Multiple resistance of Botrytis cinerea from kiwifruit to SDHIs,QoIs and fungicides of other chemical groups

BACKGROUND: Botrytis cinerea Pers.: Fr. is a high‐risk pathogen for fungicide resistance development that has caused resistance problems on many crops throughout the world. This study investigated the fungicide sensitivity profile of isolates from kiwifruits originating from three Greek locations with different fungicide use histories. Sensitivity was measured by in vitro fungitoxicity tests on artificial nutrient media. RESULTS: Seventy‐six single‐spore isolates were tested for sensitivity to the SDHI fungicide boscalid, the QoI pyraclostrobin, the anilinopyrimidine cyprodinil, the hydroxyanilide fenhexamid, the phenylpyrrole fludioxonil, the dicarboxamide iprodione and the benzimidazole carbendazim. All isolates from Thessaloniki showed resistance to both boscalid and pyraclostrobin, while in the other two locations the fungal population was sensitive to these two fungicides. Sensitive isolates showed EC₅₀ values to boscalid and pyraclostrobin ranging from 0.9 to 5.2 and from 0.04 to 0.14 mg L^?1 respectively, while the resistant isolates showed EC₅₀ values higher than 50 mg L^?1 for boscalid and from 16 to > 50 mg L^?1 for pyraclostrobin. All QoI‐resistant isolates carried the G143A mutation in cytb. Sensitivity determinations to the remaining fungicides revealed in total eight resistance phenotypes. No isolates were resistant to the fungicides fenhexamid and fludioxonil. CONCLUSION: This is the first report of B. cinerea field isolates with resistance to both boscalid and pyraclostrobin, and it strongly suggests that there may be a major problem in controlling this important pathogen on kiwifruit. Copyright © 2010 Society of Chemical Industry     

Resistance profiles of Botrytis cinerea populations to several fungicide classes on greenhouse tomato and strawberry in Lebanon

Tomato and strawberry are the most important protected crops in Lebanon and are seriously affected by grey mould disease, caused by Botrytis cinerea. In the present study, the fungicide sensitivity assays revealed medium to high frequencies of B. cinerea isolates resistant to benzimidazoles, dicarboximides, and anilinopyrimidines on tomato and strawberry. Fludioxonil- and boscalid-resistant mutants were uncommonly found at generally low frequency on both crops. Resistance to fenhexamid was detected in only one site on tomato but in most sites on strawberry with high frequencies, and the occurrence of resistance to QoI fungicides was ascertained on both crops. The majority of the tested isolates (>90%) exhibited multiple fungicide resistance, and isolates resistant to the seven antibotrydial fungicide classes were detected on strawberry in three locations. A high level of resistance was shown by B. cinerea mutants resistant to boscalid, fenhexamid, and QoI fungicides, while two levels of moderate and high resistance to anilinopyrimidines were identified. Genetic analysis revealed point mutations in the target genes commonly associated with resistance in B. cinerea isolates, with all mutants resistant to dicarboximides, fenhexamid, boscalid, and QoI fungicides carrying single-nucleotide polymorphims in BcOS1 (I365S/N, Q369P, and N373S), Erg27 (F412V/I), SdhB (H272R/Y), and cytb (G143A) genes, respectively. The general incorrect use of fungicides has caused the development and spread of fungicide resistance as a widespread phenomenon on protected tomato and strawberry in Lebanon. The implementation of appropriate antiresistance strategies is highly recommended.     

Recovery of fungicide-resistant <Emphasis Type="Italic">Botrytis</Emphasis> isolates from strawberry nursery plants

In a four-year survey of strawberry nursery plants, 340 Botrytis isolates were collected and examined for fungicide resistance. High percentages of isolates with resistance to members of all fungicide classes registered on strawberries in Germany were found, i.e. trifloxystrobin (90.3%), boscalid (53.8%), cyprodinil (41.5%), fludioxonil (28.2%) and fenhexamid (15.6%). Of these isolates, 10.3% possessed multiple resistance to all compounds. At 5.0%, resistance to the recently registered fungicide fluopyram was low. Dutch nursery material harboured significantly higher proportions of isolates with resistance to any or all fungicides than plants produced in Germany. These data point to nursery plants as a possible route of introducing fungicide-resistant Botrytis strains into commercial strawberry fields.     

A rapid and simple method for determining fungicide resistance in Botrytis

A simple test based on the germination of conidia of Botrytis on agar media augmented with various fungicides has been developed. Average concentrations causing a 50% reduction of germ-tube growth (EC₅₀) of highly sensitive isolates were determined on 1% malt extract agar (thiophanate-methyl 0.090 ppm; iprodione 0.566 ppm; fludioxonil 0.026 ppm; fenhexamid 0.144 ppm), 1% malt extract agar with 100 ppm salicyl hydroxamic acid (QoI fungicides, viz. trifloxystrobin 0.009 ppm; pyraclostrobin 0.013 ppm; azoxystrobin 0.087 ppm), 0.5% yeast extract agar (boscalid 0.069 ppm) and 0.5% sucrose agar (cyprodinil 0.053 ppm). In order to detect different levels of resistance against these various fungicides, two discriminatory concentrations were identified for each compound. A routine assay method was developed in which drops of a conidial suspension harvested directly from diseased plant material or sporulating cultures were incubated on each of 20 different agar media. Because of a very short time-span of 24–48 h between sample collection and evaluation of results, field-specific information on the occurrence, frequency and types of resistance of Botrytis against common botryticides in soft-fruit production may be generated prior to the main fungicide spray season at blossom time.

     

Pervasive fungicide resistance in Botrytis from strawberry in Norway: Identification of the grey mould pathogen and mutations

Control of grey mould, caused by Botrytis spp., is a major challenge in open field strawberry production. Botrytis was isolated from plant parts collected from 19 perennial strawberry fields with suspected fungicide resistance in the Agder region of Norway in 2016. Resistance to boscalid, pyraclostrobin and fenhexamid was high and found in 89.1%, 86.0% and 65.4% of conidia samples, respectively. Multiple fungicide resistance was common; 69.6% of conidia samples exhibited resistance to three or more fungicides. Botrytis group S and B. cinerea sensu stricto isolates were obtained from 19 and 16 fields, respectively. The sdhB, cytb, erg27 and mrr1 genes of a selection of isolates were examined for the presence of mutations known to confer fungicide resistance to boscalid, pyraclostrobin, fenhexamid and pyrimethanil plus fludioxonil, respectively. Allele-specific PCR assays were developed for efficient detection of resistance-conferring mutations in cytb. Among B. cinerea isolates, 84.7%, 86.3% and 61.3% had resistance-conferring mutations in sdhB, cytb and erg27, respectively. A triplet deletion in mrr1, resulting in ΔL497, commonly associated with the multidrug resistance phenotype MDR1h, was detected in 29.2% of Botrytis group S isolates. High frequencies of resistance to several fungicides were also detected in Botrytis from both imported and domestically produced strawberry transplants. Fungicide resistance frequencies were not different among fields grouped by level of grey mould problem assessed by growers, indicating factors other than fungicide resistance contributed to control failure, a fact that has important implications for future management of grey mould.     

Molecular characterization of benzimidazole-resistant B. cinerea field isolates with reduced or enhanced sensitivity to zoxamide and diethofencarb

Sensitivity profiles of Botrytis cinerea field isolates to zoxamide and the molecular basis of the resistance mechanism involved in cross-resistance relationships between benzamides, benzimidazoles and N-phenylcarbamates were investigated. B. cinerea isolates collected from southern, central and northern Greece were characterized based on their sensitivity to zoxamide, the benzimidazole carbendazim and the N-phenylcarbamate diethofencarb. Isolates exhibiting baseline sensitivity to carbendazim and zoxamide but no sensitivity to diethofencarb were considered wild type (S phenotype) and accounted for 44% of the total strains sampled. Thirty-three percent of the isolates had increased sensitivity (HS phenotype) to zoxamide and diethofencarb and were highly resistant to carbendazim compared to S isolates. Eight percent of the sample was highly resistant (HR phenotype) to all anti-tubulin agents studied. The rest of the isolates were moderately resistant to zoxamide (MR phenotype) and equally sensitive to benzimidazoles and N-phenylcarbamates compared to isolates of the S phenotype. Fungitoxicity tests with botrycides belonging to other chemical classes revealed no cross-resistance relationships between zoxamide and the phenylpyrrole fludioxonil, the dicarboximide iprodione, the hydroxyanilide fenhexamid, the anilinopyrimidine cyprodinil, the carboxamide boscalid and the strobilurin-type fungicide pyraclostrobin. Study of fitness characteristics did not show any significant difference between zoxamide resistant and sensitive isolates with respect to the parameters tested. PCR-RFLP analysis of a part of the β-tubulin gene sequence detected mutations in position 198 for both HS and HR zoxamide-sensitivity phenotypes. DNA sequence analysis of the B. cinerea β-tubulin gene revealed two previously described benzimidazole-resistance-conferring mutations. The first one was the glutamic acid (GAG) to alanine (GCG) change at position 198 (E198A), which was identified in all HS isolates. The second mutation (E198K) was a GAG-to-AAG substitution resulting in the replacement of glutamic acid with lysine present in all B. cinerea isolates highly resistant to all three anti-tubulin classes of fungicides. A number of mutations in other positions of the β-tubulin gene were detected in the moderately zoxamide-resistance phenotype.     

Biological and molecular characterization of field isolates of <Emphasis Type="Italic">Alternaria alternata</Emphasis> with single or double resistance to respiratory complex II and III inhibitors

Field isolates of Alternaria alternata collected from tomato processors were characterized for sensitivity to respiration inhibitors using in vitro mycelial growth assays. Pyraclostrobin (QoI), boscalid, fluopyram and isopyrazam (SDHIs) mean EC₅₀ values were 0.32, 1.43, 2.21, and 3.53 μg/ml respectively. Of the 42 isolates, 36 were sensitive to all respiration inhibiting fungicides tested whereas three isolates were less sensitive to boscalid, one to pyraclostrobin and two were simultaneously resistant to both inhibitors and isopyrazam. Correlation analysis between fungicide sensitivities revealed a positive cross-resistance between pyraclostrobin and tebuconazole, and between cyprodinil and mancozeb. There was no cross-resistance between QoIs, SHDIs or any other mode of action. Sequencing of the QoI and SDHI targets revealed the G143A cytochrome b resistance mutation in all pyraclostrobin-resistant isolates while analysis of the succinate dehydrogenase coding gene revealed point mutations in two of three of the gene subunits analyzed in boscalid-resistant isolates. Specifically, two isolates carried the H277Y and three the H133Q resistance mutations located in the sdhB and sdhD subunits of the respiration complex II, respectively. Isolates bearing the H277Y mutation also carried the G143A cytochrome b resistance mutation. Boscalid and pyraclostrobin-resistant isolates exhibited greater pathogenicity and sporulation compared to sensitive isolates, respectively. Isolates with cross-resistance exhibited greater pathogenicity and sporulation but slower mycelial growth compared to sensitive isolates. This is the first report of field isolates of A. alternata with single or double resistance to QoIs and SDHIs in Greece and should be considered in planning and implementing effective anti-resistance strategies.     

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.

     

不同机制杀菌剂对小麦白粉病的敏感性及与三唑酮的交互抗性

为明确三唑酮和氟环唑、吡唑醚菌酯、啶酰菌胺、嘧菌环胺、乙嘧酚5种不同作用机制的杀菌剂对小麦白粉病的敏感性及交互抗性,采用田间小区试验和室内喷雾离体叶段法测定了不同杀菌剂对小麦白粉病的防治效果。结果表明,5种不同作用机制杀菌剂对小麦白粉病的防治效果可达90%以上,而三唑酮最高的防治效果仅为72.17%;小麦白粉病菌群体对氟环唑、吡唑醚菌酯、啶酰菌胺、嘧菌环胺、乙嘧酚的敏感性EC50分别在0.087~1.901、0.058~1.402、0.186~3.014、0.222~6.005、0.006~1.742μg/mL之间,5种不同作用机制杀菌剂的敏感性均呈连续单峰曲线,可作为小麦白粉病菌对5种不同作用机制杀菌剂的敏感基线。研究表明,三唑酮与氟环唑、吡唑醚菌酯、啶酰菌胺、嘧菌环胺、乙嘧酚之间不存在交互抗性。     

Genetic structure and fungicide sensitivity of Botrytis cinerea populations isolated from grapevine in northern Italy

Grey mould, caused by Botrytis cinerea, is a disease severely affecting grape production in northern Italy. However, little information is available on the variability of B. cinerea populations associated with grapevine. The mode of reproduction, sensitivity to fungicides, and for the first time in Italy, the genetic structure of B. cinerea populations isolated from grapevine in a northern Italian region are reported. Botrytis cinerea isolates (317) were completely genotyped for six microsatellite loci and characterized for the presence of the transposable elements Boty and Flipper, for the mating type and for resistance to cyprodinil, fludioxonil, boscalid and fenhexamid. All the isolates were found to belong to B. cinerea Group II, indicating the absence of B. pseudocinerea in the investigated areas. The populations possess a high genotypic diversity, different frequencies of transposable elements and a mixed mode of reproduction. At a regional level, B. cinerea populations belong to a large and interconnected pathogen population that includes the major grape‐growing districts. The populations were generally sensitive to fungicides, with a low proportion (8%) of isolates resistant to cyprodinil, fludioxonil and boscalid. A small genetic distance was found between B. cinerea populations. However, the populations geographically isolated from the others by a mountain range showed a small but statistically significant genetic differentiation and a different pattern of fungicide resistance. The results show that northern Italian B. cinerea populations possess a high evolutionary potential and adaptive capacity.     

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.     

Screening of <Emphasis Type="Italic">Botrytis cinerea</Emphasis> isolates from vineyards in Israel for resistance to fungicides

Plots in two vineyards in the Golan Heights, Israel were treated with six botryticides during three growing seasons with 3 applications per season. Applications of fenhexamid, pyrimethanil and cyprodinil + fludioxonil were effective, resulting in 52–65% and 53–63% mean reduction in grey mould incidence and severity, respectively. Carbendazim, fluazinam and iprodione were ineffective or slightly effective. Five hundred and sixteen B. cinerea isolates were collected from infected berries or trapped from the air in the vineyards, and profiles of sensitivity to benomyl, fenhexamid, fluazinam, fludioxonil, iprodione and pyrimethanil were established for each of the isolates based on a mycelial growth test. Seventy-four percent of the isolates were sensitive to the six tested fungicides, and the other 26% of the isolates were classified into 10 phenotypes characterized by resistance to one or more fungicides. Resistant isolates showed fitness parameters similar or reduced in comparison to sensitive isolates. Resistance to benzimidazoles and to dicarboximides was the most frequent (up to 25%) and apparently pre-existed in the populations tested. Increased frequency of benzimidazole resistance, but not dicarboximide resistance, was observed following the 3 years of applications of the fungicides. High level resistance to pyrimethanil was present at a frequency of about 2% in both vineyards in the first 2 years of the sampling survey and reached 10% in the third year at Site 2. A few isolates were resistant to fenhexamid or fludioxonil (0.8 or 0.2%, respectively). No strong resistance to fluazinam was detected, although numerous, less sensitive isolates, presumably possessing multi-drug resistance traits, were recovered at higher frequency from the plots treated with fluazinam than from the untreated plots.     

Biological activity of the succinate dehydrogenase inhibitor fluopyram against Botrytis cinerea and fungal baseline sensitivity

BACKGROUND: Succinate dehydrogenase inhibitors (SDHIs) constitute a fungicide class with increasing relevance in crop protection. These fungicides could play a crucial role in successful management of grey mould disease. In the present study the effect of fluopyram, a novel SDHI fungicide, on several developmental stages of Botrytis cinerea was determined in vitro, and the protective and curative activity against the pathogen was determined on strawberry fruit. Furthermore, fungal baseline sensitivity was determined in a set of 192 pathogen isolates. RESULTS: Inhibition of germ tube elongation was found to be the most sensitive growth stage affected by fluopyram, while mycelial growth was found to be the least sensitive growth stage. Fluopyram provided excellent protective activity against B. cinerea when applied at 100 µg mL^?1 96, 48 or 24 h before the artificial inoculation of the strawberry fruit. Similarly, fluopyram showed a high curative activity when it was applied at 100 µg mL^?1 24 h post‐inoculation, but, when applications were conducted 48 or 96 h post‐inoculation, disease control efficacy was modest or low. The measurement of baseline sensitivity showed that it was unimodal in all the populations tested. The individual EC₅₀ values for fluopyram ranged from 0.03 to 0.29 µg mL^?1. In addition, no correlation was found between sensitivity to fluopyram and sensitivity to other fungicides, including cyprodinil, fenhexamid, fludioxonil, iprodione, boscalid and pyraclostrobin. CONCLUSIONS: The obtained biological activity, baseline sensitivity and cross‐resistance relationship data suggest that fluopyram could play a key role in grey mould management in the near future and encourage its introduction into spray programmes. Copyright © 2011 Society of Chemical Industry     

Detection and characterization of <Emphasis Type="Italic">Tomato spotted wilt virus</Emphasis> infecting field and greenhouse-grown crops in Zimbabwe

‘Long-canes’ which produce the first fruit crop within three months of being planted in spring are becoming the preferred raspberry nursery material in north-western Europe. However, infections of these canes by grey mould fungi (Botrytis spp.) are common. In a survey of Botrytis infections in 22 batches of long-cane plants from six different nurseries, high proportions of strains with resistance to fenhexamid (45.5 %), trifloxystrobin (87.3 %), boscalid (75.9 %), fludioxonil (35.0 %) and cyprodinil (46.4 %) were found. Further, 29.4 % of all strains harboured multiple resistance to all five of these fungicides. Fungicide-resistant strains increased in frequency during the first cropping season and were shown in one case to spread to an adjacent raspberry field within one season. Contaminated planting material may therefore compromise the long-term productivity of raspberry plantations.     

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.     

Resistance to polyoxin AL and other fungicides in Botrytis cinerea collected from sweet basil crops in Israel

A total of 568 B. cinerea isolates were collected from diseased sweet basil plants and the air in 10 sweet basil greenhouses. Mycelial growth tests were used to evaluate the sensitivity of these isolates to benomyl, fenhexamid, iprodione, polyoxin AL and pyrimethanil. EC₅₀ values for polyoxin, the main botryticide on sweet basil in Israel, ranged from 0.4 to 6.5 μg ml^?1 and had a bimodal distribution; the EC₅₀ values for sensitive isolates ranged from 0.4 to 1.5 μg ml^?1 and the EC₅₀ values for low-level resistant isolates ranged from 4 to 6.5 μg ml^?1. Among populations that had not been exposed to polyoxin treatments, 20 to 35 % of the collected isolates were low-level resistant for polyoxin. Polyoxin treatments in an experimental greenhouse shifted the equilibrium in favour of low-level resistant isolates, and the change occurred rapidly: from a frequency of 20 % low-level resistant isolates in the population that had never been treated with polyoxin to a frequency of 72 % after a few treatments over two seasons. Prolonged use of polyoxin in Israeli basil crops (in some sites for more than 10 years) does not appear to have led to the development of high-level resistance, but low-level resistant isolates were found in commercial greenhouses with the frequency of up to 73 %. High-level resistance to benzimidazoles was common (60 to 80 % of isolates) in greenhouses with a history of benzimidazole treatments; whereas 15–25 % of the isolates from greenhouses in which fungicides were not used were resistant. Low-level resistance to dicarboximides was fairly widespread (frequency of 30 to 80 % depending on the greenhouse) and a few cases of moderate resistance to dicarboximides were also noted (frequency of 0 to 9 %). Neither high- nor low-level resistance to anilinopyrimidines was common in sweet basil commercial greenhouses (0 to 7 %). However, 34 % of the isolates were strongly resistant in the experimental greenhouse, following a few treatments with anilinopyrimidine fungicides during the previous season. Before those treatments, the proportion of anilinopyrimidines resistant isolates had been 1 %. About 3 % of the isolates exhibited low-level resistance to fenhexamid and no isolates were found to be strongly resistant to fenhexamid. Low-level resistance to one fungicide was often associated with low-level resistances to other fungicides. Thirty-two phenotypes exhibiting resistance to one or more of the tested fungicides were noted among B. cinerea isolates. Resistant isolates showed similar or reduced fitness parameters in comparison to wild-type isolates.     

High‐resolution melting analysis for rapid detection and characterization of Botrytis cinerea phenotypes resistant to fenhexamid and boscalid

A novel, high‐resolution melting (HRM) analysis was developed to detect single nucleotide polymorphisms (SNPs) associated with resistance to fenhexamid (hydroxyanilides) and boscalid (succinate dehydrogenase inhibitors) in Botrytis cinerea isolates. Thirty‐six single‐spore isolates arising from 13 phenotypes were selected and tested for fungicide sensitivity. Germ tube elongation assays showed two distinct sensitivity levels for each fungicide. Sequencing revealed that resistance to fenhexamid was due to a nucleotide change in the erg27 gene, resulting in an amino acid replacement of phenylalanine (F) with serine (S) or valine (V) at position 412 of the protein, whereas in isolates resistant to boscalid, a nucleotide change in the sdhB gene resulted in the replacement of histidine (H) with arginine (R) or tyrosine (Y) at position 272 of the respective protein. In each case, melting curve analysis generated three distinct profiles corresponding to the presence of each nucleotide in the targeted areas. HRM analysis successfully detected and differentiated the substitutions associated with resistance to both fungicides. In vitro bioassays, direct sequencing and high‐resolution melting analysis showed a 100% correlation with detection of resistance. The results demonstrate the utility of HRM analysis as a potential molecular tool for routine detection of fungicide resistance using known polymorphic genes of B. cinerea populations.     

Characterization of genetic and biochemical mechanisms of fludioxonil and pyrimethanil resistance in field isolates of Penicillium digitatum

Genetic and biochemical mechanisms of fludioxonil and pyrimethanil resistance in isolates of Penicillium digitatum were evaluated and compared to those characterized in other fungi. Resistant isolates were naturally occurring in packinghouses and were not associated with crop losses. For the phenylpyrrole fludioxonil, EC50 values were 0.02 to 0.04 microg/ml for sensitive, 0.08 to 0.65 microg/ml for moderately resistant (MR), and > 40 microg/ml for highly resistant (HR) isolates. Two fludioxonil-sensitive isolates evaluated were also significantly more sensitive to the unrelated dicarboximide fungicide iprodione, that also disrupts osmotic regulation, than the MR and HR isolates. There was no consistent relationship, however, between the HR and MR isolates and their sensitivity to iprodione or osmotic stress. Although, two nucleotide substitutions were found in a sequence analysis of the N-terminal amino acid repeat region of the os-1-related histidine kinase gene among isolates of P. digitatum, these were not correlated with fludioxonil resistance. In mycelia not exposed to fludioxonil, the amount of phosphorylated OS-2-related protein (PdOS-2) was higher in fludioxonil-sensitive isolates and lowest in the HR isolate. An increase in PdOS-2 was observed for sensitive and resistant isolates after exposure to fludioxonil. In addition, glycerol content in untreated mycelia of the fludioxonil-sensitive isolate was significantly higher than in resistant isolates. After exposure to fludioxonil, glycerol concentrations significantly increased in the sensitive and MR isolates, but not in the HR isolate. Thus, our studies indicate that the mode of action of fludioxonil in P. digitatum is probably the mitogen-activated protein kinase pathway that stimulates glycerol synthesis in sensitive and MR isolates. The general suppression of this pathway in resistant isolates was supported by the fact that growth and sporulation of MR and HR isolates were significantly reduced from that of sensitive isolates. In studies on the mode of action of anilinopyrimidines (AP), EC50 values for mycelial growth of P. digitatum and the previously characterized Botrytis cinerea were determined for cyprodinil and pyrimethanil using a defined culture medium without and with the addition of selected amino acids and homocysteine. The addition of amino acids resulted in a reduced toxicity of the two AP fungicides in both fungi, but the effect of each additive was significantly lower for P. digitatum than for B. cinerea. This suggests that methionine biosynthesis is not the primary target site of APs in P. digitatum.     

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.     

Fungicide resistance status in French populations of the wheat eyespot fungi Oculimacula acuformis and Oculimacula yallundae

Eyespot, caused by Oculimacula acuformis and Oculimacula yallundae, is the major foot disease of winter wheat in several European countries, including France. It can be controlled by chemical treatment between tillering and the second node stage. The fungicides used include antimicrotubule toxicants (benzimidazoles), inhibitors of sterol 14α‐demethylation (DMIs) or of succinate dehydrogenase (SDHIs), the anilinopyrimidines cyprodinil and the benzophenone metrafenone. Since the early 1980s, a long‐term survey has been set up in France to monitor changes in the sensitivity of eyespot populations to fungicides. Resistance to benzimidazoles has become generalised since the early 1990s, in spite of the withdrawal of this class of fungicides. In the DMI group, resistance to triazoles is generalised, whereas no resistance to the triazolinethione prothioconazole has yet developed. Resistance to the imidazole prochloraz evolved successively in O. acuformis and O. yallundae and is now well established. Specific resistance to cyprodinil has also been detected, but its frequency has generally remained low. Finally, since the early 2000s, a few strains of O. yallundae displaying multidrug resistance (MDR) have been detected. These strains display low levels of resistance to prothioconazole and SDHIs, such as boscalid. Knowledge of the spatiotemporal distribution in France of O. acuformis and O. yallundae field strains resistant to fungicides allows resistance management strategies for eyespot fungi in winter wheat to be proposed.© 2012 Society of Chemical Industry