胶孢炭疽菌及两种疫霉菌对苯酰菌胺的敏感性与

选择对多菌灵、乙霉威和苯酰菌胺具有不同敏感性的胶孢炭疽菌Colletotrichum gloeosporioides、辣椒疫霉菌Phytophthora capsici及恶疫霉菌P. cactorum,分析其β-微管蛋白氨基酸突变与敏感性的关系。结果表明,胶孢炭疽菌对苯酰菌胺、多菌灵和乙霉威的敏感性与β-微管蛋白198位或200位氨基酸突变有关：对多菌灵敏感,对苯酰菌胺和乙霉威不敏感的胶孢炭疽菌β-微管蛋白氨基酸198位为谷氨酸（E）,200位为苯丙氨酸（F）;对多菌灵已产生抗性而对苯酰菌胺和乙霉威不敏感的菌株,其氨基酸200位由苯丙氨酸（F）突变为了酪氨酸（Y）;对多菌灵高抗,对苯酰菌胺和乙霉威敏感的菌株其氨基酸198位由谷氨酸（E）突变为了丙氨酸（A）。辣椒疫霉菌和恶疫霉菌对苯酰菌胺敏感,对多菌灵和乙霉威均不敏感。检测疫霉菌菌株β-微管蛋白未发现氨基酸突变,但发现其β-微管蛋白氨基酸在196~200位与胶孢炭疽菌差异较大,这可能是导致苯酰菌胺仅对疫霉菌有抑制效果的原因。     

胶孢炭疽菌及两种疫霉菌对苯酰菌胺的敏感性与β-微管蛋白氨基酸突变的相关性分析

选择对多菌灵、乙霉威和苯酰菌胺具有不同敏感性的胶孢炭疽菌 Colletotrichum gloeosporioides、辣椒疫霉菌 Phytophthora capsici 及恶疫霉菌 P.cactorum,采用菌丝生长速率抑制法及氨基酸序列比对法分析了其 β-微管蛋白氨基酸突变与敏感性的关系。结果表明,胶孢炭疽菌对苯酰菌胺、多菌灵和乙霉威的敏感性与 β-微管蛋白198位或200位氨基酸突变有关:对多菌灵敏感、对苯酰菌胺和乙霉威不敏感的胶孢炭疽菌 β-微管蛋白氨基酸198位为谷氨酸(E),200位为苯丙氨酸(F);对多菌灵已产生抗性而对苯酰菌胺和乙霉威不敏感的菌株,其 β-微管蛋白氨基酸200位由苯丙氨酸(F)突变为了酪氨酸(Y);对多菌灵高抗、对苯酰菌胺和乙霉威敏感的菌株其 β-微管蛋白氨基酸198位由谷氨酸(E)突变为了丙氨酸(A)。辣椒疫霉菌和恶疫霉菌对苯酰菌胺敏感,对多菌灵和乙霉威均不敏感。检测疫霉菌菌株 β-微管蛋白未发现氨基酸突变,但发现其 β-微管蛋白氨基酸在196~200位与胶孢炭疽菌差异较大,这可能是导致苯酰菌胺仅对疫霉菌有抑制效果的原因。     

Identification of a novel point mutation in the <Emphasis Type="Italic">β</Emphasis>-tubulin gene of <Emphasis Type="Italic">Botrytis cinerea</Emphasis> and detection of benzimidazole resistance by a diagnostic PCR-RFLP assay

The molecular basis of resistance to benzimidazole fungicides with laboratory and field mutant isolates of Botrytis cinerea was investigated. After chemical mutagenesis with N-methyl-N-nitrosogouanidine (NMNG) two different benzimidazole-resistant phenotypes were isolated on media containing carbendazim or a mixture of carbendazim and diethofencarb. The mutant isolates from the fungicide-mixture-containing medium were moderately resistant to carbendazim with wild-type tolerance to diethofencarb while mutant isolates from carbendazim-containing medium were highly resistant to carbendazim but sensitive to diethofencarb. The studied field isolates were highly resistant to benzimidazoles and sensitive to diethofencarb. Study of fitness characteristics of benzimidazole highly-resistant isolates showed that the resistance mutation(s) had no apparent effect on fitness-determining parameters. Contrary to this, the moderately benzimidazole-resistant strains, with no increased diethofencarb sensitivity, had a significant reduction in certain ecological fitness-determining characteristics. Analysis of the sequence of the β-tubulin gene revealed two amino acid replacements in the highly benzimidazole-resistant mutants compared to that of the wild-type parent strain. One was the glutamic acid (GAG) to alanine (GCG) change at position 198 (E198A), identified in both laboratory and field highly benzimidazole-resistant isolates, a mutation previously implicated in benzimidazole resistance. The second was a novel benzimidazole resistance mutation of glutamic acid (GAG) to glycine (GGG) substitution at the same position 198 (E198G), identified in a highly benzimidazole-resistant laboratory mutant strain. Molecular analysis of the moderately benzimidazole-resistant strains revealed no mutations at the β-tubulin gene. A novel diagnostic PCR-RFLP assay utilising a BsaI restriction site present in the benzimidazole-sensitive (E198) but absent in both resistant genotypes (E198G and E198A) was developed for the detection of both amino acid replacements at the β-tubulin gene.     

Thiophanate-methyl and carbendazim resistance in Fusicoccum amygdali,the causal agent of constriction canker of peach and almond

In light of growing environmental concerns, surveys of fungicide resistance are needed to ensure efficient control of fungi and avoid unnecessary treatments. Investigations of fungicide resistance in Fusicoccum amygdali are scarce despite the economic impacts of this pathogen in peach and almond orchards. Thiophanate-methyl has been registered for more than 20 years to control F. amygdali but no resistance has been reported to date. This propesticide is metabolized by fungi into carbendazim, a β-tubulin inhibitor. Sensitivity to carbendazim of nine populations of F. amygdali from French orchards was assessed using germination bioassays. Also, resistance levels of 63 strains isolated from four populations were evaluated using mycelial growth assays. The underlying mechanism of resistance was investigated by sequencing the β-tubulin gene, the molecular target of thiophanate-methyl, in a set of isolates with different levels of sensitivity to carbendazim. Cross-resistance to thiophanate-methyl and to another β-tubulin inhibitor, diethofencarb, was also assessed in carbendazim-sensitive and -resistant strains. Isolates highly resistant to carbendazim were found in one of the nine orchards studied. Sequencing showed that resistant phenotypes carry a mutation in the β-tubulin gene leading to E198K substitution. Positive cross-resistance to thiophanate-methyl was confirmed and no negative cross-resistance to diethofencarb was identified in the phenotyped isolates, which were all resistant to this active substance. To our knowledge, this is the first report of resistance to thiophanate-methyl in F. amygdali. The high level of resistance of isolates sampled in one population is of concern, although the limited geographical scope of resistance suggests its recent emergence.     

Cross-resistance relationships between benzimidazole fungicides and diethofencarb in Botrytis cinerea and their genetical basis in Ustilago maydis

After nitrosoguanidine- or UV-mutagenesis, three different benzimidazole-resistant phenotypes were isolated on media containing benomyl or a mixture of carbendazim and diethofencarb from wild-type strains of Botrytis cinerea Pers. ex Fr. and Ustilago maydis (D.C.) Corda. Mutants of B. cinerea with moderate (MBr) or low (LBr) resistance to benzimidazoles and high resistance to diethofencarb (Dr) were isolated from the fungicide-mixture-containing medium in low frequency (7–1 × 10^?8). Only benzimidazole-resistant strains highly sensitive to diethofencarb (HBrDs) were identified on benomyl-containing medium at a frequency of 6.6 × 10^?6. Fitness-determining characteristics such as sporulation, germination and germ-tube elongation, were found to be reduced significantly in the mutants of B. cinerea that were resistant to both benzimidazoles and diethofencarb. However, pathogenicity of a MBrDr mutant strain on cucumber seedlings was equal to that of the wild type and a carbendazim + diethofencarb mixture was found to control grey mould caused by the wild type, but was not effective when the plant cotyledons were infected by the mutant strain. Three benzimidazole-resistant phenotypes (HBrDs, HBrDr, MBrDr) were isolated easily in U. maydis from a benomyl-containing medium. In contrast with B. cinerea, only one-tenth of the benzimidazole-resistant strains were sensitive to diethofencarb. Genetic analysis of benzimidazole resistance in U. maydis showed that the three benzimidazole-resistant phenotypes were due to three allelic mutations in a single gene and one of them was responsible for the negative cross-resistance between benzimidazoles and diethofencarb.     

Distribution and fitness of isolates of Botrytis cinerea with multiple fungicide resistance in Spanish greenhouses

Forty-nine greenhouses of vegetable crops were surveyed in southeast Spain at the beginning of the disease season in December 1992 to estimate frequencies of resistance to benzimidazoles, dicarboximides and N -phenylcarbamates (NPC) in B. cinerea . Out of 261 isolates collected, 28% were sensitive to both benzimidazoles and dicarboximides, 15% were benzimidazole-resistant and dicarboximide-sensitive, 8% were benzimidazole-sensitive and dicarboximide-resistant and 46% were benzimidazole- and dicarboximide-resistant. Resistance to benzimidazole, dicarboximide and N -phenylcarbamate was determined by measuring the ability of each isolate to grow in the presence of carbendazim, procymidone and diethofencarb fungicides respectively. Carbendazim- or procymidone- resistant isolates were found in all surveyed greenhouses. Three isolates were found with resistance to carbendazim, procymidone and diethofencarb collected in two adjacent greenhouses that were sprayed with the carbendazim and diethofencarb mixture. All other isolates were sensitive to the mixture because they were either sensitive to carbendazim and resistant to diethofencarb or vice versa. Fitness of 31 isolates of B. cinerea was determined in vivo by measuring their sporulation and lesion growth rate on leaf disks. No fitness costs were associated with resistance to iprodione (dicarboximide) and benomyl (benzimidazole). Isolates with EC₅₀ values higher than 101 mg/L for benomyl and 1.6 mg/L for iprodione were considered to be field resistant (they caused visible lesions on cucumber leaf disks treated with each fungicide).     

不同类型Monilinia fructicola菌株的温度适应性及其对苯并咪唑类杀菌剂和乙霉威的交互抗性

褐腐病菌Monilinia fructicola是引起多种果树褐腐病的重要病原菌,前期研究发现该病原菌对甲基硫菌灵的抗性与Tub2蛋白的多个氨基酸变异有关。为明确不同类型菌株的温度适应性及乙霉威是否对所有抗性类型菌株均具有抑菌活性,本研究测定了敏感型菌株S及3种抗性类型包括R_(E198A)、R_(E198Q)及R_(F200Y)型菌株的温度敏感性及其对甲基硫菌灵、多菌灵和乙霉威的敏感性差异,同时分析了不同突变类型菌株对苯并咪唑类药剂和乙霉威的交互抗性。结果表明,4种类型菌株的最适生长温度为21~22 ℃,其中R_(E198Q)型菌株的最适生长温度最低,且其在低温13 ℃和高温28 ℃条件下,生长速率显著低于其他3类菌株,而S、R_(E198A)及R_(F200Y)型菌株在所有温度条件下的生长速率均无显著差异。3种抗性类型菌株对甲基硫菌灵的敏感性有所差异,其中R_(E198Q)型表现较为敏感,R_(F200Y)型在低浓度下与R_(E198A)型无明显差异,但在高浓度下对甲基硫菌灵表现较为敏感;对多菌灵的敏感性差异与甲基硫菌灵相似;只是R_(E198Q) 和R_(F200Y)型菌株对多菌灵比甲基硫菌灵更为敏感。乙霉威的敏感性测定结果表明,R_(E198A)型菌株表现敏感,S类型表现为不敏感性,R_(E198Q)及 R_(F200Y)两种类型均表现为抗性,且抗性水平差别不大。线性相关分析结果表明,仅R_(E198A)型菌株和S型菌株对苯并咪唑类杀菌剂和乙霉威敏感性存在显著负相关性 (甲基硫菌灵和乙霉威:r = ?0.992,p = 0.000;多菌灵和乙霉威:r = ?0.982,p = 0.001)。综合分析结果表明,在3类抗性菌株中,仅R_(E198A)型菌株对苯并咪唑类杀菌剂和乙霉威存在负交互抗性。     

Detection and characterization of benzimidazole resistance of <Emphasis Type="Italic">Botrytis cinerea</Emphasis> in greenhouse vegetables

From 2003 to 2006, a total of 426 single-conidial isolates of B. cinerea collected from greenhouse vegetables in China were characterized for resistance to benzimidazole fungicides and diethofencarb according to inhibition of mycelial growth. Rapid development of double-resistance to benzimidazoles and diethofencarb was observed. Three types of benzimidazole-resistant isolates, Ben R1, Ben R2 and Ben R3 were detected. A new phenotype, Ben R3, which showed low level of resistance to benzimidazole fungicides and resistance to diethofencarb, was detected with frequencies of 6.8%, 10.0%, 13.2% and 12.4% from 2003 to 2006, respectively. Further studies indicated that Ben R3 was caused by a point mutation from GAG in sensitive(S) isolates to GTG at codon 198 in the β-tubulin gene, predicted to cause a change from glutamic acid to valine. Ben R3 isolates had comparable growth, sporulation and pathogenicity ability as isolates of other phenotypes but were more sensitive at lower temperatures.     

Evolution of resistance to different classes of fungicides in <Emphasis Type="Italic">Botrytis cinerea</Emphasis> from greenhouse vegetables in eastern China

Between 2003 and 2005, 337 isolates of Botrytis cinerea collected from greenhouse vegetables were characterized for resistance to fungicides. A low level of chlorothalonil resistance was detected and in these resistant isolates there was cross-resistance to captan and thiram. To the best of our knowledge, this is the first report of chlorothalonil resistance in B. cinerea from vegetables in China. The sub-population of B. cinerea highly resistant to benzimidazoles developed quickly during the years 2003 to 2005. Rapid spread of double resistance to benzimidazoles and diethofencarb was also observed. Resistance to dicarboximides was of low-level character and no highly resistant isolates were detected. In contrast, emergence of resistance to pyrimethanil, the only anilinopyrimidine fungicide used in China at present, was detected in 2003 just 3 years after pyrimethanil introduction. Pyrimethanil-resistant isolates demonstrated fitness comparable with that of wild sensitive isolates. These results suggest that pyrimethanil has a high risk of leading to resistance development in B. cinerea in greenhouse vegetables.     

Negative cross-resistance to N-phenylanilines in benzimidazole- resistant strains of Botrytis cinerea,Venturia nashicola and Venturia inaequalis

A new series of compounds with high contra-selective activity against benomyl-resistant fungal strains was found among ring-substituted N-phenyl-anilines. Hydrophobic substitution in one of the benzene rings, together with the secondary amine character of the molecule, are important factors for high fungitoxicity. The sensitivities of benomyl-resistant isolates to some representatives of the N-phenylanilines equal or even surpass their sensitivity to diethofencarb or MDPC. The negative cross-resistance with benzimdazole fungicides is valid for strains of Botrytis cinerea, Venturia nashicola and Venturia inaequalis. The strains of B. cinerea with double insensitivity to benzimidazoles and phenyl-carbamates are insensitive also to N-phenylanilines. However, the similarly double-insensitive strain of V. nashicola was found to be just as sensitive to the N-phenylanilines tested as the strain resistant only to benzimidazoles. The latter result revealed an important bonus compared with diethofencarb. Preventive application of one of the most active representatives of the N-phenylanilines to young cucumber plants was effective against infection with benomyl-resistant isolates of B. cinerea. Moderate apical translocation after root-dipping was also observed.     

Monitoring and characterization of resistance development of strawberry Phomopsis leaf blight to fungicides

From 2005 to 2009, a total of 479 single-conidial isolates of Phomopsis obscurans were collected from strawberry. The isolates were characterized for their resistance to benzimidazole fungicides, diethofencarb, and sterol demethylation inhibitors (DMIs). Low-level DMI resistant isolates (DMI-LR) and two types of benzimidazole-resistant (Ben R) isolates, Ben R1 (benzimidazole-resistant and diethofencarb -sensitive) and Ben R2 (benzimidazole-resistant and diethofencarb -resistant), were detected. Both Ben R and DMI-LR isolates exhibited comparable growth, sporulation, and pathogenicity with the sensitive isolates. No significant difference in growth at low temperature was observed between Ben R and benzimidazole-sensitive (Ben S) isolates. Ben R1 was caused by a point mutation from GAG to GTG at codon 198 in the β-tubulin gene in Ben S isolates, predicted to cause a change from glutamic acid to valine. Ben R2 was induced by a point mutation from TTC to TAC at codon 200 in the β-tubulin gene in Ben S isolates.     

Resistance to diethofencarb (NPC) in benomyl-resistant field isolates of Botrytis cinerea

Based on the 'negative cross-resistance' phenomenon between N -phenylcarbamates and benzimidazole fungicides, the fungicidal mixture of carbendazim + diethofencarb (MBC + NPC) was introduced, to control Botrytis cinerea phenotypes which are either sensitive to benzimidazoles and resistant to NPC (wild-type: Ben^S NPC^R). or resistant to benzimidazoles and sensitive to NPC(Ben^R NPC^S). At one out of four sites where the MBC + NPC mixture was used in commercial cucumber greenhouses, grey mould control failed and a new phenotype of B. cinerea was found. The new phenotype was resistant to benzimidazoles, as was 100% of the population screened in the four sites, but retained resistance to NPC. Accordingly, the new phenotype was designated Ben^R NPC^R. It was pathogenic on cucumber seedlings.     

Characterisation of benzimidazole resistance of Cercospora beticola in Serbia using PCR-based detection of resistance-associated mutations of the β-tubulin gene

A survey to detect and characterise benzimidazole resistance within populations of Cercospora beticola in Serbia was performed. From 52 field isolates collected from sugar beet and beet root, only eight were found to be benzimidazole-sensitive based on the inhibition of mycelial growth by discriminatory concentrations of carbendazim and thiophanate-methyl. Sensitivity tests revealed the presence of three resistant phenotypes among the tested isolates: high-resistance (HR), low-resistance (LR) and moderate-resistance (MR). The benzimidazole resistant isolates were characterised based on the DNA sequence of the β-tubulin gene and temperature sensitivity. The HR isolates showed no temperature sensitivity regardless of carbendazim concentration, whereas the LR and MR isolates were sensitive at lower temperatures. Analysis of the β-tubulin gene sequence revealed two amino acid replacements in the benzimidazole-resistant isolates of C. beticola. One was a glutamic acid to alanine change at position 198 (codon GAG to GCG) that was identified in HR isolates; this mutation has previously been reported to be associated with the development of benzimidazole resistance in C. beticola. The second replacement was a novel point mutation of phenylalanine (TTC) to tyrosine (TAC) at position 167, identified in low and moderate benzimidazole-resistant isolates, sharing a single LR/MR β-tubulin genotype. A diagnostic PCR-RFLP assay utilising a BsaI restriction site present in the benzimidazole sensitive and LR/MR genotypes but absent in the HR genotype was developed for the routine detection of high resistance. A mutation-specific PCR assay was developed for the diagnosis of LR/MR genotype based on a mutation from T to A at codon 167, which is unique to this genotype.     

Negative cross-resistance of benzimidazole-resistant strains of Botrytis cinerea,Fusarium nivale and Pseudocercosporella herpotrichoides to various pesticides

Among benzimidazole-resistant strains ofFusarium nivale andPseudocercosporella herpotrichoides negative cross-resistance to N-phenylcarbamates like barban or chlorpropham was more common than to diethofencarb. Such differences were also observed with N-methylcarbamate or organophosphorus insecticides and with triazine herbicides. Several compounds belonging to these various groups of pesticides were highly toxic against the most common benzimidazole-resistant isolates ofBotrytis cinerea. They were not active, however, against isolates recently found in French vineyards that were treated with a mixture of diethofencarb and carbendazim. Some diphenylether derivatives seemed to be effective against all the benzimidazole-resistant phenotypes of this fungus.     

Fungicide resistance inBotrytis cinerea isolates from strawberry crops in Huelva (southwestern Spain)

The severity of disease caused byBotrytis cinerea in strawberries is very high and chemical control is common practice; low residue levels of chemical products are required. Thus, it is important to be aware of the development of fungicide resistance in order to choose the best strategies of chemical control. In the present study we evaluated the response of 36B. cinerea isolates against eight different fungicides. The isolates were sampled twice, at the beginning and the end of the season, in 11 commercial strawberry fields located in the area of Huelva (Spain). In addition, two reference isolates, SAS56 and SAS405, were evaluated. The proportion of isolates resistant to benomyl was very high (86%). Resistance to dicarboximides was detected in 44% of the isolates and resistance to pyrimethanil in 25% of the isolates. Different degrees of sensitivity to captan and dichlofluanid were recorded. No resistance was found to diethofencarb plus carbendazim. http://www.phytoparasitica.org posting Sept. 18, 2002.     

Differential binding of a N-phenylformamidoxime compound in cell-free extracts of benzimidazole-resistant and-sensitive isolates of Venturia nashicola,Botrytis cinerea and Gibberella fujikuroi

Venturia nashicola isolates with a high level of resistance to carbendazim showed either increased sensitivity or were resistant to theN-phenylformamidoxime compoundN-(3,5-dichloro-4-propynyloyphenyl)-N′-methoxyformamidine (DCPF). Isolates with an intermediate or low level of carbendazim resistance were resistant to DCPF. Increased sensitivity to DCPF was also associated with a high level of carbendazim resistance inBotrytis cinerea but not with a moderate resistance level. Increased sensitivity to DCPF was not observed in carbendazim resistant isolates ofGibberella fujikuroi.Binding of [¹⁴C]DCPF in cell-free mycelial extracts of the highly carbendazim-resistant and DCPF-sensitive isolate ofV. nashicola was higher than in those of DCPF-resistant isolates that were either highly-resistant, intermediately resistant, or weakly resistant to carbendazim or were sensitive to carbendazim. [¹⁴C]carbendazim binding in extracts of highly carbendazim-resistant isolates ofB. cinerea was lower than that in extracts of sensitive isolates, whereas [¹⁴C]DCFP binding was higher. A decreased [¹⁴C]carbendazim binding was also observed in extracts of carbendazim-resistant isolates ofG. fujikuroi, binding of [¹⁴C]DCPF, however, was similar in extracts of both carbendazim-resistant and sensitive isolates.     

禾谷镰孢菌对多菌灵的敏感性基线及抗药性菌株生物学性质研究

 引起小麦赤霉病的禾谷镰孢菌(Fusarium graminearum)在华东地区对多菌灵已出现了高水平抗药性。本文报道了F.graminearum对多菌灵的敏感性基线及其抗药性菌株生物学特性。离体条件下多菌灵对100个野生敏感型菌株的平均EC₅₀和MIC值分别为0.5748±0.0133 μg/ml和 < 1.4μg/ml。而对50个抗药性菌株的平均EC₅₀和MIC值则分别为9.2375μg/ml和 > 100 μg/ml。在麦穗上多菌灵对50个敏感菌株和抗药性菌株防效的平均EC₅₀分别为282.6 μg/ml和 > 2 000μg/ml。从田间获得的抗多菌灵菌株对苯菌灵、噻菌灵、甲基托布津表现交互抗药性,但不同于室内突变菌株,对乙霉威不表现负交互抗药性。抗药性菌株的无性和有性繁殖后代以及在无药培养基上菌丝体转代培养后,抗药水平保持不变。抗药性菌株的菌丝生长速率、产孢能力及致病力等与敏感菌株相比没有差异。     

In vitro and In vivo Activity of Cyprodinil and Pyrimethanil on Botrytis cinerea Isolates Resistant to other Botryticides and Selection for Resistance to Pyrimethanil in a Greenhouse Population in Greece

No cross-resistance was observed between pyrimethanil or cyprodinil and the fungicides benomyl, iprodione or carbendazim + diethofencarb. In vitro, both anilinopyrimidine fungicides were effective against strains of Botrytis cinerea resistant to benzimidazoles and/or dicarboximides and against a wild type strain insensitive to diethofencarb (EC₅₀ values ranged between 0.03–0.19 and 0.006–0.054gml^–1 for pyrimethanil and cyprodinil, respectively). Preventive applications of anilinopyrimidines completely protected young cucumber plants and fruits that were inoculated with all strains of B. cinerea. The effectiveness of pyrimethanil against grey mould was studied in greenhouse grown tomatoes in relation to (a) the type of infection and the progress of the disease on different plant parts and (b) the response of the naturally occurring B. cinerea population to the selection pressure caused by eight successive applications of this fungicide. Pyrimethanil effectively controlled grey mould on leaves, fruits and stems but did not significantly reduce the number of dead plants and fruits with 'ghost spot' symptoms. The selection pressure caused by the consecutive applications of pyrimethanil resulted in reduction of its effectiveness on leaves that became apparent after the sixth application. This was correlated with a shift of the B. cinerea population (not previously exposed to anilinopyrimidines) towards reduced sensitivity, probably due to the development of a low level of resistance (R _L = 7.7). Pyrimethanil delayed the onset of the disease but it did not reduce the infection rate.     

禾谷镰孢菌对戊唑醇抗药性的诱导及抗性菌株特性研究

通过紫外线诱导获得了8株对戊唑醇具有不同抗性水平的禾谷镰孢菌Fusarium graminearum抗药突变体,其抗性倍数均≥30倍,最高达到186.03倍。将抗药突变体在无药培养基上继代培养9代后,其抗性倍数逐渐下降,抗药性可能不能稳定遗传。与亲本菌株相比,抗药突变体在菌落生长速率、菌丝干重、对温度和pH值的敏感性方面均有所下降,推测禾谷镰孢菌对戊唑醇可能具有中等或高抗药性风险。室内交互抗性测定结果表明,戊唑醇对苯醚甲环唑、多菌灵、异菌脲、百菌清及福美双均无交互抗性。     

Activity of fungicides and modulators of membrane drug transporters in field strains of Botrytis cinerea displaying multidrug resistance

In Botrytis cinerea, multidrug resistant (MDR) strains collected in French and German vineyards were tested in vitro, at the germ-tube elongation stage, towards a wide range of fungicides. Whatever the MDR phenotype, resistance was recorded to anilinopyrimidines, diethofencarb, iprodione, fludioxonil, tolnaftate and several respiratory inhibitors (e.g., penthiopyrad, pyraclostrobin). In MDR1 strains, overproducing the ABC transporter BcatrB, resistance extended to carbendazim and the uncouplers fluazinam and malonoben. In MDR2 strains, overproducing the MFS transporter BcmfsM2, resistance extended to cycloheximide, fenhexamid and sterol 14α-demethylation inhibitors (DMIs). MDR3 strains combined the overexpression of both transporters and exhibited the widest spectrum of cross resistance and the highest resistance levels. The four transport modulators, amitriptyline, chlorpromazine, diethylstilbestrol, and verapamil, known to affect some ABC transporters, were tested in B. cinerea. In our experimental conditions, the activity of several fungicides was only enhanced by verapamil. Interestingly, synergism was only recorded in MDR2 and/or MDR3 isolates treated with tolnaftate, fenhexamid, fludioxonil or pyrimethanil, suggesting that verapamil may inhibit the MFS transporter BcmfsM2. This is the first report indicating that a known modulator of ABC transporters could also block MFS transporters.