Genetic aspects of resistance to imazalil in Aspergillus nidulans

Mutant strains of Aspergillus nidulans have been isolated which display a low level of resistance to imazalil, a recently developed systemic fungicide. Agar growth tests showed that A. nidulans is about three times as sensitive to imazalil when growing on supplemented minimal medium (SM) as compared with complete medium. This effect was reduced by adding glutamic acid to the SM.Imazalil resistance was found to be based on a multigenic system; 21 single gene mutations define 8 loci which were allocated to 6 different linkage groups. Mutations at different loci lead pleiotropically to one or more of the following properties: hypersensitivity or resistance to acriflavin, cycloheximide and neomycin, resistance to chloramphenicol and fenarimol, and to cold sensitivity. Of 120 cycloheximide-resistant strains isolated, 98 were also imazalil-resistant.Recombination analysis of different imazalil-resistant strains with mutations at three loci resulted in additive effects, giving strains with a high level of resistance to imazalil.The results indicate that imazalil may interfere either with protein synthesis like cycloheximide, chloramphenicol and neomycin or with synthesis or function of cell membranes. Interference with cell membrane synthesis might lead to altered sterol composition, resulting in selective permeability to different compounds.     

Metabolism of imazalil by wild-type and DMI-resistant isolates of Penicillium italicum

Metabolism of imazalil (1-[2-(2,4-dichlorophenyl)-2-(2-propenyloxy)ethyl]-1H-imidazole) inPenicillium italicum isolates with a wild-type sensitivity and with various degrees of resistance to sterol demethylation inhibitors was studied in liquid cultures. The metabolite 1-[2(2,4-dichlorophenyl)-2-(2,3-dihydroxypropyloxy)ethyl]-1H-imidazole (R42243) was detected in the culture filtrate after prolonged incubation. The metabolism occurred in the propenyl side chain of imazalil probably through epoxidation and hydratation. This is the first report of such a conversion of imazalil in fungi. R42243 was much less toxic toP. italicum than imazalil. Therefore, the metabolism can be regarded as a detoxification step. Both wild-type and resistant isolates metabolized imazalil, but metabolism by resistant isolates was faster than by the wild-type isolate. This is probably caused by a relatively strong inhibition of growth of the wild-type isolate by the fungicide. Results indicate that the detoxification of imazalil does not operate as a mechanism of resistance. This conclusion was confirmed by the fact that resistant isolates showed cross-resistance to miconazole and R42243, which had a similar structure as imazalil except for the propenyl side chain.     

Effects of phthalimide fungicides on the accumulation of fenarimol by Aspergillus nidulans

Phthalimide fungicides (captafol, captan and folpet) enhanced the accumulation of fenarimol by the mycelium of a wild-type strain and a fenarimol-resistant strain of Aspergillus nidulans. The accumulation is ascribed to inhibition of active efflux of fenarimol from the mycelium. It is assumed that the synergistic action observed between the phthalimide fungicides and fenarimol with respect to fungitoxicity, was caused by the increased accumulation of fenarimol.     

Thiophene carboxamide fungicides: Structure-activity relationships with the succinate dehydrogenase complex from wild-type and carboxin-resistant mutant strains of Aspergillus nidulans

Carboxin (5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilide) and the thiophene compound 3-methylthiophene-2-carboxanilide (I), inhibit succinate oxidation (succinate-ubiquinone reductase; complex II) in mitochondria from a wild-type strain and three mutant, carboxin-resistant strains of Aspergillus nidulans. Studies by White et al. [Pestic. Biochem. Physiol. 9, 165–182 (1978)] showed that certain oxathiin carboxamide structures were selectively active against particular mutated succinate dehydrogenase complexes (SDCs) of A. nidulans, significantly lowering the level of resistance. Although no oxathiin carboxamides were found to be negatively correlated to carboxin with respect to their effect on SDCs from wild-type and mutant strains of A. nidulans, several could distinguish between moderately and highly carboxin-resistant SDCs and, as shown in the present study, between the two non-allelic highly carboxin-resistant mutants cbx B-28 and cbx C-34. Variation in the molecular structure of thiophene carboxamides can also affect the phenotypic expression of mutations to carboxin resistance in the SDC of A. nidulans, with certain structures being capable of differentiating between moderately and highly carboxin-resistant mutated SDCs. With a moderately carboxin-resistant mutant, cbx A-17, a wide structural variety of thiophene carboxamides, e.g., the 2′-methyl, 2′-benzoyl, 3′-phenoxy, 4′-nbutyl and the N-nhexyl derivatives of (I), did exhibit negative activity correlation to the parent anilide (I). However, with the possible exception of the 4′-nbutyl and 4′-noctyloxy analogs of (I), thiophene carboxamides showed no negative activity correlation to carboxin or (I) for the highly carboxin-resistant mutants cbx B-28 and cbx C-34. As with carboxin-resistant mutants of Ustilago maydis [Pestic. Biochem. Physiol. 14, 26–40 (1980)], molecular selectivity for mutated carboxin-resistant SDCs of A. nidulans can be markedly influenced by substitution of an oxathiin with a thiophene heterocyclic ring. None of the thiophene carboxamides were considerably toxic to mycelial growth of the wild-type and carboxin-resistant strains of A. nidulans with permeability rather than affinity for the SDC appearing to be the limiting factor. For certain derivatives such as the 5-amino analog of (I), SDC activity and cell growth were inhibited similarly. Several thiophene carboxamides [2′-phenyl, 4′-phenoxy, and N-ndecyl analogs of (I)] showed specificity for the highly carboxin-resistant mutants cbx B-28 and cbx C-34. Thiophene carboxamide structures have been identified which inhibit spore germination of non-Basidiomycete plant pathogens, particularly Phytophthora infestans and Verticillium dahliae. In vivo experiments with late blight (P. infestans) on tomato plants have shown that a few thiophene carboxamides, e.g., the 3′-nbutyl analog of (I) give satisfactory protectant activity.     

Genetic effects of phthalimide fungicides on diploid Aspergillus nidulans

A few specimens of ‘Ridomil 25 WP’, a commercial formulation of metalaxyl, showed high genetic activity on a heterozygous diploid strain of Aspergillus nidulans. This activity was due to an impurity which was identified by mass and n.m.r. spectroscopy as captafol. This and the related fungicides captan, dichlofluanid, and tolyfluanid greatly increased the yield of euploid colour segregants from the diploid strain. At low captafol concentrations, most of the induced segregants resulted from mitotic crossing over but chromosome non-disjunction or loss was also caused at high concentrations. Mitotic crossing over could be induced also by vapour-phase action of captafol and captan. The mechanism by which these phthalimides may exert their genetic effects is discussed.     

Characterization of fenarimol-resistant mutants of Aspergillus nidulans

The fitness of twelve fenarimol-resistant mutants of Aspergillus nidulans was tested with respect to spore germination, germ tube elongation, hyphal growth and sporulation. Half of the strains tested were isolated on triarimolcontaining media. The other strains were selected on imazalil-or cycloheximide-containing media (Van Tuyl, 1977).A number of mutant strains produced spores with unimpaired viability on synthetic medium. In other strains a reduction in spore viability was noticed. The rate of germ tube elongation of all resistant mutants was significantly lower than that of the wild-type strain. Mutant strains with a low degree of resistance always had an almost normal mycelial growth rate, whereas growth of some of the strains with a relatively high degree of resistance was significantly slower. Spore production on malt agar was highest in the wild-type strain and was found to be lower in fenarimol-resistant mutants. In most of the mutant strains tested a high degree of cross-resistance between fenarimol, imazalil and triadimefon was established; in some of them cross-resistance to these chemicals was low or even absent.Possible implications of the results described for the chance of development of resistance in phytopathogenic fungi to sterol biosynthesis-inhibiting fungicides are discussed.     

Properties of an Aspergillus nidulans propanil hydrolase

Aspergillus nidulans is able to hydrolyze the herbicide propanil (3′,4′-dichloropropionanilide) with liberation of 3′,4′-dichloroaniline. When the fungus is grown with or without propanil, the hydrolytic activity is identical, but can be increased by starving the mycelium either for carbon, nitrogen, or both carbon and nitrogen. The enzyme which is responsible of this activity is of the aryl acylamidase type (EC 3.5.1 aryl acylamine amidohydrolase). It is also active on propionanilide and acetanilide, two structural analogs of propanil. A value of K_m = 0.13 mM has been obtained for propanil. Temperature optimum is 40°C, when assayed with propanil as substrate. Although the pH optimum is 8, there is a relatively high enzyme activity over a wide range of pH values between 7.8 and 10.2. Carbaryl has been found to effectively inhibit the enzyme activity on propanil (K_i = 0.03 mM). The results indicated that the properties of this aryl acylamidase from A. nidulans are very similar to those of enzymes isolated from a variety of organisms such as rice, mammals and the fungus Fusarium solani.     

Effects of imazalil on sterol composition of sensitive and DMI-resistant isolates of Penicillium italicum

Imazalil differentially inhibited dry weight increase of 10-hour-old germlings of wild-type and DMI-resistant isolates ofPenicillium italicum in liquid malt cultures. EC₅₀ values ranged from 0.005 to 0.27 μg ml^?1. In all isolates ergosterol constituted the major sterol (over 95% of total sterols) in the absence of the fungicide. Therefore, DMI-resistance cannot be associated to a deficiency of the C-14 demethylation enzyme in the ergosterol biosynthetic pathway. Imazalil treatment at concentrations around EC₅₀ values for inhibition of mycelial growth resulted in a decrease in ergosterol content and a simultaneous increase in 24-methylene-24,25-dihydrolanosterol content in all isolates. A correlation existed between the imazalil concentration necessary to induce such changes in sterol composition and the EC₅₀ values for inhibition of mycelial growth of the different isolates. The reason for the differential effects of imazalil on sterol composition in the variousP. italicum isolates may be due to decreased accumulation of the fungicide in the mycelium and to other yet non-identified mechanisms of resistance.     

Effects of imazalil on sterol composition of sensitive and DMI-resistant isolates of Penicillium italicum

Imazalil differentially inhibited dry weight increase of 10-hour-old germlings of wild-type and DMI-resistant isolates ofPenicillium italicum in liquid malt cultures. EC₅₀ values ranged from 0.005 to 0.27 g ml^–1. In all isolates ergosterol constituted the major sterol (over 95% of total sterols) in the absence of the fungicide. Therefore, DMI-resistance cannot be associated to a deficiency of the C-14 demethylation enzyme in the ergosterol biosynthetic pathway. Imazalil treatment at concentrations around EC₅₀ values for inhibition of mycelial growth resulted in a decrease in ergosterol content and a simultaneous increase in 24-methylene-24,25-dihydrolanosterol content in all isolates. A correlation existed between the imazalil concentration necessary to induce such changes in sterol composition and the EC₅₀ values for inhibition of mycelial growth of the different isolates. The reason for the differential effects of imazalil on sterol composition in the variousP. italicum isolates may be due to decreased accumulation of the fungicide in the mycelium and to other yet non-identified mechanisms of resistance.Imazalil remt differentieel de toename in drooggewicht van 10-uur-oude gekiemde sporen van wild-type en DMI-resistente isolaten vanPenicillium italicum in vloeistofcultures van moutextract. De EC₅₀ waarden voor groei van de verschillende isolaten lopen uiteen van 0,005 tot 0,27 g ml^–1. In afwezigheid van het fungicide is in alle isolaten ergosterol het belangrijkste sterol (meer dan 95% van het totaal). DMI-resistentie kan daarom niet in verband staan met deficiëntie van het C-14 demethyleringsenzym in de ergosterol biosynthese. Imazalilbehandeling van mycelium bij concentraties rond de EC₅₀ waarde voor groeiremming, resulteerde bij alle isolaten in een afname van het ergosterolgehalte en een gelijktijdige toename van het gehalte aan 24-methyleen-24,25-dihydrolanosterol. Er bestaat dus een nauwe correlatie tussen de imazalilconcentratie die noodzakelijk is om vergelijkbare veranderingen in sterolsamenstelling te induceren en de EC₅₀ waarde voor remming van myceliumgroei van de verschillende isolaten. De differentiële effecten van imazalil op de sterolsamenstelling van de verschillendeP. italicum isolaten kunnen worden veroorzaakt door verminderde accumulatie van het fungicide in het mycelium en door andere, nog niet geïdentificeerde resistentiemechanismen.     

Lack of cross resistance to benomyl and thiabendazole in some strains of Aspergillus nidulans

Samenvatting Verworven resistentie inAspergillus nidulans tegen benomyl en thiabendazol bleek te berusten op één gen, gelocaliseerd op chromosoom VIII, op een afstand van 34 eenheden van ts-D 15 en 5 eenheden van orn-B 7. Hoewel kruisresistentie de regel is, blijkt dat sommige, mutanten tegen slechts één van beide fungiciden resistent zijn, en dat resistantie tegen thiabendazol zelfs gepaard kan gaan met extra gevoeligheid voor benomyl (Fig. 1 en 2).     

Mitotic instability in Aspergillus nidulans caused by the fungicides iprodione,procymidone and vinclozolin

The fungicides iprodione, procymidone and vinclozolin were found to increase greatly the frequency of mitotic recombination in diploid colonies of Aspergillus niduluns. Although generally more potent, these fungicides do not appear to differ in their activity from members of a previously suggested ?aromatic hydrocarbon group’? with which they should be classified in this context.     

Field release of a non-toxigenic Aspergillus flavus L strain in central Benin

Journal of Plant Diseases and Protection - Non-toxigenic strains of Aspergillus flavus (Link) can be used as biological control agents to reduce the contamination of grains such as maize with...     

Effect of fenpropimorph and imazalil on sterol biosynthesis in penicillium italicum

Fenpropimorph was found to be highly active against Penicillium italicum (EC₅₀ 0.01/μg ml^?1). Conidia of P. italicum, treated with low concentrations of fenpropimorph, swelled in size and showed distorted germ tubes. During the initial stages of mycelial growth, fenpropimorph had little or no effect on the dry weight increase, which became strongly inhibited within 24 h after addition of the toxicant (0.05, 0.1 and 0.2 μg ml^?1). Irregular deposition of β–1,3 and β–1, 4 polysaccharides, probably chitin, was observed after treatment with fenpropimorph or imazalil. Fenpropimorph (0.05 and 0.2 μ ml^?1) caused the accumulation of a major demethyl-sterol that was different from ergosterol. It was identified as ergosta-8, 14, 24(28)-trien-3β-ol by mass, infrared, ultraviolet and proton nuclear magnetic resonance, spectrometric procedures. At both concentrations, the accumulation was already detected after incubation for 2 h. In contrast, imazalil (0.1 μg ml^?1) caused the accumulation of several methyl- and dimethyl-sterols which were tentatively identified as eburicol (24-methylene-24, 25-dihydrolanosterol), 4, 14α-dimethylergosta-8, 24(28)-dien-3-one, 14α-methylergosta-8, 24(28)-dien-3-one and obtusifoliol (4, 14α-dimethylergosta-8, 24(28)-dien-3α-ol). The accumulation of ergosta-8, 14,24(28)-trien-3β-ol indicates inhibition of the Δ¹⁴-reductase in P. italicum in a similar manner to that found previously in Ustilago maydis.     

保鲜剂抑霉唑在香蕉中的消解动态及残留分析

采用GC-uECD法探索了室内自然温度和12~14℃恒温贮藏条件下抑霉唑在香蕉中的消解规律和最终残留量。方法的定量限为1~2μg/kg,在添加0.02、0.2、2mg/kg范围内平均回收率为94.7%~107.6%,相对标准偏差为1.0%~7.4%。采用该方法测定抑霉唑在香蕉中的残留,将抑霉唑按有效成分300~450mg/kg在香蕉上使用后,其在香蕉上的原始沉积量为1.596~1.848mg/kg,抑霉唑在香蕉中的消解行为符合一级降解动力学方程,在室内自然温度和12~14℃恒温贮藏条件下半衰期分别为8.9~10.2d和13.3d。     

抑霉唑的毛细管电泳手性拆分及其在线富集

采用毛细管电泳法对抑霉唑进行手性拆分,研究了手性选择剂β-环糊精 (β-CD)、有机添加剂、NaH₂PO₄、NH₄H₂PO₄和分离电压对手性拆分的影响;同时,采用堆积法对抑霉唑进行在线富集,研究了进样压力和进样时间对在线富集的影响。结果表明:在分离电压为20 kV、β-CD浓度为5 mmol/L及缓冲体系为2.5% 异丙醇 + 75 mmol/L NaH₂PO₄ + 5 mmol/L NH₄H₂PO₄时,抑霉唑获得最佳分离度,分离度可达3.0;在分离电压为20 kV、β-CD浓度为5 mmol/L、缓冲体系为75 mmol/L NaH₂PO₄ + 5 mmol/L NH₄H₂PO₄ + 50 mmol/L H₃PO₄及进样条件为13.8 kPa × 99.9 s时获得最高富集倍数,富集倍数达91~92倍。     

An energy-dependent efflux mechanism for fenarimol in a wild-type strain and fenarimol-resistant mutants of Aspergillus nidulans

Uptake of [¹⁴C]fenarimol (30 μM) by mycelium of wild-type Aspergillus nidulans was characterized by a rapid initial accumulation during the first 10 min of incubation with the fungicide and a subsequent gradual release with time. Uptake appeared to be the result of influx and efflux. Influx of fenarimol could not be inhibited by low temperature, anaerobiosis, starvation of mycelium, or incubation with several respiratory inhibitors and is, therefore, a passive process. Under identical test conditions efflux activity was severely inhibited and should, therefore, be regarded as an energy-dependent mechanism. After prolonged incubation (90 min) an equilibrium between influx and efflux was established, resulting in an energy-dependent permeability barrier, since uptake could instantaneously be enhanced by addition of oligomycin or N,N′-dicyclohexylcarbodiimide. It also indicates that efflux activity is inducible; this hypothesis is supported by the observation that pretreatment of mycelium with unlabeled fungicide prevented subsequent uptake of [¹⁴C]fenarimol. Uptake by fenarimol-resistant mutants J146, M193, and R264 of A. nidulans, all possessing the imaB gene for resistance, was relatively low and almost constant in time. In this case, uptake appeared to be considerably enhanced by low temperature, anaerobiosis, starvation of mycelium, and incubation with respiratory inhibitors. Low uptake by these mutants is ascribed to a higher energy-dependent efflux activity for fenarimol compared with the wild-type strain. Upon inhibition of the barrier activity, net uptake resulted from remaining passive influx, which in that case may be as high as in the wild-type strain. The results suggest that both wild-type and fenarimol-resistant mutants possess an energy-dependent efflux mechanism with different efficiencies to excrete fenarimol and probably other chemicals to which cross-resistance or collateral sensitivity is present.     

Observations on the antisporulant activity of imazalil (enilconazole)

Imazalil, incorporated in a solid nutrient medium, had no effect on the sporulation of Penicillium italicum at concentrations that did not strongly inhibit mycelial growth. However, when the fungus was grown submerged in liquid culture, sporulation of P. italicum appeared to be more affected than vegetative growth; at 33 nM, imazalil completely prevented the formation of penicilli without adverse effects on growth. Imazalil was able to prevent sporulation in the vapour phase as shown by scanning electron microscopy on P. italicum and Aspergillus niger; the vapours of the compound were found to affect different stages of the sporulation process, depending on the timing of the experiment. Thus, in order to show antisporulant activity, imazalil had to reach the target site by means other than migration within the fungus.     

Oxathiin carboxamides highly active against carboxin-resistant succinic dehydrogenase complexes from carboxin-selected mutants of Ustilago maydis and Aspergillus nidulans

The systemic fungicide carboxin (5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilide) and structurally related analogs are powerful inhibitors of succinate oxidation in mitochondria isolated from a variety of sources. The site of action which is, apparently, also that for thenoyltrifluoracetone, is in the complex II (succinate-ubiquinone reductase) region of the mitochondrial electron transfer chain. The succinic dehydrogenase complex (SDC) in mitochondria from carbon-resistant mutant strains of Ustilago maydis and Aspergillus nidulans is resistant to the fungicide in vitro. The current study shows that certain oxathiin carboxamides are selectively active against particular mutated SDCs of U. maydis and A. nidulans. Molecular structures affecting the phenotypic expression of mutation to carboxin resistance in U. maydis do not appear to affect similarly such expression in A. nidulans and vice versa. Of particular interest was the discovery of oxathiin carboxamides, e.g., 4′-phenylcarboxin, which were more inhibitory to the enzyme complex from one category of carboxin-resistant mutants of U. maydis than from the wild-type strain. Although such negative correlation between carboxin and other carboxin analogs has not been observed in studies with other categories of mutants, structures which drastically lower the resistance level were found in all cases. It appears that for any given mutation affecting carboxin sensitivity of the SDC in fungi, a specific structural group of carboxamides (or even a specific carboxamide) may be found which will alleviate or reverse the effect of the mutation in terms of inhibition of the SDC. If the mutations alter a protein receptor site for carboxamides, such mutations might be expected to influence the binding of carboxins of different structure. In essence, then, different molecular structures can “recognize” different alterations in the mutated enzyme complex and inhibit effectively. With few exceptions, the inhibition by carboxamides of cell growth of wild-type and carboxin-resistant strains of U. maydis and A. nidulans closely paralleded the inhibition of their respective SDCs. Although the few analogs tested were found unable to control corn smut systemically in seedlings artificially inoculated with compatible carboxin-resistant strains, control of naturally occurring carboxin-resistant strains of pathogenic fungi may be possible using particular structural analogs of carboxin which selectively inhibit the mutant organisms.     

抑霉唑手性异构体及其代谢物在柑橘贮藏中的选择性行为

本研究确定了抑霉唑及其代谢产物咪唑乙醇的绝对构型,建立了一种基于超高效液相色谱-串联质谱检测柑橘不同部位抑霉唑和咪唑乙醇对映体的手性分析方法,并开展了柑橘贮藏过程中抑霉唑的选择性降解行为研究.柑橘(椪柑Citrus reticulata Blanco cv.Ponkan)果肉、全果和果皮样品通过乙腈(含体积分数1％ 的...     

The determination of imazalil on potatoes and its use in controlling potato storage diseases

Methods are described for the extraction and analysis by gas-liquid and high-pressure liquid chromatography of the fungicide imazalil, 1-(β-allyloxy-2, 4-dishlorophenethyl) imidazole, on potatoes. Before storage, over 80% was recovered from potatoes treated with 0.01–3.0 mg imazalil kg^?1, with a detection limit of 2 μg kg^?1. Imazalil applied to potatoes at 10 g t^?1 before storage decreased the incidence of gangrene (Phoma exigua), silver scurf (Helminthosporium solani), skin spot (Polyscytalum pustulans) and black scurf (Rhizoctonia solani), and was at least as effective as thiabendazole applied at 40 g t^?1. At 1 g t^?1 it also decreased skin spot and silver scurf. Incidence of black dot (Colletotrichum coccodes) was unaffected by these fungicide treatments.