Detection and characterization of fungicide resistant phenotypes of Botrytis cinerea in lettuce crops in Greece

The development of resistance to chemical control agents needs continuous monitoring in Botrytis cinerea. 790 isolates from lettuce and other vegetable crops were collected from six widely separated sites in Greece and tested for their sensitivity to 11 fungicides from nine unrelated chemical groups. 44 of the isolates exhibited multiple resistance to fenhexamid (hydroxyanilides), azoxystrobin and pyraclostrobin (QoI’s), boscalid (SDHI’s), cyprodinil and pyrimethanil (anilinopyrimidines), fludioxonil (phenylpyrroles), carbendazim (benzimidazoles) and iprodione (dicarboximides). Thirty per cent of such phenotypes were detected in an experimental glasshouse with lettuce crops, the third year after commencing fungicide applications. The average resistance factor (R_f) for mycelial growth to fenhexamid, pyraclostrobin, boscalid, cyprodinil and fludioxonil, was over 40, 1,000, 100, 700 and 50, respectively. Some strains with high resistance to anilinopyrimidines (14 %) or moderate to fludioxonil (7 %) were detected even in isolates collected from vegetable crops prior to commercial use of these fungicides in Greece. Isolates with fludioxonil moderate resistance and fenhexamid high resistance, were detected for the first time in Greece. The results suggested the high risk in chemical control of grey mould due to development of resistance to most fungicides with site-specific modes of action. Isolates with resistance to fluazinam (phenylpyridinamines) and to chlorothalonil (phthalonitriles) were not found. The inclusion of appropriate multi-site inhibitors like chlorothalonil in fungicide anti-resistance strategies was indispensable.     

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.

     

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.     

Identification of the G143A mutation associated with QoI resistance in Cercospora beticola field isolates from Michigan,United States

BACKGROUND: Cercospora leaf spot (CLS), caused by the fungus Cercospora beticola, is the most serious foliar disease of sugar beet (Beta vulgaris L.) worldwide. Disease control is mainly achieved by timely fungicide applications. In 2011, CLS control failures were reported in spite of application of quinone outside inhibitor (QoI) fungicide in several counties in Michigan, United States. The purpose of this study was to confirm the resistant phenotype and identify the molecular basis for QoI resistance of Michigan C. beticola isolates. RESULTS: Isolates collected in Michigan in 1998 and 1999 that had no previous exposure to the QoI fungicides trifloxystrobin or pyraclostrobin exhibited QoI EC₅₀ values of ?0.006 µg mL^?1. In contrast, all isolates obtained in 2011 exhibited EC₅₀ values of > 0.92 µg mL^?1 to both fungicides and harbored a mutation in cytochrome b (cytb) that led to an amino acid exchange from glycine to alanine at position 143 (G143A) compared with baseline QoI‐sensitive isolates. Microsatellite analysis of the isolates suggested that QoI resistance emerged independently in multiple genotypic backgrounds at multiple locations. A real‐time PCR assay utilizing dual‐labeled fluorogenic probes was developed to detect and differentiate QoI‐resistant isolates harboring the G143A mutation from sensitive isolates. CONCLUSION: The G143A mutation in cytb is associated with QoI resistance in C. beticola. Accurate monitoring of this mutation will be essential for fungicide resistance management in this pathosystem. Copyright © 2012 Society of Chemical Industry     

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     

Resistance to pyraclostrobin, boscalid and multiple resistance to Pristine® (pyraclostrobin + boscalid) fungicide in Alternaria alternata causing alternaria late blight of pistachios in California

Pristine® (pyraclostrobin + boscalid) is a fungicide registered for the control of alternaria late blight in pistachio. A total of 95 isolates of Alternaria alternata collected from orchards with and without a prior history of Pristine® sprays were tested for their sensitivity towards pyraclostrobin, boscalid and Pristine® in conidial germination assays. The EC₅₀ values for 35 isolates from orchards without Pristine® sprays ranged from 0·09 to 3·14 µg mL^?1 and < 0·01 to 2·04 µg mL^?1 for boscalid and Pristine®, respectively. For pyraclostrobin, 27 isolates had EC₅₀ < 0·01 µg mL^?1 and six had low resistance (mean EC₅₀ value = 4·71 µg mL^?1). Only one isolate was resistant to all three fungicides tested, with EC₅₀ > 100 µg mL^?1. Among 59 isolates from the orchard with a history of Pristine® sprays, 56 were resistant to pyraclostrobin; only two were sensitive (EC₅₀ < 0·01 µg mL^?1) and one was weakly resistant (EC₅₀ = 10 µg mL^?1). For the majority of these isolates EC₅₀ values ranged from 0·06 to 4·22 µg mL^?1 for boscalid and from 0·22 to 7·74 µg mL^?1 for Pristine®. However, seven isolates resistant to pyraclostrobin were also highly resistant to boscalid and Pristine® and remained pathogenic on pistachio treated with Pristine®. Whereas strobilurin resistance is a common occurrence in Alternaria of pistachio, this is the first report of resistance to boscalid in field isolates of phytopathogenic fungi. No cross resistance between pyraclostrobin and boscalid was detected, suggesting that Pristine® resistance appears as a case of multiple resistance.     

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.

     

Fungicide resistance in Cercospora species causing cercospora leaf blight and purple seed stain of soybean in Argentina

Cercospora species cause cercospora leaf blight (CLB) and purple seed stain (PSS) on soybean. Because there are few resistant soybean varieties available, CLB/PSS management relies heavily upon fungicide applications. Sensitivity of 62 Argentinian Cercospora isolates to demethylation inhibitor (DMI), methyl benzimidazole carbamate (MBC), quinone outside inhibitor (QoI), succinate dehydrogenase inhibitor (SDHI) fungicides, and mancozeb was determined in this study. All isolates were sensitive to difenoconazole, epoxiconazole, prothioconazole, tebuconazole, and cyproconazole (EC₅₀ values ranged from 0.006 to 2.4 µg/ml). In contrast, 51% of the tested isolates were sensitive (EC₅₀ values ranged from 0.003 to 0.2 µg/ml), and 49% were highly resistant (EC₅₀ > 100 µg/ml) to carbendazim. Interestingly, all isolates were completely resistant to azoxystrobin, trifloxystrobin, and pyraclostrobin, and insensitive to boscalid, fluxapyroxad, and pydiflumetofen (EC₅₀ > 100 µg/ml). The G143A mutation was detected in 82% (53) of the QoI-resistant isolates and the E198A mutation in 97% (31) of the carbendazim-resistant isolates. No apparent resistance mutations were detected in the succinate dehydrogenase genes (subunits sdhB, sdhC, and sdhD). Mancozeb completely inhibited mycelial growth of the isolates evaluated at a concentration of 100 µg/ml. All Argentinian Cercospora isolates were sensitive to the DMI fungicides tested, but we report for the first time resistance to QoI and MBC fungicides. Mechanism(s) other than fungicide target-site modification may be responsible for resistance of Cercospora to QoI and MBC fungicides. Moreover, based on our results and on the recent introduction of SDHI fungicides on soybean in Argentina, Cercospora species causing CLB/PSS are insensitive (naturally resistant) to SDHI fungicides. Insensitivity must be confirmed under field conditions.     

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.     

Sensitivity of<Emphasis Type="Italic">Botrytis cinerea</Emphasis> from greenhouse vegetables to DMIs and fenhexamid

Two hundred isolates ofBotrytis cinerea were collected from greenhouse vegetables between 2003 and 2006 to determine their baseline sensitivity to triadimefone, penconazole, tebuconazole and fenhexamid. Mean values of 50% effective concentrations (EC₅₀) of inhibiting growth were 4.853±5.102, 0.41±0.215, 0.19±0.099 and 0.36±0.891 mgl ⁻¹, respectively (mean±SD). Individuals ofB. cinerea in the population differed by a factor (EC₅₀ of the least sensitive isolate/EC₅₀ of the most sensitive isolate) of 6625, 20, 603 and 1800, respectively. Naturally fenhexamid-resistant isolates were detected with an unexpected high frequency of 10% although the pathogen population had never been exposed to this fungicide. The resistance level (mean EC₅₀ of resistant isolates / mean EC₅₀ of sensitive isolates) was 19.5. These naturally resistant isolates also were resistantin vivo, and there was no significant difference in growth rate, conidial production or pathogenicity ability between naturally resistant and wild sensitive isolates. These results indicated that there was a potential risk of practical resistance if fenhexamid was applied alone. Negative cross-resistance was observed between fenhexamid and tebuconazole in 90% of the naturally resistant isolates. Moreover, an obvious synergism of the antifungal activity of fenhexamid by tebuconazole was demonstrated in some of the naturally fenhexamid-resistant isolates. http://www.phytoparasitica.org posting May 9, 2007.     

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.     

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.     

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.     

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。     

Evaluation of the incidence of the G143A mutation and cytb intron presence in the cytochrome bc-1 gene conferring QoI resistance in Botrytis cinerea populations from several hosts

BACKGROUND: Previous studies have shown that resistance of Botrytis cinerea to QoI fungicides has been attributed to the G143A mutation in the cytochrome b (cytb) gene, while, in a part of the fungal population, an intron has been detected at codon 143 of the gene, preventing QoI resistance. During 2005–2009, 304 grey mould isolates were collected from strawberry, tomato, grape, kiwifruit, cucumber and apple in Greece and screened for resistance to pyraclostrobin and for the presence of the cytb intron, using a novel real‐time TaqMan PCR assay developed in the present study. RESULTS: QoI‐resistant phenotypes existed only within the population collected from strawberries. All resistant isolates possessed the G143A mutation. Differences were observed in the genotypic structure of cytb. Individuals possessing the intron were found at high incidence in apple fruit and greenhouse‐grown tomato and cucumber populations, whereas in the strawberry population the intron frequency was lower. Cultivation of QoI‐resistant and QoI‐sensitive isolates for ten culture cycles on artificial nutrient medium in the presence or absence of fungicide selection showed that QoI resistance was stable. CONCLUSIONS: The results of the study suggest that a high risk for selection of QoI‐resistant strains exists in crops heavily treated with QoIs, in spite of the widespread occurrence of the cytb intron in B. cinerea populations. The developed real‐time TaqMan PCR constitutes a powerful tool to streamline detection of the mutation by reducing pre‐ and post‐amplification manipulations, and can be used for rapid screening and quantification of QoI resistance. Copyright © 2011 Society of Chemical Industry     

Characterisation of QoI‐resistant field isolates of Botrytis cinerea from citrus and strawberry

BACKGROUND: In 2004, field isolates of Botrytis cinerea Pers. ex Fr., resistant to strobilurin fungicides (QoIs), were first found in commercial citrus orchards in Wakayama Prefecture, Japan. Subsequently, QoI‐resistant isolates of this fungus were also detected in plastic strawberry greenhouses in Saga, Ibaraki and Chiba prefectures, Japan. Biological and molecular characterisation of resistant isolates was conducted in this study. RESULTS: QoI‐resistant isolates of B. cinerea grew well on PDA plates containing kresoxim‐methyl or azoxystrobin at 1 mg L^?1, supplemented with 1 mM of n‐propyl gallate, an inhibitor of alternative oxidase, whereas the growth of sensitive isolates was strongly suppressed. Results from this in vitro test were in good agreement with those of fungus inoculation tests in vivo. In resistant isolates, the mutation at amino acid position 143 of the cytochrome b gene, known to be the cause of high QoI resistance in various fungal pathogens, was found, but only occasionally. The heteroplasmy of cytochrome b gene was confirmed, and the wild‐type sequence often present in the majority of resistant isolates, indicating that the proportion of mutated cytochrome b gene was very low. CONCLUSION: The conventional RFLP and sequence analyses of PCR‐amplified cytochrome b gene are insufficient for molecular identification of QoI resistance in B. cinerea. Copyright © 2009 Society of Chemical Industry     

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.     

Distribution and molecular characterization of Corynespora cassiicola isolates resistant to boscalid

A total of 618 isolates of corynespora leaf spot fungus (Corynespora cassiicola) collected from 24 commercial cucumber greenhouses in 12 cities in Ibaraki Prefecture, Japan, were tested for their sensitivity to boscalid. Boscalid‐resistant isolates were detected in 17 out of 19 greenhouses with a history of use of this fungicide and detection frequencies of the resistant isolates exceeded 47% in nine greenhouses. Frequencies of very highly resistant (VHR) isolates with 50% effective concentration (EC₅₀) values of boscalid exceeding 30 μg mL^?1 were higher than those of moderately resistant (MR) isolates with EC₅₀ ranging from 2·0 to 5·9 μg mL^?1 in 11 greenhouses. Additionally, highly resistant (HR) isolates with EC₅₀ from 8·9 to 10·7 μg mL^?1 were first detected. Furthermore, molecular characterization of genes encoding succinate dehydrogenase (SDH) subunits (SdhA, SdhB, SdhC and SdhD) was carried out to elucidate the amino acid substitution responsible for the resistance to boscalid. All 23 VHR isolates had the same mutation from CAC to TAC in the SdhB gene leading to the substitution of histidine with tyrosine at amino acid position 278 (B‐H278Y). At the same position, the substitution to arginine conferred by a mutation to CGC (B‐H278R) was detected in all four HR isolates. Some MR isolates showed a substitution from serine to proline at position 73 in SdhC (C‐S73P), from serine to proline or from glycine to valine at position 89 (D‐S89P) and 109 (D‐G109V), respectively, in SdhD. There was no common mutation in SDH genes of all MR isolates.     

Occurrence and distribution of resistance to QoI fungicides in populations of Podosphaera fusca in south central Spain

Cucurbit powdery mildew caused by Podosphaera fusca limits crop production in Spain. Since its management is strongly dependent on chemicals, the rational design of control programmes requires a good understanding of the fungicide resistance phenomenon in field populations. Fifty single-spore isolates of P. fusca were tested for sensitivity to three quinone-outside inhibiting (QoI) fungicides: azoxystrobin, kresoxim-methyl and trifloxystrobin. Minimum inhibitory concentration (MIC) values for QoI-sensitive isolates were found to range from 0.25 to 10 μg ml⁻¹ for azoxystrobin to 5–25 μg ml⁻¹ for kresoxim-methyl, using a leaf disc-based bioassay. High levels of cross-resistance to QoI fungicides were found. Eleven isolates showed resistance to the three QoI fungicides tested with MIC and EC₅₀ values >500 μg ml⁻¹ resulting in RF values as high as >715 and >1000 for trifloxystrobin and azoxystrobin, respectively. A survey of P. fusca QoI resistance was carried out in different provinces located in the south central area of Spain during the cucurbit growing seasons in 2002, 2003 and 2004. Examination of a collection of 250 isolates for QoI resistance revealed that 32% were resistant to the three fungicides tested; the provinces of Ciudad Real, Córdoba and Murcia being the locations with the highest frequencies of resistance (44–74%). By contrast, no resistance was found in Badajoz, and relatively low frequencies were observed in Almería and Valencia (10–13%). Nearly 50% of resistant isolates were collected from melon plants. Based on these data, recommendations about the use of QoI fungicides for cucurbit powdery mildew management in the sampled areas are made.