Inheritance and Mechanisms of Resistance to Tebuconazole, a Sterol C14-Demethylation Inhibitor, inNectria haematococca

Genetic control and mechanisms of resistance to tebuconazole, a sterol C14-demethylation inhibitor, were investigated in the phytopathogenic fungusNectria haematococca. Resistant mutants have been selected from the laboratory, following UV irradiation. They have been characterized through genetic crosses and mutations in at least three genes were found to be responsible for resistance. The genesTeb1, Teb2, andTeb3were clearly identified, a fourth gene calledTeb4could be hypothesized. Mutations at lociTeb2andTeb3induced pleiotropic effects such as reduced sporulation and growth rate, mycelium pigmentation (Teb2), or altered ascospore viability (Teb3). The resistance levels determined by mutations in the different genes were relatively low (below 10). When associated in double mutants, the additive effect was recorded. Cross-resistance toward other sterol C14-demethylation inhibitors was observed in all the resistant strains, except in theTeb4-carrying strain; moreover, for some C14-demethylation inhibitors hypersensitivity was expressed. A constitutive energy-dependent efflux seemed implicated in the mechanism of resistance for theTeb1-carrying strain and probably also for theTeb2andTeb3-carrying strains. However, theTeb4-carrying strain exhibited a kinetic of fungicide uptake similar to that of the wild-type strain. The sterol profile of theTeb4-carrying strain was similar to that of all the other resistant mutants and wild-type strains. Thus the resistance mechanism induced by mutation at theTeb4locus has not been found yet.     

Mode of action of triadimefon in Ustilago avenae

Triadimefon [1-(4-chlorophenoxy)-3,3-dimethyl-(1,2,4-triazol-1-yl)-2-butanone], 1.5–2.0 μ/ml, inhibited the multiplication of sporidia of Ustilago avenae more strongly than it did the increase of dry weight. The treated sporidia appeared swollen, multicellular, and branched. At concentrations of 1.5–100 μg of triadimefon/ml, the oxidation of glucose was not affected. Increase in dry weight and synthesis of protein, RNA, and DNA were inhibited slightly, whereas cell division was acutely arrested. After an incubation period of 9.5 hr, microscopic studies revealed that daughter cells of the treated sporidia also contained one nucleus. In sporidia treated for 6 hr with triadimefon, both the total lipid content and its composition of fatty acids were not appreciably altered. The treated cells, however, differed from control cells by a higher content of free fatty acids. Triadimefon markedly interfered in sterol biosynthesis in Ustilago avenae. Gas chromatographic (glc) analysis and [¹⁴C]acetate incorporation studies indicated that ergosterol biosynthesis was almost completely inhibited by triadimefon; on the other hand, sterol compounds representing precursors of ergosterol (probably 4,4-dimethyl and C-4-methyl sterols) accumulated in treated sporidia. As the results indicate, the inhibition of conversion of immediate sterol precursors to ergosterol may be regarded as the primary target for the action of triadimefon in Ustilago avenae.     

Characterization of Laboratory Mutants of Botrytis cinerea Resistant to QoI Fungicides

Mutants of Botrytis cinerea with moderate and high resistance to pyraclostrobin, a Qo inhibitor of mitochondrial electron transport at the cytochrome bc ₁ complex, were isolated at a high mutation frequency, after nitrosoguanidine mutagenesis and selection on medium containing pyraclostrobin and salicylhydroxamate (SHAM), a specific inhibitor of cyanide-resistant (alternative) respiration. Oxygen uptake in whole cells was strongly inhibited in the wild-type strain by pyraclostrobin and SHAM, but not in the mutant isolates. Cross-resistance studies with other Qo and Qi inhibitors (QoIs and QiIs) of cytochrome bc ₁ complex of mitochondrial respiration showed that the mutation(s) for resistance to pyraclostrobin also reduced the sensitivity of mutant strains to other QoIs as azoxystrobin, fluoxastrobin, trifloxystrobin and picoxystrobin, but not to famoxadone and to the QiIs cyazofamid and antimycin-A. An increased sensitivity of pyraclostrobin-resistant strains to the carboxamide boscalid, an inhibitor of complex II, and to the anilinopyrimidine cyprodinil, a methionine biosynthesis inhibitor, was observed. Moreover, no effect of pyraclostrobin resistance mutation(s) on fungitoxicity of the hydroxyanilide fenhexamid, the phenylpyrrole fludioxonil, the benzimidazole benomyl, and to the phenylpyridinamine fluazinam, which affect other cellular pathways, was observed. Study of fitness parameters in the wild-type and pyraclostrobin-resistant mutants of B. cinerea showed that most mutants had a significant reduction in the sporulation, conidial germination and sclerotia production. Experiments on the stability of the pyraclostrobin-resistant phenotype showed a reduction of resistance, mainly in moderate resistant strains, when the mutants were grown on inhibitor-free medium. However, a rapid recovery of the resistance level was observed after the mutants were returned to a selective medium. Studies on the competitive ability of mutant isolates against the wild-type parent strain, by applications of a mixed conidial population, showed that, in vitro, all mutants were less competitive than the wild-type strain. However, the competitive ability of high resistant mutants was higher than the moderate ones. Pathogenicity tests on cucumber seedlings showed that all mutant strains tested exhibited an infection ability similar with the wild-type parent strain. Preventive applications of the commercial product of F-500 25EC (pyraclostrobin) were effective against lesion development on cotyledons by the wild-type, but ineffective, even at high concentrations, against disease caused by the pyraclostrobin-resistant isolates. Boscalid (F-510 50WG) was found equally effective against the disease caused by the wild-type or pyraclostrobin-resistant mutants. This is the first report indicating the appearance of B. cinerea strains resistant to QoI fungicides by the biochemical mechanism of site modification and the risk for field resistance.     

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.     

In vitro selection of sterol-biosynthesis-inhibitor (SBI)-resistant mutants in Monilinia fructicola (Wint.) Honey

The inherent resistance risk forMonilinia fructicola against sterol-biosynthesis inhibitors (SBIs) was estimated inin vitro andin vivo laboratory studies. Several mutant strains were selected on media amended with the triazole fungicides penconazole, etaconazole or the morpholine fungicide fenpropimorph.The potential forM. fructicola to develop resistance to the triazoles or to the morpholines was similar.The level of resistance attained did not differ for the two classes of fungicides after a single cycle of treatment with nitrosoguanidine (NTG). Attemps to select mutants with a higher level of resistance to penconazole after successive mutagenic treatments were successful. Most of the mutants were less fit than wild-type strains. Mutants with a low level of resistance had an almost normal mycelial growth rate, whereas growth of mutants with a higher level of resistance was significantly reduced. Spore production was highest in the wild-type strains, similar to the latter in a few resistant strains and less in most others. Only one mutant with an intermediate level of resistance could successfully compete in a mixed population with a wild type strain during successive infection cycles on peaches. Resistance was not stable in highly resistant mutants. Cross resistance to the inhibitors of 14-methylsterol demethylation (DMIs) tested was confirmedin vitro andin vivo for all mutant strains. One DMI-resistant mutant was also resistant to fenpropimorph and two fenpropimorph-resistant mutants were resistant to penconazole.     

Resistance to fungicides which inhibit ergosterol biosynthesis in laboratory strains of Botrytis cinerea and Ustilago maydis

The strains of Botrytis cinerea or Ustilago maydis selected on fenarimol, triarimol, or triadimefon were also resistant to the other inhibitors of sterol C-14 demethylation; the sterol composition of the strains was normal. Among the isolates of U. maydis resistant to dodemorph, fenpropidin, fenpropimorph and tridemorph, some were resistant to the 15-azasteroid A 25822B and did not contain ergosterol. The other strains remained sensitive to A 25822B and had a normal sterol composition. All the resistant isolates and the wild-type were inhibited to the same extent by nystatin and pimaricin.     

Resistance to inhibitors of sterol biosynthesis in field isolates or laboratory strains of the eyespot pathogen Pseudocercosporella herpotrichoides

Strains of Pseudocercosporella herpotrichoides collected in France on winter wheat give either fast-growing mycelial colonies with regular margins or slow-growing mycelial colonies with irregular margins. Most of the fastgrowing isolates were sensitive to triadimenol (EC₅₀ below 2mg litre^?1), but some of them were resistant to this inhibitor of sterol C-14 demethylation. In contrast, all the slow-growing strains were highly resistant to triadimenol (EC₅₀ greater than 100 mg litre^?1). This resistance was also expressed in inhibition of germ-tube elongation. Positive cross-resistance was observed between most of the inhibitors of sterol C-14 demethylation, with the exception of some imidazole derivatives (clotrimazole, prochloraz). All the fast-growing strains were tolerant to fenpropimorph and fenpropidin whereas the slow-growing ones were susceptible; the reverse was true with piperalin and tridemorph. All the field isolates were inhibited to the same extent by the inhibitors of squalene-epoxidase, nafifine and terbinafine. Two types of mutant resistant to triadimenol have been induced under laboratory conditions from sensitive fast-growing strains. The most common mutants were resistant to all the inhibitors of sterol C–14 demethylation and also in some conditions to fenpropimorph, tridemorph and the inhibitors of squalene-epoxidase. The other mutants were characterised by a reduced spectrum of cross-resistance between triadimenol and the other inhibitors of sterol biosynthesis. The field isolates and laboratory mutants resistant to triadimenol and propiconazole were also resistant to each of the four enantiomers of these two fungicides.     

Resistance mechanisms and associated mutations in acetylcholinesterase genes in Sitobion avenae (Fabricius)

Wheat aphid, Sitobion avenae (fabricius), is one of the most important wheat pests and has been reported to be resistant to commonly used insecticides in China. To determine the resistance mechanism, the resistant and susceptible strains were developed in laboratory and comparably studied. A bioassay revealed that the resistant strain showed high resistance to pirimicarb (RR: 161.8), moderate reistance to omethoate (32.5) and monocrotophos (33.5), and low resistance to deltamethrin (6.3) and thiodicarb (5.5). A biochemistry analysis showed that both strains had similar glutathione-S-transferase (GST) activity, but the resistant strain had 3.8-fold higher esterase activity, and its AChE was insensitive to this treatment. The I₅₀ increased by 25.8-, 10.7-, and 10.4-folds for pirimicarb, omethoate, and monocrotophos, respectively, demonstrating that GST had not been involved in the resistance of S. avenae. The enhanced esterase contributed to low level resistance to all the insecticides tested, whereas higher resistance to pirimicarb, omethoate, and monocrotophos mainly depended on AChE insensitivity. However, the AChE of the resistant strain was still sensitive to thiodicarb (1.7-fold). Thus, thiodicarb could be used as substitute for control of the resistant S. avenae in this case. Furthermore, the two different AChE genes cloned from different resistant and susceptible individuals were also compared. Two mutations, L436(336)S in Sa.Ace1 and W516(435)R in Sa.Ace2, were found consistently associated with the insensitivity of AChE. They were thought to be the possible resistance mutations, but further work is needed to confirm this hypothesis.     

Vegetative compatibility grouping in Botrytis cinerea using sulphate non-utilizing mutants

Twenty-one strains of Botrytis cinerea isolated from six plant species on ten sites throughout Israel, as well as a strain from France, were tested for vegetative and mycelial incompatibility, pathogenicity, resistance to the fungicides carbendazim and iprodione, and colony morphology. Selenate-resistant mutants were isolated from the strains as spontaneous, fast-growing sectors arising from restricted colonies on medium amended with sodium selenate with a mean frequency of 0.04 sectors/colony; 81% of the sectors were sulphate non-utilizing (sul) mutants. One hundred and four sul mutants were divided into two complementary groups: resistant (66 mutants) and sensitive to chromate. Based on compatibility reactions between chromate-resistant and chromate-sensitive sul mutants, 12 strains were compatible only with themselves and were each classified as belonging to different vegetative compatibility groups (VCGs). Nine strains were each compatible with one to three other strains, and were assembled into three multi-member VCGs. Mycelial incompatibility between wild-type strains (barrage), in the form of a zone of dark pigmentation or sparse mycelium with or without dark pigmentation of the agar along the line of confrontation, was observed for 70% of the inter-strain pairings. There was no correspondence in compatibility between strains revealed by two approaches: strains in different VCGs did not necessarily produce a barrage. However, self-compatibility was observed both as heterokaryon formation between complementary sul mutants and as an absence of barrages between mycelia of wild-type strains; wild-type strains belonging to the same VCG did not exhibit strong barrages, although weak antagonistic reactions were observed. Strains in two multi-member VCGs showed the same patterns of resistance to carbendazim and iprodione; the third multi-member VCG contained isolates with different patterns of resistance. Four morphological types were revealed among wild-type strains: conidial (five strains), sclerotial (six strains), intermediate (ten strains), and mycelial (one strain). On bean leaves, conidial strains were more aggressive than sclerotial strains.     

Effect of Phenylpyrrole-resistance Mutations on Ecological Fitness of <Emphasis Type="Italic">Botrytis cinerea</Emphasis> and their Genetical Basis in <Emphasis Type="Italic">Ustilago maydis</Emphasis>

Mutants of Botrytis cinerea and Ustilago maydis highly resistant to fludioxonil were isolated at a high frequency, after nitrosoguanidine or UV mutagenesis, respectively, and selection on media containing fludioxonil. Tests on the response of mutant strains to high osmotic pressure resulted in the identification of two fludioxonil-resistant phenotypes (FLD_osm/s and FLD_osm/r), regarding the sensitivity to high osmolarity. Approximately 95% of fludioxonil-resistant mutants were found to be more sensitive to high osmotic pressure than the wild-type parent strains. Genetic analysis of phenylpyrrole-resistance in the phytopathogenic basidiomycete U. maydis, showed that fludioxonil-resistance was coded by three unlinked chromosomal loci (U/fld-1, U/fld-2 and U/fld-3), from which only the U/fld-1 mutation coded an osmotic sensitivity similar to that of the wild-types. Cross-resistance studies with fungicides from other chemical groups showed that the mutations for resistance to phenylpyrroles affect the sensitivity of mutant strains to the aromatic hydrocarbon and dicarboximide fungicides, but not to the benzimidazoles, anilinopyrimidines, phenylpyridinamines, hydroxyanilides or the sterol biosynthesis inhibiting fungicides. A study of fitness parameters in the wild-type and fludioxonil-resistant mutants of B. cinerea, showed that all osmotic sensitive (B/FLD_osm/s) isolates had significant reductions in the characteristics determining saprophytic fitness such as mycelial growth, sporulation, conidial germination and sclerotial production. Contrary to that, with the exception of mycelial growth, the fitness parameters were unaffected or only slightly affected in most of the osmotic resistant (B/FLD_osm/r) isolates. Tests on cucumber seedlings showed that the osmotic-sensitive strains were significantly less pathogenic compared with the wild-type and B/FLD_osm/r strains of B. cinerea. Preventative applications of the commercial products Saphire 50 WP (fludioxonil) and Rovral 50 WP (iprodione) were effective against lesion development on cotyledons by the wild-type and the mutant strains of B. cinerea that were resistant to the anilinopyrimidine cyprodinil (B/CPL-27) and to the hydroxyanilide fenhexamid (B/FNH-21), but ineffective, even at high concentrations, against disease caused by the fludioxonil-resistant isolates (B/FLD) and a mutant strain resistant to the dicarboximide iprodione (B/IPR-1). Experiments on the stability of the fludioxonil-resistant phenotype showed a reduction of resistance, mainly in osmotic-sensitive isolates, when the mutants were grown on inhibitor-free medium. A rapid recovery of the high resistance was observed after mutants were returned to the selection medium. Studies on the competitive ability of mutant isolates against the wild-type parent strain of B. cinerea, by applications of a mixed conidial population, showed that, in vitro, all mutants were less competitive than the wild-type strain. However, the competitive ability of osmotic-resistant mutants was higher than the osmotic-sensitive ones. Furthermore, competition tests, in planta, showed a significant reduction of the frequency of both phenylpyrrole-resistant phenotypes, with a respective increase in the population of the wild-type strain of the pathogen.     

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

A variety of thiophene carboxamide compounds have been synthesized and tested on the succinate dehydrogenase complex (SDC) in mitochondria from a wild-type strain and carboxin-resistant strains of Ustilago maydis (corn smut). The site of action of thiophene carboxamides is identical to that of carboxin (5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilide) and thenoyltrifluoroacetone, that is, the succinate-ubiquinone reductase (complex II) span in the mitochondrial electron transfer chain. This investigation reveals new molecular structures which are strong inhibitors of wild-type and carboxin-resistant SDCs. The 5-amino analog of the parent anilide, 3-methylthiophene-2-carboxanilide (I), proved to be an especially potent inhibitor of the wild-type SDC (I₅₀, 0.019 μM). Analogs of (I) such as 4′-carboethoxy, 4′-nbutyl, 4′-phenyl, and 4′-benzoyl were negatively correlated in activity to the carboxanilide (I) with respect to resistance level. A number of structures showed considerable selectivity for mutated SDCs from both highly and (particularly) moderately carboxin-resistant SDCs of U. maydis, markedly lowering the resistance level, i.e., the degree of resistance. Thus, in addition to the oxathiins, specific structural groups of thiophene carboxamides can also alleviate or reverse the effect of carboxin-selected mutation with reference to inhibition of the SDC. Of important significance was the finding that molecular selectivity for mutated, carboxin-resistant SDCs can be influenced by replacement of an oxathiin by a thiophene heterocyclic ring as well as by the substitutive group on the amide nitrogen, permitting different categories of mutant types and even mutants within a single category to be distinguished from one another. With all the structural combinations available, it appears quite possible, in terms of inhibition, to overcome any type of mutation in a fungal SDC which arises through selection by carboxin or other carboxamide compounds. A reasonable correlation generally exists between inhibition by thiophene carboxamides of the SDC and sporidial growth of wild-type and carboxin-resistant strains of U. maydis. A permeability barrier to 4′-substituted analogs of (I) was encountered in the wild-type strain but not mutant strains. Excellent protectant activity against bean rust (Uromyces phaseoli) was obtained with the 3′-nhexyl, 3′-nhexyloxy, and 4′-phenoxy analogs of (I).     

Metabolism of a phosphoramidate by Pyricularia oryzae in relation to tolerance and synergism by a phosphorothiolate and isoprothiolane

Metabolism of dibutyl N-methyl-N-phenylphosphoramidate (BPA) by mycelial cells of Pyricularia oryzae was studied to elucidate the mechanism of synergism and negatively correlated cross-resistance in fungicidal action between phosphoramidates and phosphorothiolate derivatives. Rapid metabolism of BPA by a wild-type strain through hydroxylation and N-demethylation was observed. The metabolism was inhibited by diisopropyl S-benzyl phosphorothiolate (IBP; Kitazin P) and by isoprothiolane (diisopropyl 1,3-dithiolan-2-ylidenemalonate; Fuji-One). This inhibition of BPA metabolism is probably the mechanism of synergistic fungicidal action between the phosphoramidate and the thiol derivatives. The metabolism was, however, not inhibited by S-1358 (S-butyl S′-p-tert-butylbenzyl N-3-pyridyldithiocarbonimidate; Denmert) or triarimol [α-(2,4-dichlorophenyl)-α-phenyl-5-pyrimidinemethanol; EL-273], both of which are considered to be inhibitors of hydroxylation of a methyl radical in ergosterol biosynthesis. The metabolism of BPA by P. oryzae was much slower when mutants selected for IBP resistance and for isoprothiolane resistance were used. This phenomenon probably explains the differential sensitivity to phosphoramidate of wild-type strains and mutants resistant to the thiol derivatives.     

Mechanism of resistance to fenarimol in Aspergillus nidulans

Mycelial uptake of [¹⁴C]fenarimol (10 μg/ml) by 20 fenarimol-resistant mutants of Aspergillus nidulans 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 d-serine; this phenomenon appeared not to be due to a decreased uptake of the corresponding natural amino acids. Incorporation of [³H]adenine and [¹⁴C]leucine by mycelium of mutant M193 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 M193 incorporated [¹⁴C]acetate slightly less effectively than the wild-type strain. After 2 hr of incubation with this radiochemical leakage of [¹⁴C]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.     

扫描电镜下的小麦叶毛性状与麦长管蚜生物学参数相关性分析

为明确小麦叶毛物理性状（密度和长度）与小麦抗蚜性的关系,在人工气候室内对麦长管蚜Sitobion avenae（F.）在11个小麦品种（材料）上的发育历期、成虫与幼虫的体重差、相对日均体重增长率、若蚜死亡率等生物学参数进行了测定,采用聚类分析法评价了其对麦长管蚜的抗性,利用扫描电镜技术研究了叶毛的超显微结构,并对叶毛密度和长度与蚜虫生物学参数间的相关性进行了分析。结果表明：亲本98-10-35、Amigo及杂交后代O31、L87最不适于麦长管蚜的生长,抗性最好;O63、A253、A33抗性次之;L71、1376、H23抗性较弱,H19对麦长管蚜的适应性最好,抗性最差。扫描电镜结果表明叶毛长度和发育历期呈极显著正相关,叶毛密度和相对日均体重增长率呈极显著负相关,说明叶毛长度和密度与小麦品种（材料）的抗蚜性显著相关。表明小麦物理性状是小麦抗蚜性的重要组成部分,可以作为抗虫育种的目标之一;抗感杂交小麦后代可遗传亲本的抗感特性。     

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.     

Effects of imazalil on a wild-type and fungicide-resistant strain of Aspergillus nidulans

The effects of different incubation conditions on toxicity and uptake of imazalil by mycelium of a wild-type (003) and fungicide-resistant (R264) strain of Aspergillus nidulans were determined. In agar plate and liquid shake culture, imazalil was 200 and 40 times less toxic to the R264 strain, respectively, than to the wild-type strain. Inhibition of C-4 desmethyl sterol (ergosterol) biosynthesis occurred rapidly in mycelium of both strains at minimum growth inhibitory concentrations. Imazalil was neither detoxified nor converted into a toxic compound by mycelial suspension. Increased uptake of imazalil by the two strains occurred as concentrations of the fungicide were increased. However, the percentage uptake of imazalil by the wild-type strain was highest at the lowest concentration. These results suggest that binding in the wild-type strain involved a small number of high-affinity sites which became saturated as fungicide concentrations increased; and that at higher concentrations considerable nonspecific binding occurred in both strains. Uptake of imazalil during the initial 10 min of incubation was considerably lower in resistant than in the wild-type strain. However, upon prolonged incubation, both strains took up near equal amounts of fungicide. Uptake of fungicide by both strains was not inhibited by incubation at low temperature but was stimulated by respiratory inhibitors. These data support, in part, the hypothesis that resistance to imazalil, as reported previously for fenarimol (M. A. de Waard and J. G. M. van Nistelrooy, Pestic. Biochem. Physiol. 13, 255 (1980)), is based on reduced uptake of fungicide by mycelium of the resistant mutant.     

A major gene controlling pathogenicity inBotryotinia fuckeliana(Botrytis cinerea)

Non-aggressive mutants were isolated from a single-ascospore strain ofBotryotinia fuckelianafollowing ultraviolet and chemical mutagenesis of conidia and screening on French bean leaves (Phaseolus vulgaris). Crosses with reference strain SAS56 revealed one ultraviolet induced mutant (Mp97) in which the non-aggressive phenotype segregated 1:1 indicating control by a single gene with a major effect on pathogenicity. Further crosses showed that this gene, designatedPat1, is not linked toMbc1(benzimidazole resistance),Daf1(dicarboximide resistance),nit1(nitrate non-utilising), orSel1(sodium selenate resistance). The pathogenicity ofPat1strains was compared with various wild-type strains. Mutant strains produced small, restricted lesions on leaves of French bean and soybean (Glycine max) and slowly spreading lesions on rose (Rosaspp.) flowers. The mutant was essentially non-pathogenic on tomato stems at 20 and 25 °C but produced a few invasive infections at 10 and 15 °C. Tests of radial growth rate, sporulation and sclerotial production showed thatPat1strains exhibit no gross unfitness or unusual nutrient requirement. Althoughin vivostudies showed substantially less total polygalacturonase activity inPat1lesions compared to those produced by wild-type strains, differences in polygalacturonase isozyme profiles were not correlated with the presence ofPat1.Pat1strains showed relatively low acid production in pH indicator medium. Microscopic examination showed that lesions induced by the Mp97 mutant on French and soybean leaves (4 days old), but not by its parent, were surrounded by a distinct staining zone of mesophyll cells suggesting a difference in host response. However, no differences in phytoalexin induction were found in a soybean assay.     

Selection and characterization of Phytophthora parasitica mutants with ultraviolet-induced resistance to dimethomorph or metalaxyl

Mutants of Phytophthora parasitica Dast. more or less resistant to dimethomorph or metalaxyl were obtained by treating mycelium with ultraviolet radiation. Some metalaxyl-resistant mutants exhibited levels of resistance (i.e. the ratio between the EC₅₀ values for the mutant and the wild-type strain) greater than 100, but those observed in the dimethomorph-resistant mutants never exceeded 25. Most mutants retained their resistance after sub-culturing on unamended agar medium. Single zoospore clones derived from a metalaxyl-resistant and a dimethomorph-resistant mutant, selected as amongst the most resistant, had levels of resistance similar to those of the parental strains. The metalaxyl-resistant mutants were also resistant to the related phenylamide fungicides, furalaxyl and benalaxyl, but remained sensitive to dimethomorph. The dimethomorph-resistant mutants were not resistant to phenylamide fungicides. The pathogenicity of some metalaxyl- or dimethomorph-resistant mutants on tobacco leaves was similar to that of the wild-type strains.     

Sterol content and other characteristics of pimaricin-resistant mutants of Aspergillus nidulans

Pimaricin-resistant mutants of Aspergillus nidulans were selected on a medium containing the polyene-antibiotic. Some resistant mutants contained markedly reduced amounts of ergosterol, but others contained almost normal levels of this sterol. Most resistant mutants which lacked ergosterol had a biochemical lesion in sterol C-22 desaturation. Analysis of sterols in one of these isolates showed the presence of 5,7-ergostadienol, 5,7,24(28)-ergostatrienol, and 5,8-ergostadienol. The sterol C-14 demethylation inhibitor, fenarimol, was more toxic to this mutant than to the wild type. On the other hand, mutants inactive in sterol C-22 desaturation were resistant to oligomycin but showed wild type sensitivity to carboxin. Attempts to select sterol C-14-demethylation-deficient mutants of Aspergillus nidulans, Monilinia fructicola, and Pyricularia oryzae on polyene-containing media were unsuccessful. Apparently C-14-methyl sterols do not support growth of these filamentous fungi.     

Resistance in mutagen-induced mutants of Ustilago maydis to fungicides which inhibit ergosterol biosynthesis

Resistance to a number of inhibitors of sterol C-14 demethylation, (clotrimazole, imazalil, miconazole, fenarimol, nuarimol and triadimefon), as well as resistance to inhibitors of sterol C-14(15) double bond reduction, (tridemorph and fenpropi-morph), was readily induced in Ustilago maydis. Resistant mutants were obtained after mutagenic treatment by ultraviolet irradiation, or by treatment with 1-methyl-3-nitro-1-nitrosoguanidine, of sporidia of the wild-type strain, followed by selection in the presence of the toxicant. The level of resistance of these mutants varied appreciably. Although not always reciprocal, cross-resistance to fungicides which inhibit ergosterol biosynthesis (EBIs) appeared to be present in most cases. Several of the U. maydis mutants which were resistant to inhibitors of sterol C-14 demethylation lacked cross-resistance to tridemorph and fenpropimorph, or displayed increased sensitivity to fenpropimorph (negatively correlated cross-resistance). Cross-resistance between EBIs and the antimicrobial agents climbazole and lombazole was also established. It is suggested that fungal mutants that possess a resistance mechanism based on a deficiency in sterol C-14 demethylation or sterol C-14(15) double bond reduction, have a greatly reduced chance of survival.