Protective and curative efficacy of prothioconazole against isolates of Mycosphaerella graminicola differing in their in vitro sensitivity to DMI fungicides

BACKGROUND: Septoria leaf blotch is the most important disease of wheat in Europe. To control this disease, fungicides of the 14α‐demethylase inhibitor group (DMIs) have been widely used for more than 20 years. However, resistance towards DMIs has increased rather quickly in recent years. The objective of this study was to evaluate, on plants and under controlled conditions, the protective and curative efficacy of the DMI fungicide prothioconazole against three current isolates of M. graminicola, chosen to belong to different DMI‐resistant phenotypes. Fungicide efficacy was assessed by visual symptoms and by quantitative real‐time polymerase chain reaction (PCR). RESULTS: With a protective fungicide application, prothioconazole was always effective against each isolate. This was in accordance with the EC₅₀ results. However, curative efficacy differed between the isolates. It remained at a good level, between 60 and 70% against one isolate, whereas it was strongly affected by late applications from 7 days post‐inoculation with the two other isolates. CONCLUSION: A protective application of prothioconazole in wheat crops could be the best strategy to keep a high efficacy against Septoria leaf blotch. Copyright © 2011 Society of Chemical Industry     

The drug transporter MgMfs1 can modulate sensitivity of field strains of the fungal wheat pathogen Mycosphaerella graminicola to the strobilurin fungicide trifloxystrobin

BACKGROUND: The major facilitator superfamily (MFS) drug transporter MgMfs1 of the wheat pathogen Mycosphaerella graminicola (Fuckel) J Schroeter is a potent multidrug transporter with high capacity to transport strobilurin fungicides in vitro. The data presented in this paper indicate that, in addition to the predominant cause of strobilurin resistance, cytochrome b G143A subsititution, MgMfs1 can play a role in sensitivity of field strains of this pathogen to trifloxystrobin. RESULTS: In a major part of field strains of M. graminicola (collected in the Netherlands in 2004) containing the cytochrome b G143A substitution, the basal level of expression of MgMfs1 was elevated as compared with sensitive strains lacking the G143A substitution. Induction of MgMfs1 expression in wild-type isolates upon treatment with trifloxystrobin at sublethal concentrations proceeded rapidly. Furthermore, in disease control experiments on wheat seedlings, disruption mutants of MgMfs1 displayed an increased sensitivity to trifloxystrobin. CONCLUSION: It is concluded that the drug transporter MgMfs1 is a determinant of strobilurin sensitivity of field strains of M. graminicola.     

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.     

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.     

Mode of resistance to respiration inhibitors at the cytochrome bc1 enzyme complex of Mycosphaerella fijiensis field isolates

Field isolates of Mycosphaerella fijiensis, causing black Sigatoka of banana, were characterised for their sensitivity to different inhibitors of the cytochrome bc₁ enzyme complex (Qo respiration inhibitors, strobilurin fungicides), using physiological, biochemical and molecular genetic methods. Strobilurin‐resistant isolates exhibited very high resistance factors both in mycelial growth inhibition and NADH consumption assays. Cross‐resistance was observed among all Qo inhibitors, including trifloxystrobin, azoxystrobin, famoxadone, strobilurin B and myxothiazol. However, the Qi and the cytochrome aa₃ inhibitors, antimycin A and potassium cyanide, respectively, were not cross‐resistant to Qo inhibitors. In sensitive but not in resistant isolates, mixtures of Qo inhibitors and SHAM, an inhibitor of the alternative oxidase (AOX), were more active than the components alone, indicating that the alternative pathway is essential in metabolism, but not causal for resistance. In the cell‐free NADH‐consumption assay, the Qo inhibitors affected the sensitive but not the resistant isolates, suggesting that AOX was not active in sub‐mitochondrial particles. In whole cells, however, the AOX has a basic expression level and is probably not inducible by trifloxystrobin. Sequencing of the cytochrome b gene of sensitive and resistant M fijiensis isolates revealed a difference in the nucleotide sequence leading to a single amino acid change from glycine to alanine at position 143 in the resistant isolate. This change is known to occur also in the naturally tolerant basidiomycete Mycena galopoda. It is suggested that the field isolates of M fijiensis can acquire resistance to Qo inhibitors due to a target site alteration with a single base pair change. Resistant isolates do not seem to contain a mixture of mutated and non‐mutated DNA, indicating a complete selection of resistant mitochondria and a maternally donated mode of resistance. © 2000 Society of Chemical Industry     

A critical evaluation of the role of alternative oxidase in the performance of strobilurin and related fungicides acting at the Qo site of complex III

Mitochondrial respiration conserves energy by linking NADH oxidation and electron-coupled proton translocation with ATP synthesis, through a core pathway involving three large protein complexes. Strobilurin fungicides block electron flow through one of these complexes (III), and disrupt energy supply. Despite an essential need for ATP throughout fungal disease development, strobilurins are largely preventative; indeed some diseases are not controlled at all, and several pathogens have quickly developed resistance. Target-site variation is not the only cause of these performance difficulties. Alternative oxidase (AOX) is a strobilurin-insensitive terminal oxidase that allows electrons from ubiquinol to bypass Complex III. Its synthesis is constitutive in some fungi but in many others is induced by inhibition of the main pathway. AOX provides a strobilurin-insensitive pathway for oxidation of NADH. Protons are pumped as electrons flow through Complex I, but energy conservation is less efficient than for the full respiratory chain. Salicylhydroxamic acid (SHAM) is a characteristic inhibitor of AOX, and several studies have explored the potentiation of strobilurin activity by SHAM. We present a kinetic-based model which relates changes in the extent of potentiation during different phases of disease development to a changing importance of energy efficiency. The model provides a framework for understanding the varying efficacy of strobilurin fungicides. In many cases, AOX can limit strobilurin effectiveness once an infection is established, but is unable to interfere significantly with strobilurin action during germination. A less stringent demand for energy efficiency during early disease development could lead to insensitivity towards this class of fungicides. This is discussed in relation to Botrytis cinerea, which is often poorly controlled by strobilurins. Mutations with a similar effect may explain evidence implicating AOX in resistance development in normally well-controlled plant pathogens, such as Venturia inaequalis.     

A detached seedling leaf technique to study resistance to Mycosphaerella graminicola (anamorph Septoria tritici) in wheat

A detached seedling leaf technique was developed to screen for resistance to septoria tritici blotch of wheat and to detect specific interactions between cultivars and isolates. Wheat seedlings were inoculated with spore suspensions of Mycosphaerella graminicola . Detached primary leaves were then placed in a clear plastic box such that their cut ends were sandwiched between layers of agar containing benzimidazole, with a gap below the middle of the leaves. Mean levels of disease were affected by light and temperature, and also by the concentration of benzimidazole, such that higher concentrations resulted in less disease. Second leaves were more susceptible than seedling primary leaves. However, none of these factors affected ranking of disease among cultivars or cultivar-by-isolate interactions. Kavkaz–K4500 1.6.a.4, Synthetic 6x and Triticum macha showed specific susceptibility and resistance to different isolates. The detached leaf technique could be a useful complement to field trials and an alternative to whole seedling assays in assessing cultivar resistance and investigating the genetics of the host–pathogen interaction.     

Relative importance of different types of inoculum to the establishment of Mycosphaerella graminicola in wheat crops in north‐west Europe

The contribution of wheat debris to the early stages of septoria leaf blotch epidemics was assessed in a 3‐year field experiment. First lesions were detected very early (December) in the case of an early sowing (mid‐October), showing that the first contamination could occur as soon as the seedlings emerge. The tested debris management options (chopped debris, removal of debris followed by tillage, or tillage in absence of debris) had a strong effect, although transient, on the epidemic dynamic: the more debris present on the soil surface, the more severe initial disease was. The magnitude of differences between treatments differed substantially between years. The relative production of pycnidiospores and ascospores was measured on the chopped debris. Peaks in pycnidiospore and ascospore production coincided in October–November. Both types of spores can be involved as primary inoculum in north‐west European conditions. The local amount of pycnidiospores available on debris in the field, estimated per square metre, was 1000‐fold the local ascospore production. Moreover, inoculum production was quantified on debris exposed to different environmental conditions. Autumnal conditions, characterized by moderate temperature with alternating wet and dry periods, were favourable for the production of both pycnidiospores and ascospores, as shown by the high inoculum production on debris exposed to field or outdoor conditions. By late autumn, the canopy became the most important source of pycnidiospores, and this period, characterized by the decreasing role of debris as a local source of inoculum compared to distant potential sources, can be considered as the end of the early epidemic stages.     

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     

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.     

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.     

Sensitivity profiles of Mycosphaerella graminicola and Phytophthora infestans populations to different classes of fungicides

Prior to the use of fungicides, the baseline sensitivity of individuals in a pathogen population may already differ by a factor of 10 to 100 between the least and the most sensitive isolates. In Mycosphaerella graminicola populations, this factor, measured in vitro, was 5 to 20 for both the strobilurin analogue azoxystrobin (baseline) and the triazole cyproconazole which has been in use for several years. In Phytophthora infestans populations, this factor, measured in a leaf disc assay, was about 100 for azoxystrobin (baseline), up to 1000 for the cyanoacetamide cymoxanil and >10000 for the phenylamide oxadixyl; both of the latter have been used for many years. In M. graminicola, cross-sensitivity was present between all azole fungicides for the majority of the isolates, whereas no correlation was found between triazoles and azoxystrobin. Despite the existence of cross-sensitivity between azoles, ‘box-and-whiskers’ plots revealed large variations in the sensitivity profiles of some triazoles; isolates resistant to triazoles have not been detected in M. graminicola populations. In P. infestans populations, the proportion of the phenylamide-resistant sub-population increased during the season more rapidly in treated than in untreated fields, but it was low at the beginning of the next season in all fields. During disease epidemics, the fitness of phenylamide-resistant P. infestans isolates, as characterised by lesion size, was higher than that of the sensitive isolates, but after the overwintering period, the recovery of resistant isolates was apparently lower. The presence of both A1 and A2 mating types of P. infestans in European populations, although at different frequencies, allows sexual recombination and increased genetic diversity, affecting sensitivity and fitness. Such mixed populations can still be adequately controlled by using sound anti-resistance strategies. ©1997 SCI     

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.     

QoI resistance emerged independently at least 4 times in European populations of Mycosphaerella graminicola

BACKGROUND: QoI fungicides or quinone outside inhibitors (also called strobilurins) have been widely used to control agriculturally important fungal pathogens since their introduction in 1996. Strobilurins block the respiration pathway by inhibiting the cytochrome bc1 complex in mitochondria. Several plant pathogenic fungi have developed field resistance. The first QoI resistance in Mycosphaerella graminicola (Fuckel) Schroter was detected retrospectively in UK in 2001 at a low frequency in QoI-treated plots. During the following seasons, resistance reached high frequencies across northern Europe. The aim of this study was to identify the main evolutionary forces driving the rapid emergence and spread of QoI resistance in M. graminicola populations.RESULTS: The G143A mutation causing QoI resistance was first detected during 2002 in all tested populations and in eight distinct mtDNA sequence haplotypes. By 2004, 24 different mtDNA haplotypes contained the G143A mutation. Phylogenetic analysis showed that strobilurin resistance was acquired independently through at least four recurrent mutations at the same site of cytochrome b. Estimates of directional migration rates showed that the majority of gene flow in Europe had occurred in a west-to-east direction.CONCLUSION: This study demonstrated that recurring mutations independently introduced the QoI resistance allele into different genetic and geographic backgrounds. The resistant haplotypes then increased in frequency owing to the strong fungicide selection and spread eastward through wind dispersal of ascospores.     

Analysis of the CYP51 gene and encoded protein in propiconazole‐resistant isolates of Mycosphaerella fijiensis

BACKGROUND: Mycosphaerella fijiensis Morelet causes black sigatoka, the most important disease in bananas and plantains. Disease control is mainly through the application of systemic fungicides, including sterol demethylation inhibitors (DMIs). Their intensive use has favoured the appearance of resistant strains. However, no studies have been published on the possible resistance mechanisms. RESULTS: In this work, the CYP51 gene was isolated and sequenced in 11 M. fijiensis strains that had shown different degrees of in vitro sensitivity to propiconazole, one of the most widely used DMI fungicides. Six mutations that could be related to the loss in sensitivity to this fungicide were found: Y136F, A313G, Y461D, Y463D, Y463H and Y463N. The mutations were analysed using a homology model of the protein that was constructed from the crystallographic structure of Mycobacterium tuberculosis (Zoff.) Lehmann & Neumann. Additionally, gene expression was determined in 13 M. fijiensis strains through quantitative analysis of products obtained by RT‐PCR. CONCLUSION: Several changes in the sequence of the gene encoding sterol 14α‐demethylase were found that have been described in other fungi as being correlated with resistance to azole fungicides. No correlation was found between gene expression and propiconazole resistance. Copyright © 2009 Society of Chemical Industry     

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.