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.     

Dicarboximide resistance in Botrytis cinerea in protected lettuce

In surveys of protected lettuce crops in the Humberside area of the UK during 1984-5, 47.3% of Botrytis cinerea isolates were resistant to dicarboximides and 25% to benomyl. Dicarboximide-resistant isolates obtained direct from the field had a moderate degree of resistance with ED₅₀ values for mycelial growth on agar of 1.0-2.4 μg/ml vinclozolin for all except two of 30 isolates.
Isolates which were resistant to vinclozolin were also resistant to iprodione, myclozolin and procymidone. Thirty dicarboximide-resistant isolates obtained direct from the field were capable of infecting cucumber cotyledons sprayed with field-strength Ronilan (250 μg/ml vinclozolin) whereas sensitive strains were not. Strains showing different degrees of resistance to dicarboximides developed after incubation of sensitive and resistant strains on agar supplemented with vinclozolin. Some of these strains had ED₅₀ values for mycelial growth on agar of more than 100 μg/ml vinclozolin. No highly resistant isolates were obtained direct from the field. Resistance to dicarboximides in field isolates was stable.     

Persistence of resistance to fungicides in Sphaerotheca fuliginea

Isolates ofSphaerotheca fuliginea collected in 1981–1983 in cucumber glasshouses in the Netherlands were tested for their sensitivity to benzimidazole fungicides, dimethirimol, dinocap and pyrazophos.Resistance to dinocap was not observed, although this fungicide has been used for over 30 years. Resistance to benzimidazole fungicides and dimethirimol has been persistent since these fungicides were withdrawn for control of cucumber powdery mildew more than 10 years ago. Although pyrazophos has only been used incidentally after 1977, the level of resistance has not decreased.Factors possibly involved in the persistence of resistance and implications for disease control in practice are discussed.Samenvatting De gevoeligheid voor twee benzimidazool-fungiciden en dimethirimol, dinocap en pyrazofos werd getoetst vanSphaerotheca fuliginea isolaten die in 1981–1983 verzameld waren in komkommerkassen in Nederland. Resistentie tegen dinocap werd niet waargenomen, ofschoon dit fungicide al meer dan 30 jaar wordt gebruikt.De resistentie tegen benzimidazool-fungiciden en dimethirimol, die meer dan 10 jaar geleden werden teruggetrokken voor de bestrijding vanS. fuliginea, was persistent. Hoewel pyrazofos slechts incidenteel gebruikt is sinds 1977, is het resistentieniveau niet teruggelopen. Factoren die mogelijk betrokken zijn bij de persistentie van resistentie en de gevolgen voor de ziektebestrijding worden besproken.     

Characterization of the genetic variation and fungicide resistance in Botrytis cinerea populations on rooibos seedlings in the Western Cape of South Africa

Botrytis cinerea causes severe losses of rooibos seedlings in the Clanwilliam region of the Western Cape of South Africa. A total of 207 isolates were collected from diseased seedlings representing five nurseries. The cryptic species status of these isolates was determined with restriction enzyme analysis of the Bc-hch gene with 206 of the isolates identified as B. cinerea and one isolate as B. pseudocinerea. Analysis of the B. cinerea populations using seven microsatellite loci revealed a high total gene diversity (H), with a mean of 0.67. Analysis of molecular variance indicated that 97 % of the total genetic variation is distributed within subpopulations. Genotype flow was evident between nurseries indicated by shared occurrence of clonal lineages, in agreement with the relatively low but significant population differentiation between nurseries (mean F _ST?=?0.030, P?=?0.001). The distribution of the mating type alleles MAT1-1 and MAT1-2 differed significantly from the ratio of 1:1 for the total population and in two of the five nursery subpopulations. Together with significant linkage disequilibrium found, populations appear to mainly reproduce asexually. Fungicide resistance frequency against iprodione for 198 of the genotyped isolates, showed highly varying levels of resistance between the nurseries. The mean incidence of resistance towards iprodione was 44 % in total, ranging from 0 % to 81 % for the five nurseries. This study has shown an adaptive capacity to overcome current chemical means of control in the studied B. cinerea population.     

Fungicide resistance and genetic diversity of Botrytis cinerea isolates from a vineyard in Germany

Journal of Plant Diseases and Protection - To evaluate the genetic diversity and the possibility of changes in the population of the grey mould fungus in a single vineyard, a total of 209 Botrytis...     

Adaptation to pyrrolnitrin in Botrytis cinerea and cost of resistance

The objective of this work was to estimate the risk of a decrease in the efficacy of biocontrol as a result of selection pressure exerted by biocontrol agents on Botrytis cinerea, focusing on pyrrolnitrin, an antibiotic identified in diverse biocontrol agents having an effect on B. cinerea. To evaluate a possible decrease in sensitivity to pyrrolnitrin, 10 successive generations of five isolates of B. cinerea were produced in vitro in the presence of a sublethal dose (10 μg L^?1) of the antibiotic. For one isolate, a significant reduction in the sensitivity to pyrrolnitrin at the fifth generation was observed with a resistance factor of c. 11. The production of 10 additional generations for four of these isolates, with increasing doses of pyrrolnitrin (100–4000 μg L^?1), resulted in the development of variants of B. cinerea with high levels of resistance to the antibiotic (RF > 1000) and a reduced sensitivity in vitro to a pyrrolnitrin‐producing bacterium. Reverse adaptation of resistant variants after 10 additional generations in the absence of selection pressure was not observed, suggesting stability of the resistance. Comparison of the pyrrolnitrin‐resistant generations and their sensitive parental isolates for mycelial growth, sporulation and aggressiveness on plant tissues revealed that the high level of resistance to pyrrolnitrin resulted in a high fitness cost. Mycelial growth was reduced between 1·7 to 3·6 times and sporulation reduced 3·8 to 6·6 times that of sensitive parental isolates. Similarly, aggressiveness was 7 to 10 and 3 to 10 times lower for resistant isolates on tomato and apple, respectively. This study provides evidence that a fungal plant pathogen is able to gradually build up resistance to an antibiotic produced by a biocontrol agent.     

Dicofol resistance in Tetranychus cinnabarinus: resistance and stability of resistance in populations from Antalya, Turkey

Resistance against dicofol was investigated in the carmine spider mite, Tetranychus cinnabarinus. Higher resistance levels were detected by leaf residual bioassays than by topical bioassays, both done using a Potter spray tower, in almost all populations of T cinnabarinus examined from Antalya, Turkey. For instance, the resistance level at LC₉₅ was 17.5‐fold in topical bioassays but 58.9‐fold in leaf residual bioassays for the population collected from greenhouses in the Topçular district. There were differences of resistance levels at LC₉₅, ranging between 2.6‐ and 23.9‐fold using topical bioassays and between 5.0‐ and 58.9‐fold in residual bioassays, in populations collected from greenhouses from various districts. Populations from cotton showed lower resistance levels against dicofol than populations from greenhouses. Resistance to dicofol at LC₅₀ as indicated by topical and residual bioassays increased 19.7‐ and 100.7‐fold, respectively, in a colony from the laboratory strain of T cinnabarinus selected with dicofol alone for 16 cycles. However, the dicofol resistance at LC₅₀ increased 19.4‐ and 52.0‐fold in another colony selected in rotation with dicofol and tetradifon for six and eight cycles, respectively. The changes in resistance to dicofol 5 months after the selection ceased were as follows: in the colony selected for dicofol alone, using topical and residual bioassays, the resistance levels at LC₅₀ decreased to 11.7‐ and 99.1‐fold, respectively, and in the colony selected in rotation with dicofol and tetradifon to 10.8‐ and 15.8‐fold, respectively. © 2001 Society of Chemical Industry     

Mechanisms of resistance to fungicides in field strains of Botrytis cinerea

Field strains of Botrytis cinerea Pers ex Fr, the causal agent of grey mould diseases, were collected from French vineyards between 1993 and 2000. Several phenotypes have been characterized according to the inhibitory effects of fungicides towards germ-tube elongation and mycelial growth. Two types of benzimidazole-resistant strains (Ben R1 and Ben R2) could be detected; negative cross-resistance to phenylcarbamates (e.g. diethofencarb) was only found in Ben R1. Benzimidazole resistance was related to point mutations at codon 198 (Ben R1) or 200 (Ben R2) of the beta-tubulin gene. Most dicarboximide-resistant strains were also weakly resistant to aromatic hydrocarbon fungicides (e.g. dicloran) but remained sensitive to phenylpyrroles (e.g. fludioxonil). These resistant field strains (Imi R1) contained a single base pair mutation at position 365 in a two-component histidine kinase gene, probably involved in the fungal osmoregulation. Three anilinopyrimidine-resistant phenotypes have been identified. In the most resistant one (Ani R1), resistance was restricted to anilinopyrimidines, but no differences were observed in the amino-acid sequences of cystathionine beta-lyase (the potential target site of these fungicides) from Ani R1 or wild-type strains. In the two other phenotypes (Ani R2 and Ani R3), resistance extended to various other groups of fungicide, including dicarboximides, phenylpyrroles and sterol biosynthesis inhibitors. This multi-drug resistance was probably determined by over-production of ATP-binding cassette transporters. The hydroxyanilide fenhexamid is a novel botryticide whose primary target site is the 3-keto reductase involved in sterol C-4 demethylations. Apart from the multi-drug-resistant strain Ani R3, three other fenhexamid-resistant phenotypes have been recognized. For two of them (Hyd R1 and Hyd R2) fenhexamid-resistance seemed to result from P450-mediated detoxification. Reduced sensitivity of the target site could be the putative resistance mechanism operating in the third resistant phenotype (Hyd R3). Increased sensitivity to inhibitors of sterol 14 alpha-demethylase recorded in Hyd R1 strains was related to two amino-acid changes at positions 15 and 105 of this enzyme.     

一株具有诱导抗性木霉菌株的筛选及其对黄瓜灰霉病诱导抗性的初步研究

从植物根部土壤中分离到一株能够诱导黄瓜产生系统抗性的木霉Tr-92,通过形态观察和18SrDNA序列分析对其进行了分类学鉴定,并进行了诱导黄瓜产生系统抗性能力的初步研究.结果表明,菌株Tr-92的菌落形态和显微形态与木霉一致,18SrDNA序列与哈茨木霉的相似性为99％,初步鉴定为哈茨木霉.利用该菌株孢子悬浮液对5叶期黄瓜苗进行根部诱导接种,24 h后灰霉病菌挑战接种,对灰霉病的防效达56％,叶片中抗性相关酶过氧化物酶、多酚氧化酶、β-1,3葡聚糖酶、苯丙氨酸解氨酶、几丁质酶的酶活性显著上升.表明该菌在黄瓜根部灌施可以诱导叶部系统抗性相关酶的活性升高,可为植物病害的生物防治提供适宜的菌种资源.     

北京地区番茄灰霉病菌的多重抗药性检测

2009年12月-2010年5月,在北京12个郊区县采集番茄病标样150份,分离纯化得到109个灰葡萄孢（Botrytis cinerea）单孢菌株,用最低抑制浓度法（MIC）测定了其对苯并咪唑类（多菌灵）、二甲酰亚胺类（腐霉利）和氨基甲酸酯类（乙霉威）杀菌剂的抗药性。结果表明：番茄灰霉病菌对多菌灵、腐霉利和乙霉威产生抗性菌株的频率分别为96.3%、80.7% 和58.7%;所测菌株对3类杀菌剂的抗性类型有BenRDicSNPCS、BenSDicSNPCR、BenRDicRNPCS和BenRDicRNPCR 4种,所占比例分别是19.3%、3.7%、21.1%和56.0%,表明北京地区番茄灰霉病菌对苯并咪唑类、二甲酰亚胺类和氨基甲酸酯类三类杀菌剂的抗药性严重,在生产中需慎用,应选择一些替代的新型杀菌剂和生物农药。     

灰葡萄孢抗二甲酰亚胺类杀菌剂研究进展

综述了灰葡萄孢对二甲酰亚胺类杀菌剂抗性的产生及现状,抗性菌株的生物学特性、生理生化特性以及抗药性机制,并就灰葡萄孢对二甲酰亚胺类杀菌剂抗性研究进行了展望。     

Multiple fungicide resistance to benzimidazoles, dicarboximides and diethofencarb in field isolates of Botrytis cinerea in Israel

During the period January-March 1989, 15 greenhouses at 12 sites in Israel were surveyed for the presence of fungicide-resistant strains of Botrytis cinerea , using a fungicide-amended Botrytis -selective medium. Resistance to benzimidazoles (Ben^R) and to dicarboximides (Dic^R) was frequent in most sites. Resistance to carbendazim + diethofencarb (Ben^R NPC^R) was found in all eight sites in which a mixture of these fungicides had been used against grey mould, but not in other sites. A new phenotype of multiple fungicide resistance was found among these isolates. The new phenotype, designated Ben^R Dic^R NPC^R, combines the three previously described characteristics of resistance to benzimidazole, dicarboximide and N -phenylcarbamate fungicides. It was found only in cucumber greenhouses that had been sprayed with the fungicide mixture carbendazim + diethofencarb against grey mould. Isolates of this phenotype were pathogenic in artificial inoculation of cucumber cotyledons treated with carbendazim, iprodione or carbendazim + diethofencarb.     

Studies on resistance to dichlofluanid and other fungicides in Botrytis cinerea

Amongst 489 mass isolates of Botrytis cinerea obtained from protected crops in Humberside during 1989, 67.3% were resistant to benomyl and 45.6% to vinclozolin. Two isolates resistant to dichlofluanid were obtained with EC₅₀ values for mycelial growth on agar of 21.45 and 19.78 μg dichlofluanid/ml, respectively, compared with values of 1.55-2.56 μg/ml for sensitive isolates. EC₅₀ values for germ tube emergence and length were much lower than for mycelial growth. In population studies carried out on tomatoes in a polythene tunnel, resistance to dichlofluanid and to vinclozolin declined over a 12-week period in the absence of fungicides. Field isolates of B. cinerea resistant to dichlofluanid were also resistant to vinclozolin. but strains resistant to dichlofluanid and sensitive to vinclozolin were obtained during population studies. Examination of a number of features of dichlofluanid-resistant strains revealed a degree of lack of fitness as compared with sensitive strains. There is no indication that resistance to dichlofluanid poses a great threat to the control of grey mould in Humberside at present.     

Acquired resistance to benomyl and some other systemic fungicides in a strain of Botrytis cinerea in cyclamen

A benomyl-resistant strain (R) ofBotrytis cinerea was isolated from cyclamen that had been sprayed with relatively high doses of Benlate two weeks before. In vitro mycelial growth of this strain was less inhibited on PDA containing 1000 g/ml benomyl (Benlate, 50% W.P.) than that of another, wild isolate ofB. cinerea from cyclamen on PDA with 0.5 g/ml of the fungicide.The R-strain was also resistant to methyl-thiophanate, furidazol and to a lesser extent to thiabendazole. Mycelial growth of 5 other isolates was much more inhibited by benomyl than by thiabendazole.Resistance was retained for at least 20 weeks after repeated subculturing on fungicide-free agar.Samenvatting In een kwekerij, waar bespuiting met benomyl (Benlate, 50% W.P.) drie maal was toegepast ter bestrijding vanBotrytisrot in cyclamen, bleek de laatste bespuiting niet meer effectief. Integendeel, de ziekte breidde zich sneller uit dan onder normale omstandigheden het geval is. Uit bloemstelen van de aangetaste planten werd eenB. cinerea-stam (R) geïsoleerd, die zeer resistent bleek tegen benomyl. In vitro werd de groei van deze stam op aardappel-glucose-agar met 1000 g/ml benomyl (Benlate 50% W.P.) minder geremd dan die van een willekeurigB. cinerea-isolaat van cyclamen op het medium met 0.5 g/ml van het fungicide (Tabel 1, Fig. 1).De R-stam bleek eveneens resistent tegen methyl-thiophanaat, furidazol en in mindere mate tegen thiabendazol (Tabel 2).De myceliumgroei van vijf isolaten vanB. cinerea, verkregen van verschillende waardplanten, bleek in tegenstelling tot die van de R-stam juist sterker geremd te worden door benomyl dan door thiabendazol (Tabel 3).De R-stam bleef gedurende tenminste 20 weken resistent na regelmatig overenten op voedingsbodems zonder het fungicide.     

Induced systemic resistance in Trichoderma harzianum T39 biocontrol of Botrytis cinerea

Biocontrol of Botrytis cinerea with Trichoderma spp. is generally believed to result from direct interaction of the biocontrol agent with the pathogen or from a Trichoderma-induced change in environmental conditions that affects B. cinerea development. In this work we provide arguments for the participation of induced plant defence in T. harzianum T39 control of B. cinerea. In tomato, lettuce, pepper, bean and tobacco, T. harzianum T39 application at sites spatially separated from the B. cinerea inoculation resulted in a 25–100%percnt; reduction of grey mould symptoms, caused by a delay or suppression of spreading lesion formation. Given the spatial separation of both micro-organisms, this effect was attributed to the induction of systemic resistance by T. harzianum T39. The observation that in bean the effect of T. harzianum T39 was similar to that of the rhizobacterium Pseudomonas aeruginosa KMPCH, a reference strain for the induction of systemic resistance, confirmed this hypothesis. Since B. cinerea control on tobacco leaves sprayed with T. harzianum T39 was similar to the control on leaves from T. harzianum T39 soil-treated plants, induction of plant defence might also participate in biocontrol on treated leaves.