Mutations in the CYP51 gene correlated with changes in sensitivity to sterol 14 alpha-demethylation inhibitors in field isolates of Mycosphaerella graminicola

In France, as in many other European countries, Mycosphaerella graminicola (Fuckel) Schr?ter in Cohn (anamorph Septoria tritici), the causal agent of wheat leaf blotch, is controlled by foliar applications of fungicides. With the recent generalization of resistance to strobilurins (QoIs), reliable control is mainly dependent upon inhibitors of sterol 14 alpha-demethylation (DMIs). To date, strains with reduced sensitivity to DMIs are widespread, but disease control using members of this class of sterol biosynthesis inhibitors has not been compromised. In this study, sensitivity assays based on in vitro effects of fungicides towards germ-tube elongation allowed the characterization of seven DMI-resistant phenotypes. In four of them, cross-resistance was not observed between all tested DMIs; this characteristic concerned prochloraz, triflumizole, fluquinconazole and tebuconazole. Moreover, the highest resistant factors to most DMIs were found only in recent isolates; according to their response towards prochloraz, they were classified into two categories. Molecular studies showed that DMI resistance was associated with mutations in the CYP51 gene encoding the sterol 14 alpha-demethylase. Alterations at codons 459, 460 and 461 were related to low resistance levels, whereas, at position 381, a valine instead of an isoleucine, in combination with the previous changes, determined the highest resistance levels to all DMIs except prochloraz. Mutations in codons 316 and 317 were also found in some isolates exhibiting low resistance factors towards most DMIs.     

Microscopic analysis of the effect of azoxystrobin treatments on Mycosphaerella graminicola infection using green fluorescent protein (GFP)‐expressing transformants

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     

Alternative oxidase reduces the sensitivity of Mycosphaerella graminicola to QOI fungicides

Forty-six (1.5%) of nearly 3000 isolates of Mycosphaerella graminicola assayed in vitro were resistant to the QOI fungicide azoxystrobin, but on sub-culturing only ten remained resistant. Cross-resistance extended to other QOIs, but varied between different isolates. In planta the resistant isolates were not well controlled, especially at lower azoxystrobin dose rates. Propyl gallate, an inhibitor of alternative oxidase, potentiated the activity of azoxystrobin in vitro so that resistance was no longer observed. The growth of resistant strains in the presence of azoxystrobin led to alternative oxidase activation. This increased flexibility in respiration allows resistant strains to survive in the presence of a QOI fungicide. Under these conditions, selection for target-site mutations can occur. Using QOIs preventatively reduces the risk of resistance since the alternative oxidase cannot by itself generate all the energy needed for germination and early infection.     

Resistance of wheat cultivars and breeding lines to septoria tritici blotch caused by isolates of Mycosphaerella graminicola in field trials

Isolate-specific resistance of 71 cultivars and breeding lines of wheat ( Triticum aestivum ) to septoria tritici blotch was evaluated in six field trials in the Netherlands, Switzerland and the UK between 1995 and 1997. Each plot was inoculated with one of six single-pycnidium isolates of the pathogen Mycosphaerella graminicola . There were strong interactions between wheat lines and M. graminicola and the line-by-isolate interactions were stable over the six trials. Lines with specific resistance or specific susceptibility to each of the isolates were identified. Specific resistance to isolate IPO323 was especially common, being carried by 22 lines from 10 countries. The results confirm that line-by-isolate interactions in septoria tritici blotch of wheat are effective in adult plants in field conditions, and are not simply confined to seedlings. Wheat lines with good, quantitative resistance to all or most isolates were identified, including lines from Brazil, the USA and seven European countries. These may be useful as sources of resistance in wheat breeding.     

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.     

Resistance of wheat to septoria tritici blotch (Mycosphaerella graminicola) and associations with plant ideotype and the 1BL−1RS translocation

Septoria tritici blotch (STB) is a major disease of wheat, reaching epidemic proportions in many parts of the world. In several studies, taller, later-maturing cultivars have had lower disease levels. This study was undertaken to investigate the genetic associations of natural field infection by STB with disease-escape mechanisms related to heading date and height components, mainly leaf spacing, in a population where height and maturity are not controlled by major genes. In field trials of a single seed-descent population of a cross between two nonsemi-dwarf cultivars, Apollo (with strong partial resistance to STB) and Thésée (susceptible), conducted over 3 years, there was a negative correlation between STB and heading date. There was no correlation between STB and distance from stem base to leaf 2; and there was an unexpected positive correlation between STB and distance from flag leaf to leaf 2, which contradicted the so-called 'ladder effect' postulated in STB epidemiology. No effect was detected of the presence of the 1BL−1RS translocation on STB levels. The largest single contributor to variation in STB levels was genetic variation between the progeny lines, and the narrow-sense heritability was 42%. These results suggest that breeders can select for STB resistance alongside optimal stature within the range of height which is adaptive in a particular environment.     

Presence of the Stb6 gene for resistance to septoria tritici blotch (Mycosphaerella graminicola) in cultivars used in wheat-breeding programmes worldwide

Fifty-two wheat cultivars and breeding lines, most of which have been used in breeding programmes worldwide, were tested for isolate-specific resistance to Mycosphaerella graminicola isolate IPO323, which interacts with the Stb6 gene of wheat (first identified in cvs Flame and Hereward) via a gene-for-gene relationship. Twenty-three lines were specifically resistant to this isolate. Sixteen resistant lines were crossed with Flame for a test of allelism. All progeny lines were resistant, suggesting that the 16 parental lines had Stb6 , a gene allelic to it or a gene closely linked to it. In 14 lines, resistance to IPO323 was controlled by Stb6 only. An exception was Kavkaz-K4500 L6.A.4., which has two genes for resistance to IPO323, one of which is Stb6 . The microsatellite marker Xgwm369 was used to examine genetic diversity in the region of the genome containing Stb6 , to which it is closely linked. Eleven alleles of Xgwm369 , with amplified fragments of 10 different sizes, as well as apparent nonamplification of this marker in Bulgaria 88, were detected. Through the use of information about lines' ancestry, combined with Xgwm369 alleles, it was shown that Stb6 entered world wheat-breeding programmes on a minimum of six occasions, and possibly from as many as 11 sources. The presence of Stb6 in both European and Chinese landraces suggests that this gene has been present in cultivated wheat since the earliest times of agriculture.     

Quantitative PCR monitoring of the effect of azoxystrobin treatments on Mycosphaerella graminicola epidemics in the field

Quantitative PCR and visual monitoring of Mycosphaerella graminicola epidemics were performed to investigate the effect of curative and preventative applications of azoxystrobin in wheat field crops. A non-systemic protectant and a systemic curative fungicide, chlorothalonil and epoxiconazole, respectively, were used as references. PCR diagnosis detected leaf infection by M graminicola 3 weeks before symptom appearance, thereby allowing a clear distinction between curative and preventative treatments. When applied 1 week after the beginning of infection, azoxystrobin curative activity was intermediate between chlorothalonil (low effect) and epoxiconazole. When applied preventatively, none of the fungicides completely prevented leaf infection. There was some indication that azoxystrobin preventative treatments may delay fungal DNA increase more than epoxiconazole at the beginning of leaf infection. Both curative and preventative treatments increased the time lapse between the earliest PCR detection and the measurement of a 10% necrotic leaf area. Azoxystrobin only slightly decreased the speed of necrotic area increase compared with epoxiconazole. Hence, azoxystrobin activity toward M graminicola mainly resides in lengthening the time lapse between the earliest PCR detection and the measurement of a 10% necrotic leaf area. Information generated in this way is useful for optimal positioning of azoxystrobin treatments on M graminicola.     

Impact of fungal drug transporters on fungicide sensitivity, multidrug resistance and virulence

Drug transporters are membrane proteins that provide protection for organisms against natural toxic products and fungicides. In plant pathogens, drug transporters function in baseline sensitivity to fungicides, multidrug resistance (MDR) and virulence on host plants. This paper describes drug transporters of the filamentous fungi Aspergillus nidulans (Eidam) Winter, Botrytis cinerea Pers and Mycosphaerella graminicola (Fückel) Schroter that function in fungicide sensitivity and resistance. The fungi possess ATP-binding cassette (ABC) drug transporters that mediate MDR to fungicides in laboratory mutants. Similar mutants are not pronounced in field resistance to most classes of fungicide but may play a role in resistance to azoles. MDR may also explain historical cases of resistance to aromatic hydrocarbon fungicides and dodine. In clinical situations, MDR development in Candida albicans (Robin) Berkhout mediated by ABC transporters in patients suffering from candidiasis is common after prolonged treatment with azoles. Factors that can explain this striking difference between agricultural and clinical situations are discussed. Attention is also paid to the risk of MDR development in plant pathogens in the future. Finally, the paper describes the impact of fungal drug transporters on drug discovery.     

Contributions of disease resistance and escape to the control of septoria tritici blotch of wheat

The contributions of disease escape and disease resistance to the responses of wheat to septoria tritici leaf blotch (STB) were analysed in a set of 226 lines, including modern cultivars, breeding lines and their progenitors dating back to the origin of scientific wheat breeding. Field trials were located in the important wheat-growing region of eastern England and were subject to natural infection by Mycosphaerella graminicola . STB scores were related to disease-escape traits, notably height, leaf spacing, leaf morphology and heading date, and to the presence of known Stb resistance genes and isolate-specific resistances. The Stb6 resistance gene was associated with a reduction of 19% in the level of STB in the complete set of 226 lines and with a 33% reduction in a subset of 139 lines of semidwarf stature. Greater plant height was strongly associated with reduced STB in the full set of lines, but only weakly in the semidwarf lines. Shorter leaf length was also associated with reduced STB, but, in contrast to earlier reports, lines with more prostrate leaves had more STB on average, probably because they tended to have longer leaves. Several lines, notably cvs Pastiche and Exsept, had low mean levels of STB which could not be explained by either escape traits or specific resistance genes, implying that they have unknown genes for partial resistance to STB.     

Modulators of membrane drug transporters potentiate the activity of the DMI fungicide oxpoconazole against Botrytis cinerea

Modulators known to reduce multidrug resistance in tumour cells were tested for their potency to synergize the fungitoxic activity of the fungicide oxpoconazole, a sterol demethylation inhibitor (DMI), against Botrytis cinerea Pers. Chlorpromazine, a phenothiazine compound known as a calmodulin antagonist, appeared the most potent compound. Tacrolimus, a macrolide compound with immunosuppressive activity, was also active. The synergism of chlorpromazine negatively correlated with the sensitivity of the parent strain and mutants of B. cinerea. The synergism was highest in a mutant that overexpressed the ATP-binding cassette transporter BcatrD, known to transport DMI fungicides such as oxpoconazole. The synergism of chlorpromazine positively correlated with its potency to enhance the accumulation of oxpoconazole in BcatrD mutants. These results indicate that chlorpromazine is a modulator of BcatrD activity in B. cinerea and suggest that mixtures of DMI fungicides with modulators may represent a perspective for the development of new resistance management strategies.     

Effect of azole fungicide mixtures,alternations and dose on azole sensitivity in the wheat pathogen Zymoseptoria tritici

The evolution of fungicide resistance in the cereal pathogen Zymoseptoria tritici is a serious threat to the sustainability and profitability of wheat production in Europe. Application of azole fungicides has been shown to affect fitness of Z. tritici variants differentially, so it has been hypothesized that combinations of azoles could slow the evolution of resistance. This work assessed the effects of dose, mixtures and alternations of two azoles on selection for isolates with reduced sensitivity and on disease control. Naturally infected field trials were carried out at six sites across Ireland and the sensitivity of Z. tritici isolates monitored pre‐ and post‐treatment. Epoxiconazole and metconazole were applied as solo products, in alternation with each other, and as a pre‐formulated mixture. Full and half label doses were tested. Isolates were partially cross‐resistant to the two azoles, with a common azole resistance principal component accounting for 75% of the variation between isolates. Selection for isolates with reduced azole sensitivity was correlated with disease control. Decreased doses were related to decreases in sensitivity but the effect was barely significant (P = 0·1) and control was reduced. Single applications of an active ingredient (a.i.) caused smaller decreases in sensitivity than double applications. Shifts in sensitivity to the a.i. applied to a plot were greater than to the a.i. not applied, and the decrease in sensitivity was greater to the a.i. applied at the second timing. These results confirm the need to mix a.i.s with different modes of action.     

Quantifying the effect of interactions between disease control,nitrogen supply and land use change on the greenhouse gas emissions associated with wheat production

A method for calculating the effect of disease control on greenhouse gas (GHG) emissions associated with wheat production, reported previously, was developed further to account for effects of disease control on the amount of fertilizer nitrogen (N) which should be applied and on changes in land use. Data from nine randomized and replicated field experiments from the UK and Denmark showed that the economic optimum N input to winter wheat was greater if diseases were controlled by fungicides, than for untreated wheat. The GHGs associated with this additional N largely negated the benefit to emissions per tonne of grain resulting from disease control. However, the mean grain yield obtained without fungicide treatment was 6·71 t ha^?1, compared to 8·88 t ha^?1 with fungicide treatment, if N input was optimal for each situation. In the absence of disease control by fungicides, and assuming that the optimum N rate was used, an additional 481 kha of wheat would be required to maintain UK wheat production at the current level. If the additional land area came from converting temperate grassland to arable production, the GHG emissions caused by ploughing grassland would cause emissions to rise from 503 to 713 kg CO₂e per tonne of grain produced. This would result in an additional 3·15 Mt CO₂e per year to produce the typical UK annual production of 15 Mt. This analysis reinforces the importance of winning the ‘arms race’ against pathogen evolution towards fungicide insensitivity and virulence.     

Sulphate and sulphurous acid alter the relative susceptibility of wheat to Phaeosphaeria nodorum and Mycosphaerella graminicola

The relative abundances of DNA of Mycosphaerella graminicola and Phaeosphaeria nodorum in archived wheat samples are closely correlated with UK anthropogenic emissions of oxidized sulphur over the last 160 years. To test whether this could be a causal relationship, possible modes of action of sulphur on the two fungi were examined. Mycelial growth of the two fungi in solutions of sulphurous acid was similar. Sulphurous acid at pH 4 reduced percentage germination of P. nodorum conidia more strongly than M. graminicola conidia. In spray inoculations of wheat cv. Squarehead's Master, Cappelle Desprez and Riband with water or sulphurous acid (pH 4), the ratio of leaves infected by P. nodorum to leaves infected by M. graminicola was increased by factors of 2·5, 2·1 and 0·6, respectively at pH 4. The same three cultivars of wheat were grown in sand and vermiculite and fertilized with nutrient solution containing 2·5 or 0·5 mm sulphate. Both pathogens infected less frequently at 2·5 mm sulphate, by a factor of about 2. The severity of infection by M. graminicola was reduced on all three cultivars by a factor of about 4·5 at 2·5 mm sulphate, but severity of P. nodorum was reduced only by a factor of about 2. Both elevated free sulphate concentrations in soil and sulphite in rainwater could therefore increase the prevalence of P. nodorum relative to M. graminicola, which is consistent with the historical changes in abundance.