Microscopic analysis of the effect of azoxystrobin treatments on Mycosphaerella graminicola infection using green fluorescent protein (GFP)‐expressing transformants
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Abstract Green fluorescent protein (GFP)‐expressing transformants were used to investigate the effects of strobilurin fungicide azoxystrobin on Mycosphaerella graminicola infection. Azoxystrobin treatments (125 or 250 g AI ha −1 ) were applied at various stages of the infection process under controlled conditions. GFP transformants showed conserved in vitro sensitivity to azoxystrobin and pathogenicity. Azoxystrobin controlled over 90% of M graminicola infections when applied before or during penetration of the pathogen (15% of the incubation phase). Azoxystrobin also impaired the growth of intercellular hyphae in M graminicola post‐penetration infection stages when applied at up to 50% of the incubation phase. Incubating infections observed in treated leaves were viable, but their growth was impaired and they did not induce necrosis under controlled conditions. Reduction by half of azoxystrobin dosage had little or no effect on azoxystrobin efficiency in controlling M graminicola . The contribution of post‐penetration fungistatic effect to azoxystrobin curative properties toward M graminicola in a field situation is discussed. © 2001 Society of Chemical IndustryKeywords:
Mycosphaerella graminicola
Strobilurin
Graminicola
Incubation period
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The effect of the quinone outside inhibitors (QoI) azoxystrobin and pyraclostrobin on yields of winter wheat where QoI resistant Mycosphaerella graminicola isolates were dominant was investigated in field trials in 2006 and 2007. Pyraclostrobin significantly increased yields by 1·57 t ha −1 in 2006 and 0·89 t ha −1 in 2007 when compared to the untreated controls, while azoxystrobin only provided a significant increase of 1·28 t ha −1 in 2006. These yield increases were associated with reduction in septoria tritici blotch (STB) development as determined by weekly disease assessments over a 7 week interval. The effect of pyraclostrobin on STB was studied in controlled environment experiments using wheat seedlings inoculated with individual M. graminicola isolates. Pyraclostrobin significantly reduced STB symptoms by up to 62%, whether applied 48 h pre‐ or post‐ inoculation with resistant M. graminicola isolates containing the cytochrome b mutation G143A. Extremely limited disease (<1%) was observed on similarly treated seedlings inoculated with an intermediately resistant isolate containing the cytochrome b mutation F129L, while no disease was observed on seedlings inoculated with a wild‐type isolate. Germination studies of pycnidiospores of M. graminicola on water agar amended with azoxystrobin or pyraclostrobin showed that neither fungicide inhibited germination of spores of resistant isolates containing the mutation G143A. However, pyraclostrobin significantly reduced germ tube length by up to 46% when compared with the untreated controls. Although the QoIs can no longer be relied upon to provide effective M. graminicola control, this study provides an insight into why QoIs still provide limited STB disease control and yield increases even in situations of high QoI resistance.
Mycosphaerella graminicola
Graminicola
Spore germination
Germ tube
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Azoxystrobin resistance levels of twenty two strains sampled from ten French locations and two reference isolates (IPO323 and IPO94269) of the wheat pathogen Mycosphaerella graminicola were investigated in vitro. French strains assayed were selected from twenty two genetic groups determined from three hundred sixty three strains previously characterised using microsatellites, actine and beta-tubuline markers. For the first time, the evaluation was carried out using four distinct methods: spotting on PDA medium, spore germination on PDA medium and using microtitre plates with and without Alamar blue, a growth indicator. From dose-response curve, half maximal inhibitory concentration (IC50) was determined for each strain. The results obtained using microtitre plates with the addition of Alamar blue displayed high standard deviations from the growth averages observed. Therefore, we suggest that this method is inadequate to assess M. graminicolo resistance to fungicides. However, a good correlation was observed between the rankings of strains according to their IC50 values with the three other methods used. The two reference isolates, as expected, were inhibited by low azoxystrobin concentrations. On the other hand, the IC50 values obtained showed presence of a threshold between sensitive and resistant strains that corroborates the disruptive resistance of M. graminicola against strobilurin fungicides. In addition, the strains showing resistance were those sampled mainly from northern France, where a high frequency of strobilurin resistant isolates among M. graminicola populations was reported by several studies.
Mycosphaerella graminicola
Strobilurin
Graminicola
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Summary Septoria leaf blotch caused by Mycosphaerella graminicola is one of the most important diseases of cereals. The pathogen belongs to species rapidly developing resistance to used fungicides. The increase of resistance of M. graminicola isolates to strobilurin in western Europe was already recorded in 2002, while in Poland about 10 years later. The aim of the study was to develop a PCR-RFLP (polymerase chain reaction – restriction fragment length polymorphism) assay for the identification of mutation (G143A) in cytochrome b gene of M. graminicola, which confers resistance to strobilurin. The analysis enabled to distinguish strains of M. graminicola resistant and sensitive to strobilurin, and it can be used for rapid monitoring of M. graminicola resistance to this group of fungicides.
Mycosphaerella graminicola
Graminicola
Strobilurin
Mycosphaerella
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Abstract Green fluorescent protein (GFP)‐expressing transformants were used to investigate the effects of strobilurin fungicide azoxystrobin on Mycosphaerella graminicola infection. Azoxystrobin treatments (125 or 250 g AI ha −1 ) were applied at various stages of the infection process under controlled conditions. GFP transformants showed conserved in vitro sensitivity to azoxystrobin and pathogenicity. Azoxystrobin controlled over 90% of M graminicola infections when applied before or during penetration of the pathogen (15% of the incubation phase). Azoxystrobin also impaired the growth of intercellular hyphae in M graminicola post‐penetration infection stages when applied at up to 50% of the incubation phase. Incubating infections observed in treated leaves were viable, but their growth was impaired and they did not induce necrosis under controlled conditions. Reduction by half of azoxystrobin dosage had little or no effect on azoxystrobin efficiency in controlling M graminicola . The contribution of post‐penetration fungistatic effect to azoxystrobin curative properties toward M graminicola in a field situation is discussed. © 2001 Society of Chemical Industry
Mycosphaerella graminicola
Strobilurin
Graminicola
Incubation period
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Strobilurin
Mycosphaerella graminicola
Chlorothalonil
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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).
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Mycosphaerella graminicola
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Strobilurin
Mycosphaerella graminicola
Cross-resistance
Graminicola
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The effects of varying doses of fungicides, alone or in mixtures, on selection for triazole resistance were examined under field conditions. Two experiments were conducted using the triazole fungicide fluquinconazole with the strobilurin fungicide azoxystrobin as a mixture partner. Inoculated wheat plots with a known ratio of more sensitive to less sensitive isolates of the leaf blotch fungus Mycosphaerella graminicola were sprayed with fungicide and sampled once symptoms had appeared. Selection for fluquinconazole resistance increased in proportion to the dose, up to one‐half of the full dose (the maximum tested) in both experiments. At the higher doses of fluquinconazole, the addition of azoxystrobin was associated with a decrease in selection (nonsignificant in the first experiment) for triazole resistance. Control by low doses of fluquinconazole was increased by mixture with azoxystrobin, but at higher doses mixture with azoxystrobin sometimes decreased control, so that reduced selection was obtained at the cost of some reduction in control. The effects on resistance are not necessarily general consequences of mixing fungicides, and suggest that the properties of any specific mixture may need to be demonstrated experimentally. Selection was inversely related to control in the unmixed treatments in both experiments, but the relationship was weaker in the mixtures with azoxystrobin.
Strobilurin
Mycosphaerella graminicola
Graminicola
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Septoria tritici blotch caused by the fungal pathogen Mycosphaerella graminicola (anamorph: Zymoseptoria tritici) is one of the most frequently occurring diseases on both bread and durum wheat crops worldwide. One hundred and sixty four durum wheat-adapted isolates of this fungus were sampled during the 2012 growing season from five distinct geographical locations of Tunisia (Bizerte, Béja, Kef, Jendouba and Siliana) in order to examine the status of strobilurin resistance of M. graminicola in this country. Resistance was assessed by screening the G143A substitution (Cytochrome b) which confers resistance to this class of fungicides. We used a PCR-based mismatch mutation assay allowing the amplification of either G143 (sensitive) or A143 (resistant) allele. All isolates were found to contain the sensitive wild-type G143 allele and therefore to be sensitive. Our study confirms recent reports on M. graminicola in Tunisia and shows that the Tunisian population of the fungus remains fully sensitive to strobilurins. A durability-oriented management of strobilurin applications in Tunisia is thereby recommended to prevent the development and widespread of the corresponding resistance such as in Europe, where pathogen populations are nowadays fully resistant to strobilurins.
Mycosphaerella graminicola
Strobilurin
Graminicola
Mycosphaerella
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Mycosphoerella graminicola (anamorph: Zymoseptoria tritici), causal agent of Septoria tritici blotch, is currently one of the most damaging diseases on both bread and durum wheat crops worldwide. Since wheat resistance against this pathogen is always partial at various extents in most cultivars, disease control relies mainly on the use of fungicides. However, management of fungicide applications is necessary in order to avoid the emergence and widespread of fungicide resistant genotypes within populations of the pathogen. In the present study, we investigated for the first time the resistance of M. graminicola toward strobilurin fungicides in Algeria. This was performed by identifying the G143A substitution of the cytochrome b encoding sequence (which confers resistance to strobilurins) in a collection of 120 single-conidial isolates. These isolates have been sampled during the 2012 growing season from five distinct geographical locations (Guelma, Annaba, Constantine, Skikda and Oran). We used a PCR-based mismatch mutation assay allowing the amplification of either G143 (sensitive) or A143 (resistant) allele in each isolate. This study should give valuable information regarding the management of strobilurin use in order to control in a durable manner M. graminicola epidemics in Algeria.
Mycosphaerella graminicola
Strobilurin
Graminicola
Mycosphaerella
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