Strobilurin Resistance Research Articles

First report of strobilurin resistance in field samples of Blumeria graminis f. sp. tritici, causal agent of powdery mildew in wheat, in Paraguay

First report of strobilurin resistance in field samples of Blumeria graminis f. sp. tritici, causal agent of powdery mildew in wheat, in Paraguay

Characterization of mutants with single and combined Qi and Qo site mutations in Saccharomyces cerevisiae reveals interactions between the picolinamide fungicide CAS-649 and azoxystrobin

Picolinamide and strobilurin fungicides bind to the Qi and Qo sites on cytochrome b, respectively, and target many of the same plant pathogens. Using Saccharomyces cerevisiae as a model system, we explore effects of amino acid changes at each site on sensitivity to a fungicide acting at the opposite site and examine the relationship between altered sensitivity and growth penalty. In addition, double mutants containing the G143A or F129L mutations responsible for strobilurin resistance in combination with Qi site mutations that confer resistance to picolinamides are characterized in terms of their sensitivity to QiI and QoI fungicides and growth rate. Mutants containing amino acid changes at the Qo site varied in their growth rate and sensitivity to the picolinamide CAS-649, and increased sensitivity was associated with a greater growth penalty. Conversely, changes at the Qi site affected sensitivity to azoxystrobin and also showed a correlation between increased sensitivity and reduced growth. There was no overall correlation between resistance to azoxystrobin and CAS-649 among mutants, however negative cross-resistance occurred in the case of mutations which conferred resistance to either compound and also carried a growth penalty. These results suggest the use of QoI fungicides to delay the emergence of pathogen resistance to QiIs, and vice versa. Double mutants containing G143A or F129L in combination with Qi site changes N31K, G37C/V or L198F that cause resistance to picolinamides generally exhibited lower resistance factors for both azoxystrobin and CAS-649 than corresponding resistant strains with a single mutation. Reduced growth was observed for all F129L-containing double mutants, whereas the growth rate of double mutants containing G143A was significantly reduced only by the Qi site mutations N31K and G37V that confer a larger growth penalty. Our results suggest that resistance to picolinamides in pathogens could emerge more readily in a strobilurin-sensitive genetic background than in a strobilurin-resistant one.

Thevetia peruviana (Pers.) K. Schum. Potential Antifungal Agent Against Mycosphaerella fijiensis Morelet, Fungi Responsible of Black Leaf Streak Disease (BLSD) of Plantain (Musa spp)

Alternatives to synthetic chemicals are undertaken against phytopathogens. The aim of this work is to evaluate the effect of seed extracts of Thevetia peruviana (Pers.) K. Schum. on Mycosphaerella fijiensis Morelet, fungus responsible for banana black leaf streak disease. Five extracts of T. peruviana, hexane extract (HE), ethyl acetate extract (EAE), acetone extract (AcE), methanol extract (ME) and aqueous extract (AqE), and a fungicide, Azoxystrobin were used. GC-MS of acetone extract was performed. Fifty (50) strains of M. fijiensis per sampling site were tested. Three concentrations of extracts 6.25 (C1), 12.5 (C2), and 25 (C3) μl/ml, a negative control (0 μl/ml) and 10 ppm of azoxystrobin were used for the tests. The MIC50 and MIC90 were determined. GC-MS showed chemical compounds with different molecular height such as acids, sugars, and esters. AcE and AqE significantly reduced M. fijiensis germ tube growth at C2 and C3 concentrations and with inhibition percentage respectively ranged of 60-90% and 40-80%. The growth levels of the germ tubes were above the strobilurin resistance threshold at Njombe and peasant plantation, ranging from 77.9% to 92.3%. AcE showed the same or superior efficacy as the fungicide used on conidial germination at all tested concentrations. The MIC50 totally reducing mycelial growth and conidial germination was 6.25 μl/ml. T. peruviana seeds extracts can be exploited in integrated pests management against M. fijiensis.

Fungicide Sensitivity Shifting of Zymoseptoria tritici in the Finnish-Baltic Region and a Novel Insertion in the MFS1 Promoter.

Septoria tritici blotch (STB) is caused by the ascomycete Zymoseptoria tritici and one of the predominating diseases in wheat (Triticum aestivum) in Europe. The control of STB is highly reliant on frequent fungicide applications. The primary objective of this study was to assess sensitivity levels of Z. tritici to different fungicide groups. The fungicides included in this study were epoxiconazole, prothioconazole-desthio, tebuconazole, and fluxapyroxad. A panel of 63 isolates from Estonia, Latvia, and Lithuania, and 10 isolates from Finland were tested. Fungicide sensitivity testing was carried out as a bioassay analyzing single pycnidium isolates on different fungicide concentrations. The average EC50 value in Baltic countries and Finland to epoxiconazole was high ranging from 1.04 to 2.19 ppm. For prothioconazole-desthio and tebuconazole, EC50 varied from 0.01 to 0.24 ppm, and 1.25 to 18.23 ppm, respectively. The average EC50 value for fluxapyroxad varied from 0.07 to 0.33 ppm. To explain the range of sensitivity, the samples were analyzed for CYP51 and Sdh mutations, as well as cytb G143A, CYP51 overexpression, and multidrug resistance (MDR). Frequencies of ZtCYP51 mutations D134G, V136A/C, A379G, I381V, and S524T in the Finnish-Baltic region were lower than in other European countries, but have increased compared to previous years. The frequency of cytb G143A conferring strobilurin resistance also augmented to 50–70% in the Z. tritici populations from Estonia, Finland, Latvia, and Lithuania. No Sdh mutations were found in this study, and neither strains of MDR phenotypes. However, we found a strain harboring a previously unknown transposon insertion in the promoter of the MFS1 gene, involved in drug efflux and multi-drug resistance. This new insert, however, does not confer an MDR phenotype to the strain.

Improving fungicide-based management of Cercospora leaf spot in table beet in New York, USA

Cercospora leaf spot (CLS), caused by Cercospora beticola Sacc., is the dominant foliar disease affecting the profitability of table beet production in New York, USA. The disease is controlled primarily with fungicides, but sustainability is critically threatened by strobilurin resistance within the C. beticola population and a limited suite of alternatives. This study evaluated the temporal dynamics of strobilurin resistance within the C. beticola population over three years, and quantified sensitivity to selected demethylation inhibitors (DMIs; propiconazole and difenoconazole) and succinate dehydrogenase inhibitors (SDHIs; fluopyram, penthiopyrad, boscalid and benzovindiflupyr). Resistance to azoxystrobin (EC50 > 0.2 µg mL−1) was prevalent and detected in all fields sampled in 2015 and 2016, and six of 10 fields in 2017. Within fields, the incidence of azoxystrobin-resistant isolates ranged between 4.8% and 70%. No significant differences were detected in the frequency of azoxystrobin-resistant isolates within populations from fields with conventional and organic practices in 2016 and 2017. Reduced sensitivity to propiconazole and difenoconazole was detected in less than 1% of isolates. The EC50 values for the SDHIs were higher than the maximum concentrations (≥ 20 µg mL−1) evaluated. Two small-plot replicated field trials were also conducted over two years to evaluate alternatives to the commercial standards, propiconazole and azoxystrobin. Two applications of benzovindiflupyr + difenoconazole and pydiflumetofen + difenoconazole significantly reduced CLS severity and epidemic progress compared with plots that received propiconazole and non-treated controls.

Long term field sampling in Sweden reveals a shift in occurrence of cytochrome b genotype and amino acid substitution F129L in Alternaria solani, together with a high incidence of the G143A substitution in Alternaria alternata

The incidence of the pathogenic fungus Alternaria solani on potato during the years 2009 to 2016 was analysed and the distribution of the two genotypes of cytochrome b (genotype 1, GI and genotype 2, GII) determined. Potato leaflets with lesions resembling early blight were collected in August and September 2009–2016 in the south-eastern part of Sweden and during 2010 and 2012–2014 in the central part of Sweden. In total, 2042 potato leaflet samples were analysed using diagnostic PCR methods. Alternaria solani was more commonly found in the samples collected later in the growing seasons. The occurrence of substitutions in the gene encoding cytochrome b, associated with loss of sensitivity towards strobilurins, was determined by sequencing a segment in the target gene in all confirmed A. solani samples. The proportion of A. solani GI decreased in relation to GII over the years. All except five GI samples were non-mutated. Genotype 2 was already present in the first year of sampling but only one sample possessed the F129 L substitution. A distinct shift from non-mutated GII to GII F129 L was observed between the season of 2011–2012 and a similar shift occurred in the pathogen population from GI to GII between 2012 and 2013. Identification of Alternaria alternata was performed during 2009–2013. The species was most often in co-occurrence with A. solani. Most of the samples investigated possessed the G143A substitution associated with strobilurin resistance.

The G143A Mutation Confers Azoxystrobin Resistance to Soybean Cercospora Leaf Blight in Bolivia

During the summer of 2017, 38 Cercospora spp. isolates were collected from soybean leaves displaying symptoms of Cercospora leaf blight (CLB) from commercial soybean fields at three locations in Santa Cruz, Bolivia. Portions of cytochrome b (N = 38) and calmodulin (N = 37) were amplified, sequenced, edited, and assembled. Two representative isolates from each lineage (with distinct calmodulin haplotypes, if present) were selected for sequencing of two additional loci, histone 3 and elongation factor 1-alpha, for species identification. Based on the corresponding multilocus phylogenetic analysis, the 37 isolates belong to three species: C. nicotianae (N = 14), C. sp. “P” (N = 16), and C. kikuchii (N = 7). All 38 isolates (100%) possessed the G143A mutation in cytochrome b, and none carried the F129L mutation. Results of phenotypic assays on a subset of 15 isolates (five from each species) supported a high level of resistance to azoxystrobin. This is the first report of the G143A mutation in Cercospora species associated with CLB in South America and the first report of C. nicotianae associated with CLB. Future monitoring for G143A mutants of Cercospora spp. in South America will be necessary to assess whether strobilurin resistance is widespread.

Characterization of the mechanism of action of the fungicide fenpicoxamid and its metabolite UK-2A.

BACKGROUNDFenpicoxamid is a new fungicide for control of Zymoseptoria tritici, and is a derivative of the natural product UK‐2A. Its mode of action and target site interactions have been investigated.RESULTSUK‐2A strongly inhibited cytochrome c reductase, whereas fenpicoxamid was much less active, consistent with UK‐2A being the fungicidally active species generated from fenpicoxamid by metabolism. Both compounds caused rapid loss of mitochondrial membrane potential in Z. tritici spores. In Saccharomyces cerevisiae, amino acid substitutions N31K, G37C and L198F at the Qi quinone binding site of cytochrome b reduced sensitivity to fenpicoxamid, UK‐2A and antimycin A. Activity of fenpicoxamid was not reduced by the G143A exchange responsible for strobilurin resistance. A docking pose for UK‐2A at the Qi site overlaid that of antimycin A. Activity towards Botrytis cinerea was potentiated by salicylhydroxamic acid, showing an ability of alternative respiration to mitigate activity. Fungitoxicity assays against Z. tritici field isolates showed no cross‐resistance to strobilurin, azole or benzimidazole fungicides.CONCLUSIONFenpicoxamid is a Qi inhibitor fungicide that provides a new mode of action for Z. tritici control. Mutational and modeling studies suggest that the active species UK‐2A binds at the Qi site in a similar, but not identical, fashion to antimycin A. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Equal Distribution of Mating Type Alleles and the Presence of Strobilurin Resistance in Algerian Zymoseptoria tritici Field Populations.

Zymoseptoria tritici, the causal agent of septoria tritici blotch, is an important wheat pathogen responsible for high yield losses worldwide. The assessment of the distribution of both mating type idiomorphs was studied in several wheat-growing areas in North Algeria. Both mating types occurred at all spatial scales tested and showed an equal frequency distribution at the country level, with 53% for Mat1-1 and 47% for Mat1-2. At finer scales, co-occurrence of both mating types was found in 38% of leaves analyzed and 44% of the studied lesions. Additionally, efficacy reduction of strobilurin applications prompted us to study strobilurin resistance conferred by the G143A substitution in the mitochondrial cytochrome b gene, which was found for the first time in six strains. The results suggest that the Algerian Z. tritici population undergoes frequent sexual reproduction mirrored by co-occurrence of both mating types, which likely will rapidly increase the fraction of strobilurin-resistant isolates in the Algerian population, as has been shown in all other regions where strobilurin resistance surfaced.

The use of RFLP analysis in the monitoring of Cercospora beticola strobilurin resistance occurring in sugar beet (Beta vulgaris L.)

The use of RFLP analysis in the monitoring of Cercospora beticola strobilurin resistance occurring in sugar beet (Beta vulgaris L.)

The application of next-generation sequencing (NGS) for monitoring of Zymoseptoria tritici QoI resistance

Septoria leaf blotch caused by Zymoseptoria tritici is a common and serious disease of winter wheat. In many wheat growing areas QoI (strobilurins) are widely used to control the disease. Due to the singlesite mode of action many plant pathogenic fungi develop QoI resistance. The most common mutation related with strobilurin resistance is glycine to alanine substitution at 143 codon position (G143A) in mitochondrial cytochrome b gene. In the study we describe a novel diagnostic protocol based on next-generation sequencing (NGS) to detect the G143A mutation in Z. tritici. In 2014, 2015 randomly chosen isolates (40) were collected from winter wheat in 26 independent locations in western Poland. Genomic DNA was extracted from fresh mycelium in 15 min procedure. The NGS library was prepared from PCR amplicons of 251 bp fragment of cytochrome b gene containing G143A mutation site. The sequencing in the paired-end (PE) mode was performed on Illumina MiSeq. Having done primer and quality trimming, paired reads were joined, resulting in a total of 722,692 paired sequences of ∼211 bp fragment. The obtained sequences were mapped to a complete genome reference sequence (EU090238.1) of the Z. tritici mitochondrion. The QoI sensitive cytochrome b variant (G143) was found in 45%, while the resistant variant (A143) was noted in 55% of the sequences analysed. The NGS assays proved to be very successful in detection and characterisation of drug resistance mutation. In conclusion, we postulate that NGS technology enables quick, accurate and large-scale monitoring, particularly in mixed populations of sensitive and resistant pathogens. To our knowledge, this is the first report describing the use of NGS technology for genotypic QoI resistance in crop pathogenic fungi.

Erratum to: Molecular analysis of Cercospora beticola isolates for strobilurin resistance from the central High Plains, USA

Erratum to: Molecular analysis of Cercospora beticola isolates for strobilurin resistance from the central High Plains, USA

Molecular analysis of Cercospora beticola isolates for strobilurin resistance from the Central High Plains, USA

Cercospora leaf spot (CLS), caused by Cercospora beticola, is the most destructive foliar disease and is a problem in sugar beet production areas, such as Central High Plains (states of Colorado, Montana, Nebraska and Wyoming) in the United States. The disease can be controlled by strobilurin fungicides, referred to as quinone outside inhibitors (QoIs), with a single target site on C. beticola. Strobilurin resistance has been reported in beet production areas from the United States, including the Central High Plains. Although strobilurin resistance is quantitatively inherited, it is considered that it has low to medium heritability in the population. Effective diagnostic tools are required for the rapid detection of C. beticola strobilurin resistance. The study obtained a partial nucleotide sequence of the C. beticola cytochrome b gene and determined to a putative protein with ~386 amino acid residues. Eighty C. beticola isolates (2004–2011) from the Central High Plains were analyzed for mutations. We found a single nucleotide polymorphic (SNP) site which led to G143A mutation and was present in 2 C. beticola QoI-resistant isolates. Partial sequences obtained from 82 C. beticola QoI-sensitive isolates showed identical cytochrome b gene. We developed a PCR-RFLP assay that involved an in vitro digestion using Fnu4HI restriction enzyme for the rapid molecular detection of G143A mutation in the C. beticola population. Results indicated the PCR-RFLP assay was reliable, sensitive, and can be used for the rapid detection of C. beticola strobilurin resistance.

Evidence of Selection for Fungicide Resistance in Zymoseptoria tritici Populations on Wheat in Western Oregon.

Plant pathogens pose a major challenge to maintaining food security in many parts of the world. Where major plant pathogens are fungal, fungicide resistance can often thwart regional control efforts. Zymoseptoria tritici, causal agent of Septoria tritici blotch, is a major fungal pathogen of wheat that has evolved resistance to chemical control products in four fungicide classes in Europe. Compared with Europe, however, fungicide use has been less and studies of fungicide resistance have been infrequent in North American Z. tritici populations. Here, we confirm first reports of Z. tritici fungicide resistance evolution in western Oregon through analysis of the effects of spray applications of propiconazole and an azoxystrobin + propiconazole mixture during a single growing season. Frequencies of strobilurin-resistant isolates, quantified as proportions of G143A mutants, were significantly higher in azoxystrobin-sprayed plots compared with plots with no azoxystrobin treatment at two different locations and were significantly higher in plots of a moderately resistant cultivar than in plots of a susceptible cultivar. Thus, it appears that western Oregon Z. tritici populations have the potential to evolve levels of strobilurin resistance similar to those observed in Europe. Although the concentration of propiconazole required to reduce pathogen growth by 50% values were numerically greater for isolates collected from plots receiving propiconazole than in control plots, this effect was not significant (P > 0.05).

Przyczyny odporności izolatów Cercospora beticola (chwościk buraka) na strobiluryny w Wielkopolsce

Leaf spot caused by Cercospora beticola is one of the most serious diseases of sugar beet. Various fungicide groups are used to control this disease and among them also strobilurin based products. The aim of the study was to analyze the causes of strobilurin resistance of C. beticola isolates collected in Wielkopolska region. The strobilurin inhibits mitochondrial respiratory processes and mutations occurring in the cytochrome b gene (cytb) are the most important reason of C. beticola strobilurin resistance. An increasing activity of an alternative oxidase (AOX) also plays an important role in resistance occurring in C. beticola isolates. The sequence of cytochrome b gene of C. beticola isolates were analyzed. Mutations G143 and F129L – the main determinants of strobilurin resistance were no found. However, an increased strobilurin resistance level of the isolates is caused by higher activity of alternative oxidase, which replaces the function of cytochrome b in the respiratory pathway. Chwościk wywoływany przez Cercospora beticola należy do najpoważniejszych chorób buraka cukrowego. Do ochrony upraw buraka wykorzystywane są fungicydy z różnych grup chemicznych, także strobiluryny, które hamują procesy oddechowe zachodzące w mitochondriach. Mutacje pojawiające się w genie cytochromu b (cytb) są najważniejszą przyczyną nabywania odporności na strobiluryny przez izolaty C. beticola. Wśród pozostałych przyczyn odporności wymienia się wzrost aktywności alternatywnej oksydazy. Celem badań była analiza przyczyn podwyższonej odporności na piraklostrobinę izolatów C. beticola zbieranych na terenie Wielkopolski. W badanym fragmencie genu cytochromu b izolatów C. beticola nie stwierdzono mutacji G143A oraz F129L, które wymieniane są wśród podstawowych czynników warunkujących odporność na strobiluryny. Zwiększona odporność badanych izolatów spowodowana jest wzmożoną aktywnością alternatywnej oksydazy, zastępującej funkcję cytochromu b w szlaku oddechowym.

Baseline sensitivity of Brazilian Mycosphaerella fijiensis isolates to protectant and systemic fungicides

Black Sigatoka caused by Mycosphaerella fijiensis is a foliar disease that affects banana plants and large amounts of fungicides are required to prevent crop losses. Intensive applications of single-site fungicides can select for fungicide-resistant isolates. The objective of this study was to assess the sensitivity of 60 isolates of M. fijiensis to commonly used fungicides. Using two different protocols, microtiter and Petri plate tests, the effective concentration at which mycelium growth is reduced by 50% (EC50) was determined for thiophanatemethyl, tebuconazole, chlorothalonil and mancozeb. Additionally, partial sequences of the cytochrome b gene were obtained for 46 isolates to detect the G143A mutation, commonly associated with strobilurin resistance. The EC50 values for tebuconazole and thiophanate-methyl ranged from 0.02 to 1.39 and from 0.008 to 8.22 µg mL-1, respectively. For chlorothalonil, the lowest and highest EC50 values were 0.39 µg mL-1 and 53.7 µg mL-1, respectively. For mancozeb, approximately 50% of the isolates had EC50 values greater than 1000 µg mL-1. No mutation was found in the isolates assayed for strobilurin resistance. There was no correlation between sensitivity levels to any fungicide and geographic region. Low EC50 values were estimated for most fungicides but, some isolates had high EC50 values for mancozeb.

First Report of Resistance to QoI Fungicides in North American Populations of Zymoseptoria tritici, Causal Agent of Septoria Tritici Blotch of Wheat.

The G143A mutation in cytb (cytochrome b gene) is associated with high levels of resistance to quinone outside inhibitor (QoI or strobilurin) fungicides that disrupt electron transport during cellular respiration (1). The G143A mutation in Zymoseptoria tritici (synonyms: Mycosphaerella graminicola and Septoria tritici), the causal agent of septoria tritici blotch of wheat (Triticum aestivum), was first reported in Europe in 2001 (1). Although Z. tritici has a global distribution (3), G143A mutants of Z. tritici have not been reported outside of Europe. We used PCR-RFLP (4) to estimate the frequencies of G143A mutants in Z. tritici populations at two locations in the Willamette Valley of western Oregon: the Hyslop Crop Science Field Research Laboratory (Hyslop Farm, HF), Benton County (44°37'52.85″ N, 123°11'55.19″ W) and research plots planted in a commercial wheat field in Washington County (45°33'58.53″ N, 123°00'11.78″ W) (North Valley Farm, NVF). Isolates originated from flag leaf collections from two cultivars ('Bobtail' and 'Tubbs 06') made in April and June of 2012 from plants in a replicated fungicide-treatment experiment, with isolates collected from both sprayed and unsprayed plots. Sixteen of the 169 isolates (9.5%) from HF possessed the G143A mutation (7 of 132 isolates from plots not receiving a QoI fungicide and 9 of 37 isolates collected from plots receiving two applications of the QoI azoxystrobin). One hundred forty six of the 175 isolates (83.4%) from NVF were G143A mutants (101 of 129 isolates from plots receiving no QoI fungicide and 45 of 46 isolates from plots receiving two applications of azoxystrobin). Results of phenotypic assays of a subset of 10 isolates from each location (5 mutants, 5 wild types from each location; 20 isolates altogether) supported a high level of resistance to azoxystrobin only in the G143A mutants. All 10 G143A mutants developed colonies after 8 days of growth on YMA plates amended with SHAM (2) and 1 ppm or 10 ppm azoxystrobin, with nine and eight G143A mutant isolates developing colonies on plates amended with 1 ppm and 10 ppm azoxystrobin, respectively. None of the wild-type isolates developed colonies on plates amended with SHAM and 1 ppm azoxystrobin, nor on plates amended with SHAM and 10 ppm azoxystrobin. All 20 isolates developed colonies on YMA plates lacking azoxystrobin, and treatments produced identical results across three replicates. These results are consistent with findings of higher levels of azoxystrobin resistance in G143A mutants compared to wild types in European populations (1). Isolates from HF and NVF differ in their previous exposure to QoI fungicides. The majority of the wheat area at HF is planted to breeding plots that are not sprayed with fungicide. Plots at NVF were planted in a commercial wheat field in a county where most wheat fields were treated with two to three applications of strobilurins each year over the past 4 years. Future monitoring for G143A mutants of Z. tritici throughout its range in North America will be necessary to assess whether strobilurin resistance will spread via wind-dispersal of ascospores or emerge de novo in treated fields. In Europe, stobilurins were first applied to wheat in 1996. G143A mutants of Z. tritici emerged de novo several times (4) and were widespread by 2007.

Microdochium nivale and Microdochium majus in seed samples of Danish small grain cereals

Microdochium nivale and Microdochium majus are two of fungal species found in the Fusarium Head Blight (FHB) complex infecting small grain cereals. Quantitative real-time PCR assays were designed to separate the two Microdochium species based on the translation elongation factor 1α gene (TEF-1α) and used to analyse a total of 374 seed samples of wheat, barley, triticale, rye and oat sampled from farmers' fields across Denmark from 2003 to 2007. Both fungal species were detected in the five cereal species but M. majus showed a higher prevalence compared to M. nivale in most years in all cereal species except rye, in which M. nivale represented a larger proportion of the biomass and was more prevalent than M. majus in some samples. Historical samples of wheat and barley from 1957 to 2000 similarly showed a strong prevalence of M. majus over M. nivale indicating that M. majus has been the main prevalent Microdochium species in Danish cereals for at least 50 years. PCA analysis of the two quantified Microdochium species in wheat, barley and triticale samples generally showed co-existence of M. majus and M. nivale in all three cereal species. Strobilurin resistance in M. nivale/majus was analysed in selected wheat samples from 2003 to 2007, selected barley samples from 2007 as well as in historical samples from 1957 to 2000 using CAPS analysis to detect the G143A substitution. The results confirm strobilurin resistance from 2003 in the Microdochium populations of wheat and also confirmed resistance in barley for the first time. The presence of strobilurin resistance should be considered in future fungicide control strategies.

Frequency of mutations associated with fungicide resistance and population structure of Mycosphaerella graminicola in Tunisia

The occurrence of fungicide resistance in Mycosphaerella graminicola populations from Tunisia was investigated by examining mutations known to be associated with strobilurin and azole resistance. Few mutations associated with fungicide resistance were detected. No evidence for strobilurin resistance was found among 357 Tunisian isolates and only two among 80 sequenced isolates carried mutations associated with azole resistance. A network analysis suggested that these mutations emerged independently from mutations found in previously described European populations. The population genetic structure of M. graminicola in Tunisia was analyzed using variation at 11 microsatellite loci. Populations in Tunisia were characterized by high gene and genotype diversity. All populations were in gametic equilibrium and mating type proportions did not deviate from the 1:1 ratio expected under random mating, consistent with regular cycles of sexual reproduction. In combination with a high degree of gene flow among sampling sites, M. graminicola must be considered a pathogens with high evolutionary potential. Thus, control strategies against Septoria blotch in Tunisia should be optimized to reduce the emergence and spread of resistant isolates.

Real‐time PCR Assay with SNP‐specific Primers for the Detection of a G143A Mutation Level in Venturia inaequalis Field Populations

AbstractFourteen primers were designed specific to SNP (G→C mutation) in the cytochrome b gene of Venturia inaequalis, corresponding to G143A substitution related to strobilurin resistance. Specificity of the primers and amplification efficiency were preliminarily tested in conventional PCR at different annealing temperatures. The performance of several preselected primer sets was verified in real‐time PCR with SYBR Green I dye. Finally, two primer sets (‘2Wt’ and ‘5Mt’) were successfully applied for discrimination between wild‐type and mutated allele. Different sensitivity of the detection of homologous and non‐homologous DNA corresponded to the difference in Ct values equalled 10.5 for ‘2Wt’ and 12.0 for ‘5Mt’ primer set. Primers specific to Mycosphaerella fijensis cytochrome b sequence (Wille et al. 2002) were applied for additional control of PCR inhibitors. The reliability of the new method was evaluated and verified using two series of reference DNA dilutions and artificial mixtures of both types of DNA. Developed real‐time PCR assay was applied to measure the ratio of mutated to non‐mutated allele in field samples, collected in Poland in 2009, using two series of reference DNA in every run. The measured mutation level for the samples derived from orchards with conventional chemical control was very high (50–100%). For two populations originating from one organic orchard, the measured level of mutation was 1% and 46%. The combination of molecular and traditional tests for the evaluation of mutation level and monitoring of resistance levels in orchards is recommended.