations which affect amino acid transport in Aspergillus nidulans, J. Gen. Microbial. 86, 174 (1975). 23. M. Piotrowska, P. P. Step&, E. Bartnik, and E. Zakrzewska, Basic and neutral amino acid transport in Aspergillus nidulans, J. Gen. MicrobioJ. 92, 89 (1976). 24. L. Wolfinbarger Jr., Mutations in Neurospora crassa which affect multiple amino acid transport, Biochim. Biophys. Acta 436, 774 (1976).
IN
ASPERGILLUS
229
NIDULANS
25. H. van den Bossche, Biochemical effects of miconazole on fungi: I, Effects on the uptake and/or utilization of purines, pyrimidines, nucleosides, amino acids and glucose by Candida albicans.
Biochem.
Pharmacol.
23, 887 (1974).
26. K. T. Huang, T. Misato, and H. Asuyama, Selective toxicity of blasticidin S to Pyricularia oryzae and Pellicularia sasakii, J. Antibiot. SPY. A. 17, 71 (1964). 27. M. Hori, K. Kakiki, and T. Misato, Mechanism of polyoxin-resistance in Altemaria kikuchiana, .I. Pesfic.
Sci.
1, 31 (1976).
28. I. F. Brown and H. D. Sisler, Mechanisms of fungitoxic action of n-dodecylguanidine acetate, Phytopathology 50, 830 (1960). 29. L. P. Miller, Uptake and innate toxicity of dodine (n-dodecylguanidine acetate) to fungus conidia. Phytopathology 50, 64 (1960). 30. E. Somers, The uptake of dodine acetate by Neurospora hogesch.
crassa, Optoekingssta.
Meded. Staat
LandbouwGent 28, 580
(1963). 31. R. G. Owens and L. P. Miller, Intracellular distribution of metal and organic fungicides in fungus spores, Contrib. Boyce Thompson Intt. 19, 177 (1958). 32. N. W. Dunn and B. W. Holloway, Pieiotropy of p-fluorophenylalanine-resistant and antibiotic hypersensitive mutants of Pseudomonas aeruginosa,
Genet.
Res.
18,
185 (1971).
33. J. M. T. Hamilton-Miller, Fungal sterols and the mode of action of polyene antibiotics. Advan. Appl.
Microbial.
17,
109 (1974).
34. S. G. Georgopoulos, Development of fungal resistance to fungicides, in “Antifungal Compounds” (M. Siegel and H. D. Sisler, Eds.), Vol. 2, p. 439, Dekker, New York, 1977. 35. J. A. Bartz and J. E. Mitchell, Evidence for the metabolic conversion of n-dodecylguanidine acetate by ungerminated macroconidia of Fusarium solani f. sp. phaseoli. Phytoparbology 60, 350 (1970).
BIOCHEMISTRY
Mechanism Laboratory
AND
PHYSIOLOGY
10,219-229
(1979)
of Resistance
to Fenarimol
M. A. DE
AND J. G. M. VAN NISTELROOY
of Phytopathology,
WAARD
Binnenhaven
9, 6709 PD
in Aspergilhs Wageningen.
nidulans
The Netherlands
Received April 13, 1978; accepted June 21, 1978 Mycelial uptake of [‘4C]fenarimol (10 &ml) by 20 fenarimol-resistant mutants of Aspergiks was compared with uptake by wild-type strain 003. Uptake of the fungicide during the initial 10 min of incubation was significantly lower in all mutant strains than in the wild-type strain indicating that resistance is related with reduced uptake. Upon prolonged incubation a gradual decrease of accumulated radioactivity in the wild-type strain was observed. A few mutants displayed resistance to unrelated chemicals such as p-fluorophenylalanine or o-serine; this phenomenon appeared not to be due to a decreased uptake of the corresponding natural amino acids. Incorporation of [Wladenine and [‘4C]leucine by mycelium of mutant Ml93 was hardly inhibited after 5 hr of incubation with the fungicide, whereas a distinct effect was found with the wild-type strain. At this time also fungitoxicity to the wild-type strain became apparent. Probably, this effect is indirectly caused by inhibition of ergosterol biosynthesis. Mycelium of mutant Ml93 incorporated [‘PC]acetate slightly less effectively than the wild-type strain. After 2 hr of incubation with this radiochemical leakage of [‘4C]acetate metabolites from mycelium of the mutant strain was observed. This indicates that resistance might be correlated with increased excretion of fungal metabolites, which in turn may be related with reduced fitness of fenarimol-resistant mutants. nidulans
INTRODUCTION
Fenarimol [EL-222; cu-(Zchlorophenyl)a-(4-chlorophenyl)-5pyrimidinemethanoll is a new systemic fungicide for control of diseases such as powdery mildews and apple scab (1). It is a broad-spectrum fungicide with antifungal in vitro activity against several Ascomycetes, Basidiomycetes, and Fungi imperfecti (Technical Report on EL222). Also some Oomycetes are sensitive (2). Both fenarimol and the structurally related chemical triarimol [EL-273; a-(2,4dichlorophenyl)-cY-phenyl-5-pyrimidinemethanol] are highly toxic to, among others, Aspergillus nidulans (3). The chemicals primarily inhibit germ tube elongation and mycelial growth, while spore germination is significantly less sensitive. Triarimol is a potent inhibitor of ergosterol biosynthesis in Ustilago maydis (4-6). Fenarimol has a similar effect in A. nidulans (2) and in U. avenue (7). Therefore, fenarimol can be classified in the group of ergosterol biosynthesis-inhibiting fungicides such as triforine (5, 8)) Denmert (S-1358) (9), triadimefon (lo-12), flue-
trimazole (lo), imazalil (13), and nuarimol (7). Development of resistance in fungi to systemic fungicides with a specific mechanism of action is quite common ( 14). Therefore, it is not surprising that in vitro resistance to ergosterol biosynthesisinhibiting fungicides has been observed (3, 8, 15- 17). Up until now no development of resistance to these fungicides under practical conditions has been reported ( 17, 18). Absence of resistance under practical conditions may be due to decreased pathogenicity of resistant strains; this phenomenon, related with diminished spore germination, germ tube elongation, mycelial growth, and sporulation has been described for triarimol- and triforine-resistant mutants of Cladosporium cucumerinum (17, 19). Decreased fitness of fenarimoland imazalil-resistant strains of A. nidufans appeared to be due to the same phenomena (3, 16). Since the practical importance of ergosterol biosynthesis-inhibiting fungicides is 219 0048-3575179/020219-11$02.00/O Copyright @ 1979 by Academic Press. Inc. AU rights of reproduction in any form rrwrved.
220
DE
WAARD
AND
still increasing, it seemed worthwhile to investigate the biochemical mechanism of resistance. According to previous studies with fenarimol and A. nidulans, resistance proved not to be caused by enhanced metabolism of fenarimol (2) nor was it accompanied with a major change in sterol patterns (20). This paper deals with other possible mechanisms of fenarimol resistance . MATERIALS
AND
METHODS
Organism. Aspergillus nidulans biA1 acrA1 requiring biotin, resistant to acriflavin, and with wild-type sensitivity to fenarimol and related chemicals (strain 003) was used in all experiments. Fenarimolresistant strains were obtained by selection of untreated (sp) or uv-irradiated (uv) conidia on fenarimol-containing media (15, 16). The genetic basis for resistance was due to an imaB gene in strain 5146 (sp) and Ml93 (uv) and an imaD gene in strain 5250 (sp). Strain 5146’ is a new subculture of 5146 selected on fenarimol-containing agar. Strain R264 is a recombinant strain carrying imaA and imaB genes and a gene which fortifies the action of the imaA gene (15). The genetic basis of all other strains used is unknown. Chemicals. Fenarimol and [a-14C]fenarimol (sp act 19 @i/mg) were gifts of Eli Lilly Company, Indianapolis, Illinois. The radiochemicals [U-14C]acetate, [8-3H]adenine, L-[U-14C]leucine, L-[U-14C]phenylalanine, and t.-[U-14C]serine were obtained from the Radiochemical Centre, Amersham, England. Culture methods. Conidia of A. nidulans strains were grown on a 2% malt extract agar medium, supplemented with 1 mg biotin/liter. Conidial suspensions with approximately IO9 conidia/ml were added to Erlenmeyer flasks which contained 100 ml glucose-nitrate medium (21) supplemented with biotin, to give a final concentration of 10’ conidia/ml. Flasks were incubated at 37°C on a Gallenkamp orbital shaker at 180 rpm for about 16 hr. Then, the mycelium was harvested by filtration on a Biichner
VAN
NISTELROOY
filter and washed two times by resuspending 5 g of wet mycelium in 250 ml of glucose-nitrate medium. The final standard mycelial suspension was made by resuspending 1 g of mycelium in 50 ml of fresh medium. Thereafter, incorporation experiments were mostly started after 30-45 min preincubation of the suspension at 37°C in a reciprocal water-bath shaker. Toxicity of fungicides. Fungitoxicity of chemicals was usually determined in mycelial growth tests. Chemicals were added as a solution in methanol or water to molten glucose-nitrate agar medium. The final methanol concentration in the medium, identical in controls and treatments, never exceeded 1%. The medium was poured in 9-cm Petri dishes and inoculated by placing three inverted 5-mm agar disks with young mycelium on the agar. Radial growth was measured in duplicate after 3 days of incubation at 37°C. Toxicity of fungicides in shake cultures was established by adding the chemicals to standard mycelial suspensions in Erlenmeyer flasks. Growth was measured by determining increase in fungal dry weight of the cultures. Three IO-ml samples per treatment were filtered using preweighed filter papers. After washing with distilled water the filter papers were dried overnight at 60°C and reweighed. Uptake and incorporation of radiochemicals by mycelium. Standard mycelial suspensions were incubated in a reciprocal shaker at 37°C. Radiochemicals were added to the suspension at final concentrations of 100 /.L&! acetate (sp act 215 &i/mmol), 10 @V adenine (sp act 5 mCi/mmol), 30 w fenarimol (sp act ca. 67 &i/mmol), 100 m L-leucine (sp act 116 ,&i/mmol), 100 m L-phenylalanine (sp act 230 &i/mmol), and 100 /..N L-serine (sp act 447 &ilmmol). After various incubation times 5-ml samples of mycelial suspensions were filtered over Whatman GF/A glass filter paper using a Millipore sampling manifold filter apparatus. The mycelial residues were washed five times within 30 set with 5 ml
RESISTANCE
TO
FENARIMOL
glucose-nitrate medium. Radioactivity was determined by transferring the filters with mycelial residue into vials containing 10 ml scintillation- liquid (3 g PPO in a mixture of 750 ml xylene and 250 ml Triton X-100; Lumac B.V., Amsterdam, The Netherlands) and by counting in a Nuclear Chicago Mark I liquid scintillation spectrometer. Linear regression analysis and calculation of correlation coefficients (Y) of the relation between uptake and incorporation of radiochemicals versus incubation time were carried out with a Diehl algotronic calculator. RESULTS
Toxicity
of Fenarimol
The ED5,, value of fenarimol for radial mycelial growth of the wild-type strain 003 of A. nidulans was found to be about 3.8 pg/ml (Fig. 1). ED,,, values for radial growth of the resistant strains tested were maximally ca. fivefold as high as that of the wild-type strain, indicating that the degree of resistance was relatively low. Differences between minimal inhibitory concentrations, on the other hand, were much larger since 30 &ml inhibited growth of the wild-type strain completely, while some resistant mutants even grew at 500 &ml. Fenarimol added to standard mycelial suspensions hardly inhibited growth during the initial 3.5 hr of incubation irrespective
IN
ASPERGILLUS
221
NIDULANS
of the concentration used (Fig. 2). Thereafter, growth was inhibited by a concentration of at least 10 &ml. This concentration was used in most of the following experiments. Cross-resistance
In previous studies (2, 3, 5, 8, 15, 16) fenarimol-resistant strains of A. nidulans often displayed cross-resistance to other inhibitors of sterol biosynthesis such as imazalil and triadimefon. Cross-resistance to a number of unrelated chemicals was investigated in this study in radial growth tests. Four out of 12 resistant mutants tested showed a pronounced change in sensitivity to chemicals such as 8-azaguanine (J146), p-fluorophenylalanine (M 193 and J250), n-serine (M193), thiourea (Ml93 and 5146) and 3-aminotyrosine (M193) (Table I). Uptake
of Amino
Acids
Resistance to antimetabolites like p-fluorophenylalanine and n-serine is often ascribed to a deficiency in amino acid transport (22-24). Strain J250 and Ml93 with resistance to p-fluorophenylalanine and n-serine, respectively, might therefore be unable to take up L-phenylalanine and L-serine. This was tested by measuring the
100 -g ’
8
z 5 ,” 2
90 80 70 60 50 LO
g 30 z r, 20 e 10
0)
0 1 FIG.
10 concentration 1. Dosage
radial growth fenarimol-resistant
100 (jdg/ml
500 1
-response curves of fenarimol for of the wild-type strain 003 and mutants of Aspergillus nidulans.
0
4 incubation
time
8 (h 1
FIG. 2. Toxicity offenarimol for mycelial growth of the wild-type strain 003 of Aspergillus nidulans in liquid cultures. Treatments: control (0). 5 (0). 10 fO), 20 (m), and 40 f x) pglml fenarimol.
222
DE
Cross-Resistance
WAARD
of Fenarimol-Resistant
Compound
I-Azaguanine p-Fluorophenylalanine n-Serine Thiourea
AND
VAN
NISTELROOY
TABLE
1
Mutants
of Aspergillus
nidulans
to Antimetabolites
003
Ml93
5146
5250
R264
10 24 112 70
EDso G.&N 10 12 MOO 180
4 16 120 240
12 2100 85 -
9 30 70 -
82 60 41
107 52 36
Growth as percentage of control 3-Aminotyrosine (3 mg/ml) L-Ethionine (5 mgknl) Thiourea (100 &ml)
88 47 47
uptake of L-[ 14C]phenylalanine and L-[ ‘4Clserine in standard mycelial suspensions. Uptake of both amino acids by the mutant strains proved to be similar to that of the wild-type strain during 1 hr of incubation (Fig. 3). Utilization of the amino acids was also tested by growing the wild-type and mutant strains on synthetic agar media with L-phenylalanine and L-serine as sole nitrogen source (22). Results showed that radial growth of all strains tested did not differ much (Table 2), indicating again normal ability to take up the amino acids.
F 5 1601 1LO 120 100 80 60 LO 20 0 0
10
20 30 incubation
LO 50 60 time ( min 1
FIG. 3. Uptake and incorporation of L-[Tlserine by mycelium of Aspergillus nidulans wild-type strain 003 (O), and D-serine-tolerant mutant Ml93 (0); ditto of [‘4C]phenylalanine by the wild-type strain (0) and p-jiuorophenylalanine-tolerant mutant 5250 (U). Dry weights of IO-ml mycelial suspension (0) 27.7. (0) 33.2, (0) 30.0, and (m) 31 .I mg.
33 66 86
94 67 83
Effect of Fenarimol on Uptake and Incorporation of Radiochemicals The fungicide miconazole inhibits uptake of adenine, guanine, and hypoxanthine by cells of Candida al&cans (25). The fungicide is chemically related to imazalil. Both fenarimol and imazalil inhibit ergosterol biosynthesis in fungi (2,7). Therefore, it was of interest to examine whether fenarimol affected the uptake of the radiochemicals [ ‘4C]acetate, [ 3H]adenine, and [‘4C]leucine by mycelium of the wildtype strain and of mutant M193. Uptake and incorporation of [‘T]acetate was investigated by addition of the radiochemical to mycelial suspensions with 10 Mm1 fenarimol. Every 5 min radioactivity in washed mycelium of 5-ml samples was measured. In both strains uptake and incorporation proved to be almost linear with time during the first hr of incubation and was unaffected by the presence of fenarimol (Fig. 4). The observed relatively low uptake and incorporation of [%]acetate by mutant strain Ml93 (Fig. 4) might also be due to increased excretion of [ Ylacetate metabolites. Therefore, in a following experiment it was examined whether after 2 hr of preincubation of mycelium with [%]acetate when maximal uptake and incorporation of the radiochemical was found, addition of 10 pug/ml fenarimol induced a leakage of [%]acetate and [ 14C]acetate metabolites. Figure 5
RESISTANCE
TABLE
TO
FENARIMOL
IN
ASPERGILLUS
223
NIDULANS
2
Effect of the Nitrogen Source in Synthetic Nutrient Medium on Radial Growth ofAspergillus nidulans 003 (wild-type strain), Ml93 (D-serine tolerant), and J250 (p-fluorophenylalanine tolerant) Radial growth Strain
NaNO,
003 Ml93
25.0 21.3 24.0
J250 ” Relatively
(mm)
L-Phenylalanine
+ 0 + 1.2 + 0
19.7 + 0.6 24.0 A 1.0’ 22.0 ‘- 1.0
thin mycelium
L-Serine
003-control 003-fenarmol
lo1 (01
r 012 032
19.7 e 0.6
M193-control M193-fenarlmol
IO) (=I
-0Ll -051
18.0 k 1.0 f 2.1
22.3
8
I
0
mat.
10
I
20
I
1
I
I
30
LO
50
60
incubation
shows that radioactivity in mycelium of the wild-type strain remained essentially constant during 1 hr of incubation, while in strain Ml93 a slight decrease of radioactivity was noticed. The latter results might indicate that low amounts of fungal constituents were excreted by mycelium of strain M193. No differences were observed between controls and fenarimol treatments. Uptake and incorporation of [W]adenine and [‘CJleucine were investigated as described before for [‘4C]acetate and measured after 1, 3, and 5 hr of preincubation 5 2201
timt,
( mm 1
5. Effect offenarimol(l0 Hplml) on content of [Tlacetate and [Wlacetate metabohtes in myrelium of Aspergillus nidulans strain 003 (wild type) and Ml93 (fenarimol resistant). Fenarimol added at zero time after2 hr preincubation with [‘Tlacetate. Dry weights of IO-ml suspension strain 003: 28.4 and Ml93: 25.6 w. FIG.
with 10 CLg/ml fenarimol. Pronounced differences with both chemicals were only observed with mycelium of the wild-type strain 003, preincubated with fenarimol for 5 hr; under similar conditions such differences were not found with mycelium of strain Ml93 (Figs. 6 and 7). Uptake of [14C]Fenarimol
Uptake of [ “‘Qfenarimol was determined by measuring radioactivity in mycelium of 5-ml samples after filtration over glass filter paper and five consecutive washings with
003-fencmmol M 193 -control M193-fenanmol
0
10
20 30 incubation
(*I 099 (01 0 99 (*I 098
LO 50 60 time (mm )
FIG. 4. Effect offenarimol (IO pglml) on uptake and incorporation of [‘Tlacetate by mycelium of Aspergillus nidulans strain 003 (wild-type) and Ml93 (fenarimol-resistant). [‘*C]Acetate added at zero time after I hr preincubation with fenarimol. Dry weight of JO-ml suspension strain 003: 32.4 and MJ93: 34.1 mg.
FIG. 6. Effect offenarimol (JO pglml) on uptake and incorporation of [3H]adenine in mycelium of Aspergillus nidulans strain 003 (wild type) and MJ93 (fenarimol resistant). [3H]adenine added at zero time after I (0.a). 3 (0,m). and 5 (V.V) hr ofpreincubation with fenarimol. Controls (O,O, V). fenarimol treatments (0, n , V). Dry weights of IO-ml suspension’strain 003: 28.7 and M193: 31.6 mg.
224
DE
0
10
20
30
LO 50 60 0 mcubation time
WAARD
10 20 ( min I
30
LO 50
AND
60
FIG. 7. Effect offenarimol (IO CLglml) on uptake and incorporation of L-[W]/eucine in mycelium of Aspergillus nidulans strain 003 (wild type) and Ml93 (fenarimol resistant). L-[W]Leucine added at zero time after I (O,O), 3 (O,m), and 5 (0.v). hr ofpreincubation with fenarimol. Controls (0, q ,V), fenarimol treatments (0, H, V). Dry weights of IO-ml suspension strain 003: 30.3, and M193: 32.6 mg.
synthetic medium as described under Materials and Methods. Results showed that [‘4C]fenarimol was rapidly taken up by mycelium of the wildtype strain 003 as compared with mycelium of the fenarimol-resistant mutants J146’, M193, and R264 (Fig. 8). With the wild-type strain an evident maximum in uptake was found after 5- 10 min of incubation. At that time generally about 13- 16% of the radioactivity added was present in the washed mycelial pellet. Unwashed pellets generally contained about 30-40% of the
o!
0
1
0
3
10
20
30
1
LO
incubation
FIG. 8. Uptake
I
50 time
,“1
60
120 210 (mm)
of [‘4Clfenarimol by mycelium of the strain ooj (0) and fenarimoi-resistant strains 3146’ (O), Ml93 (0). and R264 R) of Aspergillus nidulans. [‘T]Fenarimol (10 pglml: sp act 447,100 dpml mg) added at zero time. Dry weight of IO-ml suspension strain 003: 21.9, J146’: 27.2, M193: 28.1, and R264: 21.7 mg. wild-type
VAN
NISTELROOY
radioactivity added. After 10 min of incubation radioactivity in mycelium of strain 003 decreased almost linearly with time, until after about I hr a minimum level of about 2% of the radioactivity added was reached. Usually radioactivity was only measured in washed mycelial pellets. In one separate experiment, however, radioactivity w,as also measured in the culture medium and successive rinse waters. With three out of four samples of strain 003 and Ml93 about 70-75% of the added radioactivity could be recovered in the culture medium; the rest of the radioactivity was almost totally found in the first three water rinses (Table 3). In contrast, recovery of radioactivity in samples of strain 003 taken after 10 min of incubation showed a different pattern: only about 60% of the added radioactivity was present in the culture medium while in the last two rinse waters and the mycelial pellet still a significant amount of radioactivity was found. Radioactivity remaining in the mycelial pellet of this particular sample was about half of the amount mentioned above; this is probably due to the longer lasting rinsing procedure in this experiment. With boiled mycelium of 003 no difference in uptake of [‘4C]fenarimol after 10 and 60 min of incubation was found (Table 3). In further experiments uptake of [‘4C]fenarimol was investigated in a large number of fenarimol-resistant mutants ofA. nidulans. First, new mutant strains were selected by plating lo8 viable conidia of the wild-type strain 003 on synthetic medium containing 50, 100, and 500 pug/ml fenarimol. Isolates from developing colonies (Fig. 9) were transferred at least three times to fungicide-free media and tested for resistance in radial growth tests. EDsO values of fenarimol for these isolates were of the same magnitude as values for strains M 193 and R264 (Table 4). Uptake of [Ylfenarimol by these strains was tested by measuring radioactivity in 5-ml mycelium samples after 10 and 60 min of incubation with the fungicide. All strains
RESISTANCE
TO
FENARIMOL
IN
ASPERGILLUS
FIG.
275
NIDULANS
of fenarimol-resistant
9. Development
of Aspergillus
nidulans
on
and 500 conidia
fenarimol. wild-type
Per Petri dish ca. strain 003 M’ere seeded
agar
pg/m/
of the
media
containing
strains 50. 100. 5
): 10’ OII ihe
surflcrcr.
tested in this way showed a significantly lower uptake after 10 min of incubation than the wild-type strain 003 (Table 4). Complete time-course experiments were performed with four of these strains (Fig. 10). Again only the wild-type strain 003 showed a distinct optimum in the uptake of the fungicide. DISCUSSION
am -.*m Ad--d-; +I ti tl 0-0000 v;v;v;v;-;
q 0 +I
fl -
tl d ti -
The main difference found in this study between the wild-type strain and fenarimol-resistant mutants of A. niduhs concerns mycelial the uptake of [ ‘4Clfenarimol. Under the test conditions used radioactivity recovered in unwashed and washed mycelium of the wild-type strain 003 after 10 min of incubation amounted to about 30-40% and about 13- 16% of the quantity of fungicide added, respectively. Since the mycelial volume was estimated to be maximally 2% of the suspension, the fungicide was assumed to accumulate to a certain degree in the mycelium. Uptake may also have been partly due to adsorption on the mycelial cell surface. However, no attempts were made to discriminate between these different processes. Under the same test conditions uptake of [ ‘?]fenarimol was much lower in all resistant strains tested than in the wildtype strain (Figs. 8 and 10; Table 4). Therefore, the mechanism of resistance may be a reduced uptake of fenarimol. Reduced uptake has also been suggested as the mechanism of resistance to the fungicides
DE
226
WAARD
AND
VAN
TABLE Uptake
NISTELROOY
4
of [W]Fenarimol (10 uglml; spec act 489,300 dpmlmg) Aspergillus nidulans Carrying Spontaneous Mutations
Strain
EDm radial growth (&ml)
Dry weight (mg/lO ml supension)
3.1 18.0 18.0 22.0 19.0 12.5 18.0 17.0 18.0 12.0 12.5 18.0 17.0 14.5 14.0 14.5 13.5 19.5 12.0 15.0 14.5
30.4 34.8 27.9 28.2 27.9 35.8 28.9 32.3 30.4 30.7 33.8 31.0 33.5 34.5 31.9 35.8 33.3 32.1 33.7 29.7 35.4
003 5146’ Ml93 R264 003.50.20 003.50.23 003.50.24 003.100.11 003.100.18 003.100.19 003.100.20 003.100.21 003.500.1 003.500.3 003.500.4 003.500.10 003.500.14 003.500.16 003.500.22 003.500.23 003.500.24
by Mycelial Suspensions of 17 Isolates for Resistance to Fenarimola
Uptake of [ 14C]fenarimol (dpm)
of
10 min
60 min
Ratio of uptake after 10 and 60 min
4336 567 767 13% 1302 1091 788 1285 1200 1327 1268 2200 1172 687 701 835 1005 1071 906 1309 640
411 587 616 405 438 434 493 522 518 403 388 733 720 310 181 605 242 606 360 602 303
10.5 1.0 1.2 3.4 3.0 2.5 1.6 2.5 2.3 3.3 3.3 3.0 1.6 2.2 3.9 1.4 4.3 1.8 2.5 2.2 2.1
u [W]Fenarimol, 10 &ml, sp act 489,300 dpm/mg. Strains 003, J146’, M193, and R264 were used for comparison. Uptake of [Wlfenarimol indicated as dpm in washed mycelium of 5-ml samples.
gE tzLooo-l - ‘o A LOO0 a tc 3000s %
2 2000-
3000
_
aAx \ o 0
2000
120 2LO lncubotlon
FIG. 10. Uptake
tlme(mfnl
of [‘Tlfenarimol by mycelium of the wild-type strain 003 (0) and fenarimolstrains 003.500.16 ( l ), 003.50.24 (x), 003.500.10 (o), and 003.500.24 (W) of Aspergillus nidulans. [‘*C]Fenarimol (10 uglml; sp act exp. A 489.300 dpmlmg, exp. B 453,100 dpmllmg) added at Zero time. Dry weights of IO-ml suspension exp. A strain 003: 28.5, 003.500.16: 29.0 and 003.50.24: 30.6 mg; ditto exp. B strain 003: 28.9, 003.500.10: 33.1, and 003.500.24: 33.0 mg. resistant
RESISTANCE
TO
FENARIMOL
blasticidin in Pyricularia oryzae (26) and to polyoxin in Alternaria kikuchiana (27). In the latter study transport in the wild-type strain was supposed to be partly active, sensitive to, among others, sodium azide and blasticidin, and partly passive caused by adsorption to cellular debris. In resistant strains only the latter process might operate. The rapid uptake of [Wlfenarimol by mycelium of the wild-type strain 003 is probably mainly caused by a physicochemical partitioning between lipid fractions of mycelium and the culture medium. This hypothesis is supported by the fact that radioactivity in culture medium of boiled mycelium of strain 003 incubated for 10 min with [Wlfenarimol was at least as low as that in fresh mycelium (Table 3); furthermore, sodium azide did not inhibit the initial uptake of fenarimol in the wild-type strain (to be published). A similar physicochemical uptake has been described for other fungicides such as dodine (28-30) and glyodin (31). Reduced active transport has frequently been shown to be involved in resistance of fungi and bacteria to toxic amino acid analogs. Kinghorn and Pateman (22) reported that mutation in A. nidulans in at least four different loci can result in impaired transport of certain amino acids. Impaired transport of natural amino acids such as L-serine and L-phenylalanine resulted in resistance to D-serine and p-fluorophenylalanine and poor growth on media with the natural amino acid as sole nitrogen source. However, fenarimol-resistant mutants of A. nidulans with cross-resistance to D-serine (M193) and p-fluorophenylalanine (5250) showed almost normal uptake patterns of natural amino acids (Fig. 3) and growth on media with the same amino acids as sole nitrogen source (Table 2). This type of p-fluorophenylalanine resistance has also been described for mutants of Pseudomonas aeruginosa (32). Therefore, in these particular strains of A. nidufans resistance to fenarimol seems not related with a deficiency in amino acid transport. This
IN
ASPERGILLUS
NIDULANS
227
view is corroborated by the supposed physicochemical uptake of fenarimol mentioned earlier. Major sterol patterns in mycelia of resistant mutants tested are similar to that of the wild-type strain (20). Hence, a mechanism of resistance as described in yeast for polyene antibiotics [cf. (33)] does not apply. Other mechanisms of resistance related with the decreased uptake of [ WJfenarimol by resistant mutants of A. nidulans might be reduced (passive) permeability of the cell membrane or a decrease in the number, affinity, or activity of intracellular receptors of the compound [cf. (34)]. Furthermore, an apparent decreased uptake may also be due to a fast detoxification and efflux of the fungicide from the mycelium. However, this possibility seems improbable since in culture media of neither the wildtype strain nor fenarimol-resistant mutants of A. nidulans could conversion products of fenarimol be detected (2). Most of the [‘Flfenarimol present in or on mycelium of the wild-type strain after 10 min of incubation can be washed away in successive rinse waters (Table 3), indicating that the fungicide is not strongly bound. In contrast dodine taken up by conidia of several fungi is firmly bound (29). Fenarimol is also released from mycelium of the wild-type strain upon prolonged incubation. The mechanism of this phenomenon is not clear. Release of glyodin or dodine from mycelial cells after accumulation has not been reported, although these fungicides affect cell membrane permeability with the resulting loss of cellular constituents (28, 30). In the case of macroconidia of Fusarium solani f. sp. phaseoli incubated with [ ‘%]dodine, release of [‘“Cllabeled material was caused by metabolic detoxification (35). In our studies, other efflux mechanisms might operate, since no conversion products were found (2). Another possibility might be that fenarimol taken up by mycelium of the wild-type strain disrupts cell membrane permeability and as a consequence will be
228
DE
WAARD
AND
leached from the mycelium. In that case, however, leakage should be very selective for fenarimol, since under the same test conditions fungitoxicity only became apparent after 3-4 hr of incubation (Fig. 2). Furthermore, uptake and incorporation of [Ylacetate (Fig. 4), [-ladenine (Fig. 6), and L-[%]leucine (Fig. 7) appeared hardly affected. Therefore, another more plausible explanation might be that fenarimol, possibly complexed with cellular constituents, is passively or actively released into the medium. Van den Bossche et al. (25) reported that miconazole, a fungicide related to imazalil, which like fenarimol affects ergosterol biosynthesis, inhibits the rate of uptake of adenine, guanine, and hypoxanthine by Candida afbicuns cells and hence inhibited incorporation of these purines into nucleic acids. In our experiments fenarimol also inhibited incorporation of [ 3H]adenine and, though to a lesser extent, [‘4C]leucine into mycelium of the wild-type strain. Since these effects only became apparent after 5 hr of preincubation with the fungicide, fenarimol evidently does not inhibit uptake of the radiochemicals directly, but does so indirectly by affecting membrane structure through its interference with ergosterol biosynthesis. Incorporation of [%I]adenine and [%]leucine was much less affected in the fenarimol-resistant mutant Ml93 (Figs. 6 and 7). This might be due to uninhibited ergosterol biosynthesis which in turn results in normally functioning cell membranes . Uptake and incorporation of [ %]acetate by mycelium of the fenarimol-resistant strain Ml93 proved to be slightly less effective than by mycelium of the wild-type strain (Fig. 4). Furthermore, mycelium of strain Ml93 released [14C]acetate metabolites, both in the absence and presence of fenarimol (Fig. 5). This might indicate that resistance is correlated with increased excretion of fungal constituents. With strain Ml93 this also became apparant in uv spectra of culture media, since untreated
VAN
NISTELROOY
mycelium of this mutant secreted significantly more substances absorbing at 260 nm than that of the wild-type strain (to be published). These phenomena might indicate a less effective functioning of the cell membrane, which in turn may be related with the relatively low fitness of fenarimolresistant mutants of A. niduluns (3, 16). ACKNOWLEDGMENTS
The authors are grateful to Dr. J. Dekker and Dr. A. Fuchs for a critical reading of the manuscript. REFERENCES
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T. Kato, S. Tanaka, M. Ueda, and Y. Kawase, Effects of the fungicide S-1358 on general metabolism and lipid biosynthesis in Monilia fructigena. Agr. Biol. Chem. 38, 2377 (1974). 10. H. Buchenauer, Hemmung der Ergosterinbiosynthese in Ustilago aveliae durch Triadimefon und Fluotrimazol, Z. Pj7anzenkr. Pflanzenschutz. 83, 363 (1976). 11. P. Leroux and M. Gredt, Comparaisons des modes d’action fongitoxique du triadimefon (Meb 6447). du triarimol et de la triforine, Phyropathol. Z. 86, 276 (1976). 12. H. Buchenauer, Mode of action of triadimefon in 9.
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RESISTANCE
TO
FENARIMOL
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IN
ASPERGILLUS
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crassa, Optoekingssta.
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