Evolution of fungicide resistance in the cereal eyespot fungi Tapesia yallundae and Tapesia acuformis in France

Field isolates of the cereal eyespot pathogen can be divided into two groups which are now considered as two species: Tapesia yallundae and Tapesia acuformis. In both species the first case of acquired resistance was observed with benzimidazole fungicides in the early 1980s. At the same time, a number of sterol C-14 demethylation inhibitors (DMIs), such as the imidazole prochloraz and several triazoles, including flusilazole, were introduced. Surprisingly T. acuformis appeared intrinsically resistant to the triazole derivatives in comparison to T. yallundae, but both species were sensitive to prochloraz. The intensive use of these DMIs led to the development of acquired resistance towards triazoles in T. yallundae and towards prochloraz in T. acuformis. Today all the strains in both species appear equally sensitive to the anilinopyrimidine cyprodinil. ©1997 SCI     

Sensitivity of Mycosphaerella graminicola (anamorph: Septoria tritici) to DMI fungicides across Europe and impact on field performance

Since the occurrence and spread of resistance to quinone outside inhibitors (QoI) in Mycosphaerella graminicola in the early 2000s in Europe, demethylation inhibitors (DMIs) form the backbone for control of Septoria leaf blotch. European monitoring studies, carried out by various research institutes and DMI manufacturers, have shown a shift of the European M. graminicola population towards increased ED₅₀ values for DMI fungicides. Populations of M. graminicola consist of very heterogeneous isolates within a region, and even within a field, in terms of DMI sensitivity. Sensitivity to DMIs is influenced by the haplotype of CYP51, the target of DMIs. New CYP51‐haplotypes have emerged and the frequency of less sensitive haplotypes in Europe has increased in recent years. Studies with efflux transporter inhibitors showed that not only CYP51, but also enhanced efflux, may play a role in the DMI sensitivity response. Sensitivity studies with 5 DMIs registered for Septoria leaf blotch control indicated that sensitivity of isolates to the 5 DMIs is heterogeneous and the overall correlation of sensitivity to the different DMIs is poor. A key requirement for sustainable control and resistance management of Septoria leaf blotch is therefore the continued availability of different DMIs.     

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.     

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     

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

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

Characterization of CbCyp51 from field isolates of Cercospora beticola

The hemibiotrophic fungus Cercospora beticola causes leaf spot of sugar beet. Leaf spot control measures include the application of sterol demethylation inhibitor (DMI) fungicides. However, reduced sensitivity to DMIs has been reported recently in the Red River Valley sugar beet-growing region of North Dakota and Minnesota. Here, we report the cloning and molecular characterization of CbCyp51, which encodes the DMI target enzyme sterol P450 14α-demethylase in C. beticola. CbCyp51 is a 1,632-bp intron-free gene with obvious homology to other fungal Cyp51 genes and is present as a single copy in the C. beticola genome. Five nucleotide haplotypes were identified which encoded three amino acid sequences. Protein variant 1 composed 79% of the sequenced isolates, followed by protein variant 2 that composed 18% of the sequences and a single isolate representative of protein variant 3. Because resistance to DMIs can be related to polymorphism in promoter or coding sequences, sequence diversity was assessed by sequencing >2,440 nucleotides encompassing CbCyp51 coding and flanking regions from isolates with varying EC(50) values (effective concentration to reduce growth by 50%) to DMI fungicides. However, no mutations or haplotypes were associated with DMI resistance or sensitivity. No evidence for alternative splicing or differential methylation of CbCyp51 was found that might explain reduced sensitivity to DMIs. However, CbCyp51 was overexpressed in isolates with high EC(50) values compared with isolates with low EC(50) values. After exposure to tetraconazole, isolates with high EC(50) values responded with further induction of CbCyp51, with a positive correlation of CbCyp51 expression and tetraconazole concentration up to 2.5 μg ml(-1).     

甘肃省枸杞炭疽病菌对4种甾醇脱甲基抑制剂的敏感性

为明确甘肃省枸杞炭疽病菌对甾醇脱甲基抑制剂类药剂 (DMIs) 的敏感性,采用菌丝生长速率法测定了采自甘肃省靖远县3个地区及景泰县3个地区共102株枸杞炭疽病菌对苯醚甲环唑、戊唑醇、丙环唑及氟硅唑的敏感性,分别就不同年份、不同地区间胶孢炭疽复合种和尖孢炭疽复合种对4 种 DMIs 杀菌剂的敏感性差异进行了分析。结果表明:供试46 株枸杞胶孢炭疽复合种整体上对苯醚甲环唑、戊唑醇、丙环唑和氟硅唑仍表现为敏感,EC₅₀值分别在0.28~1.20、0.11~2.98、0.32~2.84和0.35~3.85 μg/mL之间;而56株尖孢炭疽复合种对4种药剂的敏感性则出现了不同程度分化,部分菌株疑似已出现敏感性下降现象,其中,对苯醚甲环唑、戊唑醇、丙环唑和氟硅唑敏感性最低的菌株EC₅₀值分别为1.63、3.80、6.21和4.74 μg/mL。不同年份间采集的枸杞胶孢炭疽复合种和尖孢炭疽复合种对4种杀菌剂的敏感性均存在显著差异,2017年采集的菌株敏感性相对更低,4种杀菌剂对胶孢炭疽复合种的平均EC₅₀值分别为 (0.84 ± 0.03)、(1.23 ± 0.13)、(1.19 ± 0.09) 和 (1.69 ± 0.17) μg/mL,对尖孢炭疽复合种的平均EC₅₀值分别为 (1.06 ± 0.03)、(2.25 ± 0.15)、(2.43 ± 0.20) 和 (2.85 ± 0.19) μg/mL。不同地区枸杞炭疽病菌对4种杀菌剂的敏感性表现不同,其中靖远县五合镇的胶孢炭疽复合种对4种杀菌剂敏感性最低,平均EC₅₀值分别为 (0.79 ± 0.12)、(1.28 ± 0.87)、(1.39 ± 1.05) 和 (1.74 ± 1.04) μg/mL,景泰县草窝滩镇的胶孢炭疽复合种对苯醚甲环唑和丙环唑敏感性最高,平均EC₅₀值为 (0.28 ± 0.10) 和 (0.46 ± 0.10) μg/mL,对戊唑醇和氟硅唑敏感性最高的胶孢炭疽复合种来自景泰县寺滩乡,平均EC₅₀值为 (0.42 ± 0.16) 和 (0.65 ± 0.09) μg/mL;不同地区间采集的尖孢炭疽复合种对4种杀菌剂的敏感性则不存在显著差异。研究结果可为甘肃省枸杞炭疽病防治中杀菌剂的合理使用及延缓抗药性发展提供依据。     

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

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

Detection and Quantification of Resistance of Venturia inaequalis Populations to Sterol Demethylation Inhibitors

ABSTRACT Monoconidial isolates of Venturia inaequalis were collected in 1990 and 1991 from orchards in New York, Michigan, and Nova Scotia that had never or only sporadically been treated with fungicides acting as sterol demethylation inhibitors (DMIs). Sensitivities of isolates to two representative DMIs (fenarimol and myclobutanil) were determined by a sensitivity test based on the relative growth (RG) of mycelial colonies at one discriminatory dose. Mean isolate sensitivities were not significantly different (P > 0.2) for the majority of the populations tested, and all sensitivity data obtained from these sites were combined to provide a baseline distribution of isolate sensitivities for both fenarimol and myclobutanil. The baseline distributions were compared with isolate sensitivities determined for an experimental orchard in Nova Scotia with a documented case of DMI resistance and for a commercial orchard in Michigan with a long history of DMI use and first evidence of practical DMI resistance. For both DMIs tested and in both treated orchards, frequencies of isolates with RG values <80 had decreased or only slightly increased compared to the baseline population. In contrast, frequencies of isolates with RG values >80 had increased more than 20-fold over baseline levels. Thus, isolates with RG values >80 were rated DMI resistant. The validity of a qualitative isolate classification was tested in controlled infection studies. At doses of fenarimol and myclobutanil recommended for commercial control of apple scab, reproduction of a typical sensitive isolate on treated apple leaves was suppressed completely. Lesions caused by a resistant isolate continued to expand and produced abundant conidia. Statistical analysis of orchard sensitivities revealed that the analysis of isolate counts grouped into the categories DMI sensitive or resistant was most indicative in comparisons of orchard sensitivities aimed at detection of practical DMI resistance. A high degree of cross-resistance between fenarimol and myclobutanil indicated that sensitivity tests with one of the DMIs employed as the diagnostic tool in this study can serve as a test for other DMIs.     

Sensitivity of Drechslera teres and Septoria nodorum to sterol-biosynthesis inhibitors

The sensitivity of 282 isolates ofSeptoria nodorum was tested on prochloraz, propiconazole and guazatine, and of 129 isolates ofDrechslera teres on prochloraz, propiconazole, imazalil and triadimefon. There was a great variation in sensitivity between isolates particularly at relatively low concentrations of the fungicides. However, none of the isolates was considered resistant towards the sterol biosynthesis inhibitors tested. Some isolates ofS. nodorum grew on concentrations of guazatine that may present difficulties in controlling them under practical conditions.Samenvatting Isolaten vanSeptoria nodorum (n=282) werden getoetst op gevoeligheid voor prochloraz, propiconazool en guazatine en vanDrechslera teres (n=129) voor prochloraz, propiconazool, imazalil en triadimefon. Er waren grote verschillen in de gevoeligheid van de isolaten, in het bijzonder bij lage concentraties van de fungiciden. Geen van de isolaten kan echter als resistent tegen sterolbiosynthese-remmers worden opgevat. Het feit, dat sommige van de isolaten vanS. nodorum zich ontwikkelden op media die hoge concentraties guazatine bevatten, kan echter duiden op moeilijkheden bij de bestrijding onder praktische omstandigheden.     

葡萄胶孢炭疽菌对3种麦角甾醇脱甲基抑制剂类杀菌剂的敏感性

系统比较了从云南省主要葡萄产区采集的57株葡萄胶胞炭疽菌Colletotrichum gloeosporioides对三唑类脱甲基抑制剂烯唑醇、腈菌唑及咪唑类脱甲基抑制剂咪鲜胺的敏感性、交互敏感性及敏感性与生存适合度的相关性等指标。结果表明:供试菌株对咪鲜胺的EC50值在0.01~1.58 mg/L之间,高于其对烯唑醇(EC50值为0.05~25.45 mg/L)和腈菌唑(EC50值为0.49~192.93 mg/L)的敏感性;部分菌株对烯唑醇和腈菌唑的敏感性显著降低,而对咪鲜胺仍保持较高的敏感性;供试菌株对烯唑醇和腈菌唑的敏感性具有显著的相关性,而对咪鲜胺的敏感性与对烯唑醇和腈菌唑的敏感性之间则无显著相关性。低敏感性菌株的致病力和菌落生长速率与敏感菌株无显著差异,具有较高的生存适合度。     

福建省玉米小斑病菌对丙环唑、烯唑醇和咪鲜胺的敏感性

为明确玉米小斑病菌对丙环唑、烯唑醇和咪鲜胺的敏感性,采用菌丝生长速率法测定了自福建省15个市、县分离的185株玉米小斑病菌对3种药剂的敏感性。结果显示:供试菌株对丙环唑、烯唑醇和咪鲜胺的EC50值分别在0.0186~7.779、0.0416~22.32和0.0053~7.930μg/mL之间。敏感性频率分布图显示,玉米小斑病菌群体中已出现对丙环唑、烯唑醇和咪鲜胺敏感性降低的亚群体。以连续单峰频次分布的大多数菌株群体的EC50平均值(0.5199±0.0284)、(0.4079±0.0204)和(0.3818±0.0202)μg/mL分别作为玉米小斑病菌对丙环唑、烯唑醇和咪鲜胺的相对敏感基线,发现各有15.14%、20.00%和17.30%的菌株对丙环唑、烯唑醇和咪鲜胺产生了抗性,抗性倍数最高分别为14.96、54.71和20.77。玉米小斑病菌对3种杀菌剂之间具有显著的交互抗性,而丙环唑和烯唑醇之间的交互抗性相关系数最高,为0.8662。本研究结果可为田间玉米小斑病防控杀菌剂的合理使用提供理论依据。     

Sensitivities to DMI fungicides in populations of Podosphaera fusca in south central Spain

BACKGROUND: Cucurbit powdery mildew elicited by Podosphaera fusca (Fr.) U Braun & N Shishkoff limits crop production in Spain. Disease control is largely dependent on fungicides such as sterol demethylation inhibitors (DMIs). Fungicide resistance is an increasing problem in this pathogen. To overcome such risk, it is necessary to design rational control programmes based upon knowledge of field resistance. The aim of this study was to investigate the state of DMI sensitivity of Spanish P. fusca populations and provide tools for improved disease management. RESULTS: Using a leaf‐disc assay, sensitivity to fenarimol, myclobutanil and triadimenol of 50 isolates of P. fusca was analysed to determine discriminatory concentrations between sensitive and resistant isolates. As no clearly different groups of isolates could be identified, discriminatory concentrations were established on the basis of maximum fungicide field application rate, 100 mg L^?1 for the three fungicides tested. Subsequently, a survey of DMI 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 revealed that 23% were resistant to fenarimol and 7% to triadimenol, the provinces of Almería, Badajoz and Murcia being the locations with the highest frequencies of resistance. By contrast, no resistance to myclobutanil was found. CONCLUSION: Results show that fenarimol and, to a lesser extent, triadimenol have become less efficient for controlling cucurbit powdery mildew in Spain. These are important observations that should lead to reconsideration of the current disease management programmes. Copyright © 2010 Society of Chemical Industry     

Changes in fungicide sensitivity and relative species abundance in Oculimacula yallundae and O. acuformis populations (eyespot disease of cereals) in Western Europe

Changing fungicide sensitivities in populations of Oculimacula yallundae and O. acuformis , the species responsible for cereal eyespot in Western Europe, were determined over a 17 year period between 1984 and 2000. The data were collected by Aventis Crop Science as part of their long-term survey to monitor changes in sensitivity to prochloraz and the methyl benzimidazole carbamate (MBC) fungicides in eyespot populations. The results show evidence for reduced sensitivity to both fungicides over the period of the survey. The decline in MBC sensitivity is in agreement with reports of practical resistance (a detectable loss of disease control in the field) to this fungicide which were widely reported from the mid 1980s onward. Prochloraz sensitivity was more complex, with the emergence of a higher resistance category of isolates in the late 1980s and early 1990s which then decreased in frequency towards the end of the survey. This may be partly explained by the introduction and increased use of cyprodinil in the mid 1990s. Although all trends were similar across Europe, differences were observed between the two eyespot species. A higher frequency of O. yallundae isolates showed decreased sensitivity to MBC, whereas decreased sensitivity to prochloraz was at a higher frequency in O. acuformis populations. The relative abundance of the two eyespot species was influenced by their differential levels of fungicide sensitivity, with the ratio increasing toward the species with the highest level of resistance to the prevailing fungicide.     

Resistance to inhibitors of sterol biosynthesis in field isolates or laboratory strains of the eyespot pathogen Pseudocercosporella herpotrichoides

Strains of Pseudocercosporella herpotrichoides collected in France on winter wheat give either fast-growing mycelial colonies with regular margins or slow-growing mycelial colonies with irregular margins. Most of the fastgrowing isolates were sensitive to triadimenol (EC₅₀ below 2mg litre^?1), but some of them were resistant to this inhibitor of sterol C-14 demethylation. In contrast, all the slow-growing strains were highly resistant to triadimenol (EC₅₀ greater than 100 mg litre^?1). This resistance was also expressed in inhibition of germ-tube elongation. Positive cross-resistance was observed between most of the inhibitors of sterol C-14 demethylation, with the exception of some imidazole derivatives (clotrimazole, prochloraz). All the fast-growing strains were tolerant to fenpropimorph and fenpropidin whereas the slow-growing ones were susceptible; the reverse was true with piperalin and tridemorph. All the field isolates were inhibited to the same extent by the inhibitors of squalene-epoxidase, nafifine and terbinafine. Two types of mutant resistant to triadimenol have been induced under laboratory conditions from sensitive fast-growing strains. The most common mutants were resistant to all the inhibitors of sterol C–14 demethylation and also in some conditions to fenpropimorph, tridemorph and the inhibitors of squalene-epoxidase. The other mutants were characterised by a reduced spectrum of cross-resistance between triadimenol and the other inhibitors of sterol biosynthesis. The field isolates and laboratory mutants resistant to triadimenol and propiconazole were also resistant to each of the four enantiomers of these two fungicides.     

The effect of dose and mobility on the strength of selection for DMI fungicide resistance in inoculated field experiments

Plots of spring wheat cv. Baldus were inoculated at GS 13 with four Mycosphaerella graminicola isolates, two relatively susceptible and two relatively resistant to DMI fungicides. Changes in the ratio of relatively susceptible to resistant types following fungicide or water sprays were measured. Three fungicides were compared: flutriafol, which is very mobile within leaves, fluquinconazole, which is less so, and prochloraz, which is almost immobile. All are inhibitors of sterol demethylation. In 1996, fungicide-treated plots were sprayed once with half the recommended dose at GS 39–47. In 1997, three doses were used: one-quarter and one-eighth of the recommended dose and a dual application of two one-eighth recommended doses, a week apart. Isolates were classified using a discriminating dose assay and the ratio of relatively susceptible to relatively resistant isolates in each field plot before and after fungicide application calculated. In both years, the numbers of relatively susceptible and relatively resistant isolates were equal just before fungicide application. All fungicides caused significant selection towards resistance, but the strength of selection varied with fungicide, dose and position in the crop canopy. Fluquinconazole selected most strongly and gave the best control of disease. Interactions between fungicide and dose were not significant. Selection was equally strong all along leaves sprayed with prochloraz, but increased smoothly from base to tip of leaves sprayed with fluquinconazole or flutriafol. Averaged over fungicides, reducing the dose of a single fungicide application from one-quarter to one-eighth slightly reduced selection towards resistance on both leaf layers. The dual one-eighth dose caused twice the change of the single one-eighth dose on the flag leaf, but was similar to a single spray on leaf 2.     

Inhibition of sterol biosynthesis in cell-free extracts of Botrytis cinerea by prochloraz and prochloraz analogues

The sensitivity of cytochrome-P450-dependent sterol 14α-demethylase (P450_14DM) to prochloraz and several prochloraz analogues was studied in a cell-free assay of Botrytis cinerea Pers. ex Fr. The EC₅₀ values (concentrations which inhibited radial growth of B. cinerea by 50%) of the compounds tested ranged from 3.3 × 10 ?8 to 1.7 × 10 ?5 M. The IV₅₀ values (concentrations which inhibited cell-free C₄-demethyl sterol synthesis by 50%) in cell-free assays of B. cinerea ranged from 2.6 × 10 ?9 to 4.4 × 10 ?7 M. Ranking compounds in terms of their relative inhibitory potencies showed quite similar trends to the order of fungitoxicity, but the IC₅₀ values did not quantitatively reflect the differences in toxicity. Therefore, the differential inhibition of cell-free P450_14DM activity by these compounds cannot fully account for their differences in activity towards B. cinerea. Additional mechanisms must be involved. The compounds tested were generally more potent in the B. cinerea assay than in similar assays developed for Penicillium italicum Wehmer and, in particular, Saccharomyces cerevisiae Meyen. This correlated with the relatively higher activity of most test compounds to B. cinerea. Results suggest that the cell-free assay of B. cinerea is more useful to evaluate candidate fungicides as inhibitors of sterol 14α-demethyiase activity than similar assays from model organisms. The present study confirms that the affinity of prochloraz analogues for P450_14DM depends on the nature of the N-1 substituent of the imidazole and the azole ring. It was also found that addition of an amino group at C-2 of the imidazole moiety of prochloraz gave a compound (6) which inhibited 4, 4-demethyl sterol biosynthesis in B. cinerea at a different site from the P450_14DM. This was confirmed by the observation that laboratory-generated triadimenol-resistant isolates of B. cinerea showed reduced sensitivity to triadimenol and prochloraz, but not to compound 6.     

Fungicide resistance status and chemical control options for the brassica pathogen Pyrenopeziza brassicae

Pyrenopeziza brassicae causes leaf spot disease of Brassicaceae in Europe/Oceania (lineage 1) and North America (lineage 2). In Europe, fungicides currently used for disease management are sterol 14α-demethylase (CYP51) inhibitors (azoles), quinone outside inhibitors (QoIs), and succinate dehydrogenase inhibitors (SDHIs); methyl benzimidazole carbamates (MBCs) are no longer applied. In this study, in vitro screening revealed European populations (collected 2018–2020) had shifted towards decreased azole sensitivity, but the North American population (2014–2016) was highly sensitive. Genotyping revealed CYP51 substitutions G460S or S508T were prevalent in European populations, often with a CYP51 promoter insert. Compared to wildtype CYP51 isolates, those with G460S plus an insert (44/46/151/210/302 bp) were c.25–32-fold and c.50-fold less sensitive to tebuconazole and prochloraz, respectively; those with S508T plus an insert (44/46/151/233 bp) were c.9–15-fold and c.25–40-fold less sensitive to tebuconazole and prochloraz, respectively. Selection for G460S (quantified via pyrosequencing) under different fungicide regimes was investigated in UK field trials, but G460S levels were high (c.76%) before treatment, so further selection during the trials was unclear. Despite the high G460S frequency and low disease pressure, yield data indicated measurable benefit for both azole- and non-azole-based programmes. In vitro screening against the MBC carbendazim showed European populations were predominantly moderately resistant/resistant; the North American population was sensitive. European and North American populations were sensitive to QoI (pyraclostrobin) and SDHI (penthiopyrad) fungicides. Results support an azole plus QoI/SDHI mixing partner for robust disease control and decreased risk of resistance, with continued sensitivity monitoring to ensure optimal strategies are deployed.     

Biochemical Mechanisms Involved in Selective Fungitoxicity of Two Sterol 14α-Demethylation Inhibitors,Prochloraz and Quinconazole: Accumulation and Metabolism Studies

The selective fungitoxic actions of prochloraz (an imidazole) and a triazole fungicide, quinconazole (3-(2,4-dichlorophenyl)-2-(1 H- 1,2,4-triazol-1-yl)- 4(3H)-quinazolinone: II ), were studied with selected phytopathogenic fungi. With the exception of Ustilago maydis, all the fungi tested were more sensitive to prochloraz than to II. A number of DMI-resistant mutants of Penicillium digitatum and P. italicum showed positive cross-resistance to both DMIs, but except for P. italicum isolate H17, the levels of resistance to II were much higher than to prochloraz. The generally higher toxicity of prochloraz to the fungi investigated, as compared to II , could not be ascribed to the slightly higher accumulation of prochloraz. With regard to prochloraz, there was no general correlation between the sensitivity of the fungi tested and the amount of fungicide accumulated. A similar situation was evident for II. However, the DMI-resistant mutants of P. italicum did show a reduced accumulation of both azoles, which may account for a low level of acquired DMI-resistance in this fungus. Since accumulation levels of the test compounds in the isolates with different degrees of resistance were the same, additional mechanisms of resistance may be involved in isolates with relatively high degrees of DMI-resistance. No detectable amounts of fungicide metabolites were found in most fungi tested over a 16-hour incubation period. Therefore, fungal metabolism is not generally responsible for the differences in sensitivity between fungi to each azole tested. It also does not generally explain the differential toxicities of prochloraz and II to each individual species. The exception to this was Rhizoctonia solani which metabolized prochloraz to a non-fungitoxic compound. This correlated with its low prochloraz sensitivity.     

Stepwise development of laboratory resistance to DMI-fungicides in Penicillium italicum

Isolates ofPenicillium italicum with differential levels of resistance to imazalil were obtained via step-wise mass selection of conidia of the fenarimol-resistant isolate E300-3 on imazalilamended PDA. Three out of five selection steps were successful. The resistance level to imazalil of isolates acquired after the two last selection steps was on average 122 and 197. The differential level of resistance was also apparent in decay control on oranges by imazalil inoculated with the various isolates. The isolates showed a similar cross-resistance pattern to other fungicides which inhibit C-14 demethylation of sterols (DMIs), although the level of resistance to these fungicides was significantly higher. All isolates displayed negatively-correlated cross-resistance to tridemorph and dodine. Most isolates had a normal virulence on oranges. In competition experiments with mixed-inocula of the wild-type and a resistant isolate, the proportion of the wild-type increased in successive generations on untreated oranges and the proportion of the resistant isolate increased on imazalil-treated oranges. The lower competitive ability of the resistant isolate on untreated oranges may be due to a decrease in spore production as compared with the wild-type.Since isolate E300-3 was obtained in two selection steps on fenarimol-amended PDA, the isolates obtained in the last selection steps on imazalil-amended PDA may have at least five different genes for resistance to DMIs. This is consistent with resistance to DMIs being under polygenic control, with the genes involved having an additive interaction, although this is not the only possible explanation of the results.