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.     

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 of Erysiphe graminis on barley and wheat to sterol C-14-demethylation inhibitors1

Isolates of Erysiphe graminis f. sp. hordei and tritici with decreased sensitivity to triadimefon showed cross-resistance to other inhibitors of sterol C-14-demethylation, such as triadimenol, propiconazol, diclobutrazol, prochloraz and nuarimol. The isolates exhibited a moderate degree of resistance to these compounds. No cross-resistance was detected to tridemorph, fenpropimorph and pyrazophos. The resistant hordei isolates were more sensitive to ethirimol than the sensitive isolate. The competitive abilities of resistant hordei and tritici isolates were inferior to that of the sensitive isolates. In the presence of the fungicides no differences in germination, appressorium formation and penetration between the sensitive and resistant isolates were observed; 48 h after inoculation the sensitive isolate showed several morphological alterations and further fungal development was arrested. At four to five times higher doses of triadimefon, similar morphological alterations were detected in the resistant isolate. Low concentrations of triazole fungicides which slightly affected mycelium growth of both the sensitive and the resistant isolate of f.sp. hordei severely inhibited development of conidiophores of the sensitive isolate whereas that of the resistant isolate was hardly affected.     

Genetic control of resistance to tridemorph inustilago maydis

Mutants ofUstilago maydis with low resistance to tridemorph isolated in a mutation frequency of 7x 10^-6 after UV-irradiation and selection on media containing 25 μg ml^-1 tridemorph. Genetic analysis with nine such mutant isolates resulted in the identification of two unlinked chromosomal loci,U/tdm- 1 andU/tdm- 2. TheU/tdm mutations are responsible for low resistance levels to tridemorph (resistance factor, Rf, of 3 or 5 based on effective concentration causing a 50% reduction in the growth rate (EC₅₀) or minimal inhibitory concentration (MIC) values, respectively) and low to moderate level of resistance to fenpropimorph (Rf 10 or 16 based on MIC or EC₅₀, respectively) and fenpropidin (Rf 5 or 11 based on MIC or EC₅₀, respectively). Haploid strains carrying bothU/tdm mutations exhibit higher levels of resistance to the above fungicides, indicating interallelic interaction between nonallelic genes. Crosses between mutants carrying theU/tdm- genes with compatible isolates carrying theU/fpm- 1 orU/fpm- 2 mutations, which were found in previous work to carry fenpropimorph resistance, yielded in all cases a large number of recombinants with wild-type sensitivity, indicating that the mutant genes involved were not allelic. Cross-resistance studies with the inhibitors of C-14 demethylase showed that. the U/tdm-mutations were responsible for increased sensitivity to the triazoles triadimefon, triadimenol, propiconazole and flusilazole, and to the pyridine pyrifenox. Study of gene effect on the fitness ofU. maydis showed thatU/tdm-mutations appeared to be pleiotropic, having more or less adverse effects on growth rate in liquid culture and pathogenicity on young corn plants.     

Impact of fludioxonil resistance on fitness and cross-resistance profiles of Altrenaria solani laboratory mutants

Sensitivity and inherent resistance risk of Alternaria solani to fludioxonil, cross-resistance profiles and the potential implications of resistance mutations on fitness parameters were investigated. Fludioxonil was highly effective against a wild type A. solani field strain both in vitro (EC₅₀?=?0.05 μg/mL) and in preventive applications on artificially inoculated tomato fruit. Mutants with low [Resistance factor (Rf): 15 based on EC₅₀], medium (Rf: 150–300) and high (Rf: > 1000) levels of phenylpyrrole resistance were isolated from the wild type strain at high frequencies following mutagenesis with UV irradiation and selection on fludioxonil containing medium. Resistant isolates retained their resistance levels even after 9 subcultures on fungicide-free growth medium while they could express their resistant phenotypes in planta. Investigation of cross-resistance relationships showed that fludioxonil resistance mutations also reduce the sensitivity of mutant strains to the aromatic hydrocarbon fungicide quintozene as well as the dicarboximides iprodione and vinclozolin. No cross-resistance was observed between fludioxonil and fungicides with different modes of action such as the sterol biosynthesis inhibitors (DMIs) imazalil and flusilazole and the carboxamide boscalid. All fludioxonil resistant isolates were more sensitive to the anilinopyrimidine pyrimethanil, while only two isolates were less sensitive to the QoI pyraclostrobin compared to the wild-type strain. Study of fitness determining parameters showed that resistance mutation(s) had no adverse effects on mycelial growth, conidial germination and sensitivity to osmotic stress while they had a pleiotropic effect on virulence and conidia production in resistant mutants. Results of the present study indicate that fludioxonil is a highly effective fungicide against A. solani, while the risk of resistance development to this fungicide is considered to be medium making fludioxonil an ideal alternative to high risk fungicides such as boscalid and pyraclostrobin whose performance against early blight has already been compromised by resistance development.

     

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.     

Sensitivity of populations ofBotrytis cinerea to triazoles,benomyl and vinclozolin

Sensitivity of field isolates (121) ofBotrytis cinerea from France (1992), Germany (1979–1992), Israel (1990) and the Netherlands (1970–1989) to the triazoles tebuconazole and triadimenol, the benzimidazole benomyl and the dicarboximide vinclozolin were tested in radial growth experiments. Resistance to benomyl (in 21 to 100% of isolates tested) and vinclozolin (in 25 to 71% of isolates tested) was common in most countries. EC₅₀s (concentrations of fungicides inhibiting radial mycelial growth ofB. cinerea on B5-agar by 50%) for tebuconazole and triadimenol ranged between 0.01–1.64 and 0.4–32.6g ml^–1, respectively, and were log-normally distributed. The variation factor (ratio between EC₅₀s of the least and most sensitive isolate tested) amounts 164 and 82 for tebuconazole and triadimenol, respectively. These values are comparable to those for azole fungicides applied in control of other pathogens. Hence, variation in sensitivity to triazoles can probably not explain limited field performance of triazoles towardsB. cinerea. Isolates from south west Germany (1992) were significantly less sensitive to tebuconazole than isolates collected earlier in Germany, Israel and the Netherlands. Such less sensitive populations may contribute to the limited field performance of DMI fungicides towardsB. cinerea. The sensitivity of isolates from south west Germany to tebuconazole was similar to that of DMI-resistant mutants generated in the laboratory. These mutants displayed stable resistance with Q-values (ratio between EC₅₀ of resistant mutant and wild type isolate) between 5 and 20. Sensitivity of field isolates and laboratory mutants to tebuconazole and triadimenol was correlated.     

Cross-Resistance Patterns Among Sterol Biosynthesis Inhibiting Fungicides (SBIs) in Cercospora beticola

Thirty single-spore isolates of Cercospora beticola, collected from several fields in northern Greece, representing a broad spectrum sensitivity to the sterol demethylation-inhibiting (DMIs) fungicide flutriafol, were tested for sensitivity to eleven other sterol biosynthesis-inhibiting (SBI) fungicides and to the guanidine fungicide dodine. Sensitivity was measured as EC₅₀ values for each fungicide and log-transformed EC₅₀ values to each fungicide were pairwise correlated and the correlation coefficient estimated. These pairwise comparisons showed high correlation coefficients between the DMIs suggesting a cross-resistance relationship between these fungicides. However, the degree of cross-resistance between DMIs varied greatly. Conversely, low correlation coefficients were obtained for the pair-wise comparisons with the morpholine fungicide fenpropimorph suggesting a lack of cross-resistance between morpholines and DMIs in C. beticola. Similarly, there was no correlation between the sensitivity (EC₅₀ values) to dodine and all the other fungicides tested, indicating that there was no negative cross-resistance relationship between dodine and SBIs in C. beticola. Based on these results, combinations or alternations of fungicides which show no cross-resistance relationship should be used to control the disease in areas where reduced sensitivity to DMIs has been already observed.     

Studies on the Inherent Resistance Risk to Fenhexamid in Botrytis cinerea

After chemical mutagenesis with N-methyl-N-nitrosoguanidine (MNNG) two phenotypes that were highly or moderately resistant to fenhexamid, were isolated from a wild-type strain of Botrytis cinerea, at a mutation frequency of 0.9 × 10^–5. Resistance factors, based on EC⁵⁰ values, were 460–570 and 10–15, respectively. The mutation(s) for resistance to fenhexamid did not affect the sensitivity of mutant strains to the benzimidazole benomyl, the phenylpyridinamine fluazinam, the anilinopyrimidine cyprodinil, the guanidine iminoctadine or to the sterol-biosynthesis-inhibiting fungicides fenarimol, fenpropimorph and tridemorph. On the contrary, an increased sensitivity (EC⁵⁰ ratios of 0.2–0.6) of fenhexamid-resistant strains to the phenylpyrrole fludioxonil and the dicarboximide iprodione was observed. Study of fitness parameters of fenhexamid-resistant isolates of both phenotypic classes showed that these mutation(s) had no effect on mycelial growth and sensitivity to high osmolarity, but they did affect one or more of some other characteristics, such as sporulation, conidial germination and sclerotia production. In tests on cucumber seedlings under greenhouse conditions, all highly fenhexamid-resistant isolates tested presented decreased infection ability compared with the wild-type. Preventive applications of a commercial formulation of fenhexamid, Teldor 50 WP, were effective against lesion development on cotyledons by the wild-type, but ineffective, even in high concentrations, against disease caused by the fenhexamid-resistant isolates. The risk of resistance problems arising during commercial use of fenhexamid is 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.     

Sensitivity ofFusarium graminearum to fungicide JS399-19:In vitro determination of baseline sensitivity and the risk of developing fungicide resistance

The baseline sensitivity ofFusarium graminearum Schwade [teleomorph =Gibberella zeae (Schweinitz) Petch] to the fungicide JS399-19 (development code no.) [2-cyano-3-amino-3-phenylacrylic acetate] and the assessment of risk to JS399-19 resistancein vitro are presented. The mean EC₅₀ values for JS399-19 inhibiting mycelial growth of three populations of wild-typeF. graminearum isolates were 0.102±0.048, 0.113±0.035 and 0.110±0.036 μg ml⁻¹, respectively. Through UV irradiation and selection for resistance to the fungicide, we obtained a total of 76 resistant mutants derived from five wild-type isolates ofF. graminearum with an average frequency of 1.71 × 10⁻⁷% and 3.5%, respectively. These mutants could be divided into three categories of resistant phenotypes with low (LR), moderate (MR) and high (HR) level of resistance, determined by the EC₅₀ values of 1.5–15.0 μg ml⁻¹, 15.1–75.0 μg ml⁻¹ and more than 75.0 μg ml⁻¹, respectively. There was no positive cross-resistance between JS399-19 and fungicides belonging to other chemical classes, such as benzimidazoles, ergosterol biosynthesis inhibitors and strobilurins, suggesting that JS399-19 presumably has a new biochemical mode of action. Although the resistant mutants appeared to have comparable pathogenicity to their wild-type parental isolates, they showed decreased mycelial growth on potato-sucrose-agar plates and decreased sporulation capacity in mung bean broth. Nevertheless, most of the resistant mutants possessed fitness levels comparable to their parents and had MR or HR levels of resistance. As these studies yielded a high frequency of laboratory resistance inF. graminearum, appropriate precautions against resistance development in natural populations should be taken into account. http://www.phytoparasitica.org posting August 7, 2008.     

Distributions of Sensitivities to Three Sterol Demethylation Inhibitor Fungicides Among Populations of Uncinula necator Sensitive and Resistant to Triadimefon

ABSTRACT Single-conidial isolates of Uncinula necator from (i) a population representing two vineyards with no previous exposure to sterol demethylation inhibitor (DMI) fungicides ("unexposed," n = 77) and (ii) a population representing two vineyards in which powdery mildew was poorly controlled by triadimefon after prolonged DMI use ("selected," n = 82) were assayed to determine distributions of sensitivities to the DMI fungicides triadimenol (the active form of triadimefon), myclobutanil, and fenarimol. Median 50% effective dose (ED(50)) values (micrograms per milliliter) in the selected versus unexposed populations were 0.06 versus 1.9 for triadimenol, 0.03 versus 0.23 for myclobutanil, and 0.03 versus 0.07 for fenarimol, respectively. Isolates were grouped into sensitivity classes according to their ED(50) values, and those from the selected population were categorized as resistant if the frequency of their sensitivity class had increased significantly relative to levels found in the unexposed population (ED(50) values exceeding 0.56, 0.18, and 0.18 mug/ml for triadimenol, myclobutanil, and fenarimol, respectively). Of the 76 isolates defined as resistant to triadimenol, 64% were classified as cross-resistant to myclobutanil, 18% were classified as cross-resistant to fenarimol, and 17% were classified as resistant to all three fungicides; 25% of the isolates classified as resistant to myclobutanil also were classified as resistant to fenarimol. Similar cross-resistance relationships were revealed when all isolates were examined by regressing log ED(50) values for each fungicide against those for the remaining two fungicides to determine the correlation coefficients (e.g., r = 0.85 for triadimenol versus myclobutanil and 0.56 for triadimenol versus fenarimol). The restricted levels of cross-resistance indicated by these data, particularly between fenarimol and the other two fungicides, is in sharp contrast to the high levels of cross-resistance among DMIs reported for some other pathogens and has significant implications with respect to programs for managing grapevine powdery mildew and DMI resistance.     

New Cases of Negative Cross-resistance between Fungicides,Including Sterol Biosynthesis Inhibitors

A survey of fungicide resistance in Mycosphaerella graminicola and Tapesia acuformis, two major pathogens of winter wheat in France, respectively responsible for speckled leaf blotch and eyespot, led to the characterization of two types of resistant strains to sterol 14α-demethylation inhibitors (DMIs). Most of the strains of M. graminicola collected in France in 1997–1998 were resistant to all DMIs, and only in a few strains was the resistance to several triazoles associated with increased susceptibility to pyrimidine derivatives (i.e., fenarimol, nuarimol) and triflumizole. On the other hand, in T. acuformis the most prevalent strains were those which exhibited negative-cross resistance between DMIs. In both fungi such a phenomenon could be related to changes in cytochrome P450 sterol 14α-demethylase, the target site of these fungicides. For Botryotinia fuckeliana, the causal agent of grey mould, the extensive monitoring conducted in French vineyards before the marketing of fenhexamid revealed the presence of highly resistant strains to this promising botryticide (only in tests involving mycelial growth measurements). Negative cross-resistance to edifenphos and several sterol biosynthesis inhibitors, such as prochloraz and fenpropimorph, was observed in fenhexamid resistant strains. Synergism of the antifungal action of fenhexamid by cytochrome P450 inhibitors, such as the DMI fungicides, was only recorded in fenhexamid resistant strains. These data and those previously obtained with edifenphos resistant strains of Magnaporthe grisea (rice blast pathogen) suggest that in fenhexamid resistant strains of B. fuckeliana the same cytochrome P450 monooxygenase could be involved in detoxification of fenhexamid and activation of edifenphos. Received 6 September 1999/ Accepted in revised form 13 September 1999     

Shift of sensitivity of <Emphasis Type="Italic">Botrytis cinerea</Emphasis> to azoxystrobin in greenhouse vegetables before and after exposure to the fungicide

From 2004 to 2006, 213 isolates of Botrytis cinerea never exposed to Q_O center inhibitors (Q_OIs) were collected to determine the baseline sensitivity to azoxystrobin. In the absence of salicylhydroxamic acid (SHAM), the mean EC₅₀ values were 10.49 ± 13.12 and 0.36 ± 0.48 mg l⁻¹ for inhibiting mycelial growth and conidium germination, respectively. In the presence of SHAM, the mean EC₅₀ values were 2.24 ± 1.29 and 0.22 ± 0.11 mg l⁻¹. In 2010, five azoxystrobin-resistant isolates were detected with the resistance frequency of 2.25% in greenhouse tomatoes after 4 years of continuous exposure. These resistant isolates showed cross-resistance to other Q_OIs but not to boscalid. In addition, these resistant isolates had comparable growth, sporulation and pathogenicity ability as sensitive isolates and maintained resistance in plants and the presence of SHAM. The G143A point mutation predicted to cause a change from glycine to alanine at codon 143 of cyt b gene was found in all resistant isolates.     

Detection of resistance to sterol demethylation-inhibiting (DMI) fungicides in<Emphasis Type="Italic">Cercospora beticola</Emphasis> and efficacy of control of resistant and sensitive strains with flutriafol

Single-lesion isolates ofCercospora beticola (n=150) were collected in 1998 from sugar beet fields in the area of Serres, N. Greece. In this area, sterol demethylation-inhibiting (DMI) fungicides have been used for almost 20 years to control sugar beet leaf spot. The sensitivity of these isolates to the DMI fungicides flutriafol and difenoconazole (EC₅₀ values) was determined on the basis of inhibition of mycelial growth at several fungicide concentrations. The relative growth (RG) of isolates was correlated at all tested concentrations with the respective EC₅₀ values, indicating that RG provides a reliable estimate for the sensitivity of the isolates. The highest correlation coefficients were obtained for concentrations of 1 μg ml⁻¹ flutriafol and of 0.05 μg ml⁻¹ difenoconazole, respectively. Consequently, they are proposed for monitoring of DMI sensitivity inC. beticola populations, as single discriminatory concentrations in a simplified test method. Based on the RG values at the discriminatory concentration of 1 μg ml⁻¹ flutriafol,C. beticola isolates were classified as either resistant or sensitive. The efficacy of flutriafol, applied at the commercially recommended dose, in controlling Cercospora leaf spot was examined in field experiments conducted during 1999 and 2000. Disease incidence in plots artificially inoculated with resistant isolates and treated with flutriafol was significantly higher than in similar plots inoculated with sensitive strains. These results suggest that poor disease control after application of flutriafol may be based on the presence of resistant strains within the pathogen population in northern Greece. This emphasizes the risk of the development of practical resistance if there is increased frequency of such strains within the population. http://www.phytoparasitica.org posting July 13, 2003.     

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.     

番茄灰霉病菌对咯菌腈的抗性诱变及抗药突变体的生物学特性

为评估番茄灰霉病菌Botrytis cinerea对咯菌腈的抗性风险,就室内经紫外照射获得抗药突变体的方法及抗性突变体的生物学性状进行了研究。结果表明:番茄灰霉病菌分生孢子的紫外照射亚致死时间为90~120 s;经亚致死时间紫外照射后,4个亲本菌株中有2个菌株共产生了6个抗咯菌腈的突变体,其EC50值是亲本菌株的310倍以上,抗性突变频率为3.13×10-7;经紫外照射诱变获得的所有抗性突变体在菌丝生长速率、产孢量、产菌核能力及其在番茄果实上的致病性方面均比其亲本菌株明显降低。相关分析显示,所得抗咯菌腈突变体对氟啶胺、啶菌唑、啶酰菌胺和嘧霉胺无交互抗性。表明番茄灰霉病菌对咯菌腈的抗药性风险较低。     

Sensitivity of <Emphasis Type="Italic">Botrytis cinerea</Emphasis> to propamidine: in vitro determination of baseline sensitivity and the risk of resistance

The baseline sensitivity of Botrytis cinerea to propamidine and assessment of the risk of propamidine resistance in vitro are presented in this article. The baseline sensitivities of 41 wild-type strains were distributed as a unimodal curve with EC₅₀ values of mycelial growth ranging from 0.182 to 1.460 μg ml⁻¹, with a mean of 0.79 ± 0.27 μg ml⁻¹. A total of 10 resistant mutants, obtained from one parental strain, were induced by UV irradiation and selected for resistance to propamidine with an average frequency of 1.98 × 10⁻⁹ and 0.025 respectively. These mutants were divided into three classes of resistant phenotypes with low (LR), moderate (MR) and high (HR) levels of resistance, determined by the EC₅₀ values of 5.0–15.0 μg ml⁻¹, 15.1–75.0 μg ml⁻¹ and more than 75.0 μg ml⁻¹ respectively. Neither positive cross-resistance nor negative cross-resistance was detected between propamidine and the fungicides, benzimidazole carbendazim, anilino-pyrimidine pyrimethanil, dicarboximide iprodione or procymidone. All 10 propamidine-resistant mutants showed reduced mycelial growth in vitro, sporulation, spore germination and pathogenicity when compared with the parental strain. These studies demonstrated that propamidine possesses a low risk of resistance developing. However, as B. cinerea is a high-risk pathogen, appropriate precautions against resistance development should be taken.     

Effects of Cytochrome P450-Interacting Plant Growth Retardants,Fungicides and Related Compounds on Cell Development and Phase-I Biotransformation Capacity of Unicellular Photoautotrophic Green Algae

Fourteen compounds (paclobutrazol, triadimenol, BAS111..W, propiconazole, tetcyclacis, prochloraz, metyrapone, piperonyl butoxide, 1-aminobenzotriazole, fenpropimorph, propham, prohexadione, mepiquat chloride and chlormequat chloride), most of them established inhibitors of cytochrome P450-dependent mixed function oxygenases and used as pesticides, especially plant growth regulators or fungicides, were applied to the non-target organisms Chlorella fusca and Chlorella sorokiniana, two species of photoautotrophic unicellular green algae. The inhibitory properties of these compounds were evaluated by comparing concentration/response relationships for the integral parameters of cell volume growth and cell division with those for the P450-dependent O-dealkylase activity measured in vivo using 7-ethoxycoumarin and 7-ethoxyresorufin as xenobiotic model substrates for phase-I biotransformation. The results obtained indicate a strong algicidal activity for some of these compounds, with differential sensitivity of the order: cell division>O-dealkylation>cell volume increase. EC₅₀ values for cell division of C. fusca ranged from 0·1 to 9·3 μmol litre⁻¹ for prochloraz and paclobutrazol, respectively. Furthermore, in most cases, concentrations around 10 μmol litre⁻¹ limited significantly the capacity for cytochrome P450 O-dealkylase activity.     

The lipid compositions of two isolates of Cladosporium cucumerinum do not explain their differences in sensitivity to fungicides which inhibit sterol biosynthesis

Isolate 840905 of Cladosporium cucumerinum, when grown on agar or in liqiud medium, was sensitive to triadimenol, HWG 1608 (tebuconazole), fenpropimorph and pimaricin but relatively resistant to terbinafine. Conversely, isolate 49628 was sensitive to terbinafine but relatively resistant to the other fungicides. Changes in sterol composition following treatment with the fungicides reflected the known modes of action of each fungicide. When individual enantiomers of triadimenol were tested against isolate 840905 the order of activity in reducing mycelial growth was 1 S, 2R > 1R, 2R > 1R, 2S ≈? 1S, 2S, and this was paralleled by the depletion of ergosterol and the appearance of 14α-methyl sterols. Isolate 49628 had a greater saturated:unsaturated fatty acid ratio than did isolate 840905 but no major changes in fatty acid composition of either isolate were induced by fungicide treatment. There appears to be no obvious explanation for the differences in fungicide sensitivity of the isolates in terms of their lipid compositions.