No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Evolution of fungicide resistance in the cereal eyespot fungi Tapesia yalundae and Tapesia acuformis in France
Abstract
Field isolates of the cereal eyespot pathogen can be divided into two groups which are now considered as two species: Tapesia yallundae and Tapesia acuformis. In both species the first case of acquired resistance was observed with benzimidazole fungicides in the early 1980s. At the same time, a number of sterol C-14 demethylation inhibitors (DMIs), such as the imidazole prochloraz and several triazoles, including flusilazole, were introduced. Surprisingly T. acuformis appeared intrinsically resistant to the triazole derivatives in comparison to T. yallundae, but both species were sensitive to prochloraz. The intensive use of these DMIs led to the development of acquired resistance towards triazoles in T. yallundae and towards prochloraz in T. acuformis. Today all the strains in both species appear equally sensitive to the anilinopyrimidine cyprodinil. ©1997 SCI
... In practical terms, the emergence of resistance to MBC fungicides led to withdrawal of these compounds for cereal eyespot control in France (Leroux & Gredt, 1997) and other European countries, and the use of alternative chemistry for management of the disease. Among the available sterol 14ademethlylase Inhibitor (DMI) fungicides then available, none of the triazoles showed good activity against the eyespot pathogens, especially O. acuformis, but the imidazole prochloraz was effective against both species. ...
... Prochloraz was consequently widely used in cereal fungicide programs in Europe, mainly for eyespot control. However, isolates of O. acuformis with reduced sensitivity to prochloraz were reported from Northern France in 1991 (Leroux & Marchegay, 1991) followed by detection of a highly resistant phenotype of O. yallundae the following season (Leroux & Gredt, 1997). The latter was at low frequency in the population, but resistant strains were shown to be selected by treatment with prochloraz or the triazole flusilazole in field trials. ...
... The sampling of eyespot populations in NW France started in the mid-1980s and has continued until the present, with results summarized by Leroux and Gredt (1997) and Leroux et al. (2013). The latter publication covers the period from 1997 until 2010. ...
Fungicides are widely used in developed agricultural systems to control disease and safeguard crop yield and quality. Over time, however, resistance to many of the most effective fungicides has emerged and spread in pathogen populations, compromising disease control. This review describes the development of resistance using case histories based on four important diseases of temperate cereal crops: eyespot (Oculimacula yallundae and Oculimacula acuformis), Septoria tritici blotch (Zymoseptoria tritici), powdery mildew (Blumeria graminis), and Fusarium ear blight (a complex of Fusarium and Microdochium spp). The sequential emergence of variant genotypes of these pathogens with reduced sensitivity to the most active single-site fungicides, methyl benzimidazole carbamates, demethylation inhibitors, quinone outside inhibitors, and succinate dehydrogenase inhibitors illustrates an ongoing evolutionary process in response to the introduction and use of different chemical classes. Analysis of the molecular mechanisms and genetic basis of resistance has provided more rapid and precise methods for detecting and monitoring the incidence of resistance in field populations, but when or where resistance will occur remains difficult to predict. The extent to which the predictability of resistance evolution can be improved by laboratory mutagenesis studies and fitness measurements, comparison between pathogens, and reconstruction of evolutionary pathways is discussed. Risk models based on fungal life cycles, fungicide properties, and exposure to the fungicide are now being refined to take account of additional traits associated with the rate of pathogen evolution. Experimental data on the selection of specific mutations or resistant genotypes in pathogen populations in response to fungicide treatments can be used in models evaluating the most effective strategies for reducing or preventing resistance. Resistance management based on robust scientific evidence is vital to prolong the effective life of fungicides and safeguard their future use in crop protection.
... hordei (Delye et al., 1998) where, in both diseases, the resistance was found to be correlated with the Y136F substitution in the CYP51 gene. Reduced sensitivity was also found in other cereal pathogens such as O. yallundae and O. acuformis as a result of intensive use of DMIs (Leroux and Gredt, 1997). ...
... The results obtained by in vitro (EC 50 ) trials demonstrate that there was not always a cross-resistance between all tested triazoles (R3+, R4, R5, R6, and R8), as some were sensitive to a triazole but were resistant to another one. The same phenomenon was observed with the imidazole fungicide, prochloraz, as previously observed in Oculimacula sp., the causal agents of wheat eye spot (Leroux and Gredt, 1997). However, in a study determining the in vitro sensitivity of over 120 M. graminicola isolates, from throughout England and Wales, to 8 DMI fungicides and to examine cross-sensitivity relationships, Elcock et al. (2000) found a positive cross-sensitivity between some of the DMI fungicides tested. ...
Barley net blotch (BNB) caused by Pyrenophora teres, and Septoria tritici blotch (STB) caused by Mycosphaerella graminicola, are destructive cereal diseases worldwide on barley and wheat respectively. Due to the lack of highly resistant cultivars, both diseases are widely controlled using fungicides. Systemic, site-specific modern fungicides have played an essential role in disease management in cereals. Triazole-based fungicides, which inhibit the C14 demethylation step in fungal ergosterol biosynthesis, known as demethylation inhibitors (DMIs) and strobilurins, known as quinine outside inhibitors (QoIs), which interfere with energy production in the fungal cell, by blocking electron transfer at site of quinone oxidation in the cytochrome bc1 complex, are two major site-specific systemic groups of fungicides, currently used to control cereal diseases. Multiple, consecutive and extensive use of these fungicides has led to the emergence of fungicide resistance in these plant pathogens. The existence of G143A and F129L mutations has been found to be associated with resistance of many plant pathogens to QoIs. However, in P. teres only F129L was found to confer insensitivity. The presence of an intron in several fungi (including rusts and P. teres) determines that it is impossible for the G143A mutation to survive and thus be selected for. Alterations in CYP51 gene in plant pathogens has also been found to be one of the major mechanisms resulting in reduced sensitivity towards DMIs. The aim of this research was to investigate the impact of the F129L mutation in isolates of P. teres, and mutations in the CYP51 gene in M. graminicola isolates on the activity of QoI and DMI fungicides respectively.
Results revealed a high frequency of the F129L mutation within recent UK P. teres isolates. Furthermore, the common change (G143A) in cytochrome b was not found in P. teres strains. The results also showed a lack of any fitness penalty associated with the mutation. Bioassay tests indicated that inhibition of net blotch by QoIs was variable. Single QoI fungicides such as pyraclostrobin and picoxystrobin were found to be highly inhibitory whilst the efficacy of other QoIs was less pronounced. It has been found that efficacy of QoI fungicides varied amongst a population of isolates with the F129L mutation. This might suggest that some QoIs were compromised by the F129L mutation to some degree. However, the results obtained were in agreement with previous reports that the F129L mutation in the cytochrome b gene generates lower levels of resistance and was not as serious as that posed by the G143A mutation in other plant pathogens. In addition, fungicide mixtures, comprising QoIs and DMIs or the novel SDHI formulations, were found to have great efficacy in net blotch disease management.
Sequence results of CYP51 gene fragment indicated existence of 15 alterations in recent UK and German isolates of M. graminicola. Some of these mutations, such as Y137F, were found to be rare whilst the I381V mutation was found to be increasing with time. However, investigations indicated a lack of phenotypic fitness penalties associated with these alterations. Apical germ tube growth measurement was found an effective method to assess in vitro activity of DMI fungicides against M. graminicola isolates. Based on bioassay studies, six categories within M. graminicola isolates were detected, showing different sensitivities to azole fungicides. In general, genotypes characterised S, R3+ and R4 were sensitive to most azole fungicides. The R3+ variant, however, showed less sensitivity to tebuconazole and prochloraz. In in vitro studies, the R5 variants, exhibited sensitivity to many DMIs but were less sensitive to prochloraz. This supporting the results obtained from in planta assays, where this genotype was found to be sensitive to tebuconazole but less sensitive to prochloraz. On the other hand, genotypes characterised R6a, R7 and R8, containing I381V mutation, were resistant to tebuconazole but sensitive to prochloraz. The latter variant, however, were more sensitive to prochloraz. It can be suggested from results obtained in this study that CYP51 alterations were differentially selected by different members of the azole class of fungicides.
Q-PCR was also used to evaluate in planta fungicide activity on both diseases. The method indicated similar pattern to that observed in visual assessments. Detection of medium to high correlation values between both assessments confirmed the validity of q-PCR assessment. This suggests that q-PCR assays may serve as an alternative method for accurate assessment of the fungicide effects on cereal diseases. The method can be a valuable tool to evaluate disease occurrence in pathogens with a long latent period, such as M. graminicola, as q-PCR could readily detect the pathogen during the asymptomatic latent period.
... Both surveys (Lucas et al. , 2000) and field experiments (Bateman et al. , 1990a) have suggested that use of prochloraz has selected for T. acuformis in the UK; this appears to result from isolates of T. acuformis having a greater range of sensitivity to prochloraz than have isolates of T. yallundae (Bateman et al. , 1994). Furthermore, isolates resistant to prochloraz have been reported in France (Leroux & Gredt, 1997), although the proportion of resistant isolates has been decreasing there (Birchmore et al. , 2001). The anilinopyrimidine cyprodinil is now widely used (Nicholson & Turner, 2000). ...
... Furthermore, there were no significant differences between expected and observed numbers of co-occurrences on stems in plots sampled in 2000 (Table 2). Similar results were found by Bateman (1993), whilst Leroux & Gredt (1997) found that T. yallundae and T. acuformis co-occurred on up to 56% of the stems in a survey of wheat crops in France. These cooccurrences suggest that T. yallundae and T. acuformis do not prevent each other from colonizing the stem, implying large niche differences. ...
Effects of regular treatments with the fungicides carbendazim and prochloraz applied to whole plots divided into subplots with different initial population mixtures of carbendazim-sensitive or carbendazim-resistant Tapesia yallundae or T. acuformis were studied in successive crops of winter wheat from 1984/85 to 1999/2000. In unsprayed and carbendazim-sprayed whole plots, a stable coexistence of about 50% each of T. yallundae and T. acuformis developed within five seasons, but in whole plots sprayed with prochloraz or prochloraz plus carbendazim, the proportion of T. acuformis increased to > 80%. A discrete time difference equation model was derived from knowledge of the biology of eyespot and competition theory to describe the population changes. The model was fitted to the data from treatments where coexistence occurred [subplots in unsprayed (1985–92) and carbendazim-sprayed (1985–89) whole plots], using nonlinear least squares regression. The optimized value of the resource overlap coefficient was small, suggesting niche differences between the two species. Populations were nearly 100% carbendazim-resistant in carbendazim-sprayed whole plots by July 1985 (one season) and in whole plots sprayed with prochloraz plus carbendazim by July 1986 (two seasons). In prochloraz-sprayed whole plots, the proportion of carbendazim-resistant isolates decreased more rapidly than in unsprayed whole plots in the 1980s, but by July 1992 a shift in populations in unsprayed and prochloraz-sprayed whole plots towards predominantly carbendazim-resistant strains had occurred.
... In experiments conducted on eyespot in the United Kingdom by Jones (1994), prochloraz and flusilazole gave significant reductions in eyespot disease and increases in yield. Leroux and Gredt (1997) reported irregular efficacies of flusilazole because of acquired resistance following years of application in France. Further research will now have to be conducted to determine if flusilazole is still effective under field conditions. ...
... Further evidence of this is needed. Irregular efficacies because of acquired bromuconazole resistance to T. yallundae in France have also been reported from areas where other triazoles were previously used (Duvert et aI., 1997;Leroux & Gredt, 1997). ...
In the Western Cape province of South Africa, eyespot disease (Tapesia yallundae) of wheat is primarily controlled by fungicide applications. Previous studies have shown, however, that isolates of T. yallundae vary in their response to fungicides. In the present study, 20 isolates from each of 15 fields with different fungicide histories were screened against carbendazim at 1 ¿g/ml; propiconazole, tebuconazole and flutriafol each at 0.1, 0.5, 1 and 2 ¿g/ml; and flusilazole and bromuconazole each at 0.05, 0.1, 0.5 ¿g/ml. No isolates were resistant to carbendazim. Flusilazole proved to be most effective of all the triazole fungicides tested. Based on the analysis of EC50 values compared to that of a field at Gouda that is at baseline sensitivity, shifts in sensitivity were detected to all triazoles. This suggests that measures for prevention of build-up of resistance need to be employed.
... These compounds include triazoles, imidazole prochloraz, prothioconazole etc. The anilinopyrimidine cyprodinil was registered in France in the 1990s, when the resistance to triazoles and prochloraz was encountered in practice (Leroux and Gredt 1997). Later two new active ingredients were introduced -boscalid and metrafenone (Avenot et al. 2010;Opalski et al. 2006). ...
Winter wheat samples infected by eyespot were collected during the years 2015–2017. In total 143 Oculimacula spp. isolates were obtained and determined by PCR. Oculimacula yallundae isolates prevailed (122) over Oculimacula acuformis isolates (12) and 9 mixed isolates of both species. O. acuformis predominated only on one locality. The reaction of 104 Oculimacula spp. isolates to ten different fungicides was evaluated in laboratory tests on Petri dishes. The highest efficacy showed the combination of epoxiconazole + fluxapyroxad + pyraclostrobin and the lowest efficacy was evaluated after application of prothioconazole + trifloxystrobin. The decrease of efficacy of fungicide no. 6 (prothioconazole + trifloxystrobin) has been observed since 2015. The sensitivity of 114 Oculimacula spp. isolates to prochloraz has been tested. More than half of the isolates (58%) showed low to medium resistance. One isolate of O. yallundae from the year 2017 showed high resistance to prochloraz (ED 50 ranged 1.7 µg ∙ ml –1 ). The rest of the isolates (42%) were sensitive to prochloraz.
... In the study of Ramanauskiene and Gaurilčikiene (2016) all 122 samples of cereal stem bases in Lithuania were colonised by both species o. yallundae and o. acuformis together except for one example. Previously Leroux and Gredt (1997) found that o. yallundae and o. acuformis co-occurred on up to 56% of the stems in a survey of wheat crops in France. Bierman et al. (2002) described a stable coexistence of about 50% of both species in unsprayed and carbendazim sprayed plots for five seasons in the UK. ...
The reaction of ten winter wheat cultivars grown in the Czech Republic to inoculation with Oculimacula yallundae and Oculimacula acuformis was evaluated in a small plot trial. In a parallel field trial the natural occurrence of stem-base disease complex in six of the tested cultivars was assessed. Lower severity of eyespot (and/or stem-base diseases) was observed in cultivars possessing the resistance gene Pch1 (cvs Hermann, Annie, Princeps, Manager, and Rebell) in plots inoculated with Oculimacula spp. as well as in natural field conditions. A total of 468 wheat stem bases from the Czech Republic was screened by PCR to study the frequency of eyespot causal agents. The plants were colonised significantly more often by both species Oculimacula yallundae and O. acuformis together than separately.
... The use of fungicides against eyespot is expensive for farmers. Furthermore, the evolution of resistance to specific fungicides was reported (Leroux and Gredt 1997). Differential fungicide sensitivity influenced the relative abundance of both Oculimacula species in Western Europe and led to a recent increase of relative abundance of O. yallundae (Parnell et al. 2008). ...
Key message:
Genotypes with recombination events in the Triticum ventricosum introgression on chromosome 7D allowed to fine-map resistance gene Pch1, the main source of eyespot resistance in European winter wheat cultivars. Eyespot (also called Strawbreaker) is a common and serious fungal disease of winter wheat caused by the necrotrophic fungi Oculimacula yallundae and Oculimacula acuformis (former name Pseudocercosporella herpotrichoides). A genome-wide association study (GWAS) for eyespot was performed with 732 microsatellite markers (SSR) and 7761 mapped SNP markers derived from the 90 K iSELECT wheat array using a panel of 168 European winter wheat varieties as well as three spring wheat varieties and phenotypic evaluation of eyespot in field tests in three environments. Best linear unbiased estimations (BLUEs) were calculated across all trials and ranged from 1.20 (most resistant) to 5.73 (most susceptible) with an average value of 4.24 and a heritability of H (2) = 0.91. A total of 108 SSR and 235 SNP marker-trait associations (MTAs) were identified by considering associations with a -log10 (P value) ≥3.0. Significant MTAs for eyespot-score BLUEs were found on chromosomes 1D, 2A, 2D, 3D, 5A, 5D, 6A, 7A and 7D for the SSR markers and chromosomes 1B, 2A, 2B, 2D, 3B and 7D for the SNP markers. For 18 varieties (10.5%), a highly resistant phenotype was detected that was linked to the presence of the resistance gene Pch1 on chromosome 7D. The identification of genotypes with recombination events in the introgressed genomic segment from Triticum ventricosum harboring the Pch1 resistance gene on chromosome 7DL allowed the fine-mapping of this gene using additional SNP markers and a potential candidate gene Traes_7DL_973A33763 coding for a CC-NBS-LRR class protein was identified.
... Intensive use of fungicides in many countries resulted in rapid spread of resistance of Oculimacula spp. first to the MBC and later to DMI class fungicides (Murray, 1996;Moreau and Maraite, 1996;Leroux and Gredt, 1997;Bierman et al., 2002). Different sensitivity and resistance level of O. acuformis and O. yallundae to the MBC and DMI fungicides caused predomination of O. acuformis in many countries (Bateman et al., 1990;Bierman et al., 2002). ...
The aim of this study was to estimate the effect of fungicides on the occurrence of Oculimacula spp. on winter wheat stems using real-time polymerase chain reaction (Real-time PCR) method. The ratio of Oculimacula yallundae and Oculimacula acuformis in the population was assessed from 2009 to 2011 in the winter wheat crops after 2, 3, and 4 years of use of fungicides prochloraz, cyprodinil, prothioconazole, metrafenone, and boscalid. After 4 years of fungicide use, the highest relative reduction of DNA of both fungi, compared with the unsprayed control, was detected in the samples from cyprodinil-applied plots. Relatively less O. yallundae DNA was identified in the samples from prochloraz- and that of O. acuformis from prothioconazole-applied plots. Metrafenone and boscalid had little effect on the changes in Oculimacula population. Fungicide effect on O. acuformis and O. yallundae pathogenesis slightly differed. In Oculimacula population under the fungicide treatment, 10 days after application (BBCH 37), the ratio of O. acuformis in all treatments was reduced, but the ratio of O. yallundae was increased in prevalence compared to O. acuformis in cyprodinil and prothioconazole treatments. Later in the season (BBCH 75), the ratio of O. acuformis increased in metrafenone and O. yallundae in prochloraz treatments. The effect of fungicides used for eyespot control on winter wheat productivity was inconsistent. Prochloraz use led to a significant grain yield increase in 2010, while prothioconazole use showed a similar increase in 2009 and 2010. Cyprodinil, boscalid, and metrafenone did not have any effect on the yield in any of the experimental years. A significant increase in thousand grain weight was obtained in prochloraz, prothioconazole, and boscalid treatments.
... In the past, the control of eyespot has relied largely on chemical protection [12]. However, due to the development of resistance to the main available fungicides in O. yallundae and O. acuformis populations, adaptation of the entire cropping system to control eyespot on wheat is a sound alternative [13,14]. Furthermore, growing concerns about the impact of pesticides on the environment and human health has led to attempts to limit pesticide use [15,16]. ...
IPSIM (Injury Profile SIMulator) is a generic modelling framework presented in a companion paper. It aims at predicting a crop injury profile as a function of cropping practices and abiotic and biotic environment. IPSIM's modelling approach consists of designing a model with an aggregative hierarchical tree of attributes. In order to provide a proof of concept, a model, named IPSIM-Wheat-Eyespot, has been developed with the software DEXi according to the conceptual framework of IPSIM to represent final incidence of eyespot on wheat. This paper briefly presents the pathosystem, the method used to develop IPSIM-Wheat-Eyespot using IPSIM's modelling framework, simulation examples, an evaluation of the predictive quality of the model with a large dataset (526 observed site-years) and a discussion on the benefits and limitations of the approach. IPSIM-Wheat-Eyespot proved to successfully represent the annual variability of the disease, as well as the effects of cropping practices (Efficiency = 0.51, Root Mean Square Error of Prediction = 24%; bias = 5.0%). IPSIM-Wheat-Eyespot does not aim to precisely predict the incidence of eyespot on wheat. It rather aims to rank cropping systems with regard to the risk of eyespot on wheat in a given production situation through ex ante evaluations. IPSIM-Wheat-Eyespot can also help perform diagnoses of commercial fields. Its structure is simple and permits to combine available knowledge in the scientific literature (data, models) and expertise. IPSIM-Wheat-Eyespot is now available to help design cropping systems with a low risk of eyespot on wheat in a wide range of production situations, and can help perform diagnoses of commercial fields. In addition, it provides a proof of concept with regard to the modelling approach of IPSIM. IPSIM-Wheat-Eyespot will be a sub-model of IPSIM-Wheat, a model that will predict injury profile on wheat as a function of cropping practices and the production situation.
... However, the extensive use of synthetic fungicides has raised concerns regarding decreases in the fungicide sensitivity of pathogens. A number of studies have reported the emergency of fungal resistance to prochloraz [Leroux and Credt, 1997; Gea et al., 2005]. ...
The fungicide prochloraz was subjected to degradation by the pathogen causing rice Bakanae disease, Fusarium fujikuroi, in order to gain an insight into the mechanisms of sensitivity and resistance to the fungicide. Growth-inhibiting assays of pathogens conducted on potato dextrose agar (PDA) plates by a paper-disc agar-diffusion method. Significant growth inhibition of the sensitive strain CF106 was observed at the recommended treatment level of prochloraz, whereas negligible growth inhibition of the resistant strain CF245 was observed at the same treatment level. The strain CF245 was shown to be able to grow on PDA with 500 mg/L of the fungicide, which is significantly higher than its recommended treatment level. Growth-inhibiting assays of pathogens were also conducted in potato dextrose broth (PDB) medium supplemented with prochloraz at different concentrations, measuring their biomass weights over the incubation period. Significant growth inhibition was observed in the strain CF106 at a level of 0.5 mg/L, but negligible growth inhibition was observed in the strain CF245 at the same treatment level with the strain CF106. The strain CF245 could grow in PDB supplemented with 1.0 mg/L of prochloraz. The degradation of prochloraz by the two strains was evaluated by gas liquid chromatography and mass spectrometry analyses. The strain CF245 completely degraded 1.0 mg/L of prochloraz in 5 days after incubation, whereas no degradation of prochloraz was observed by the strain CF106 at the same treatment level. Liquid chromatography Q-TOF MS detected N-(2-(2,4,6-trichlorophenoxy)ethyl)propan-1-amine as a major degradation product of prochloraz by the strain CF245. These results indicated that the degradation of prochloraz may account for the reduced sensitivity of the strain CF245 to prochloraz.
... cyprodinil) (Russell, 2005). However, resistance to these chemicals has presented a considerable challenge to disease control and there are many reported cases of practical resistance (a detectable loss of disease control in the field), in particular with the MBCs (King & Griffin, 1985;Schreiber & Schlesinger, 1985;Leroux & Migeon, 1993;Leroux & Gredt, 1997). Where resistance has occurred, the two species have shown differential levels of resistance to the same fungicides (Bateman et al ., 1990). ...
Changing fungicide sensitivities in populations of Oculimacula yallundae and O. acuformis, the species responsible for cereal eyespot in Western Europe, were determined over a 17 year period between 1984 and 2000. The data were collected by Aventis Crop Science as part of their long-term survey to monitor changes in sensitivity to prochloraz and the methyl benzimidazole carbamate (MBC) fungicides in eyespot populations. The results show evidence for reduced sensitivity to both fungicides over the period of the survey. The decline in MBC sensitivity is in agreement with reports of practical resistance (a detectable loss of disease control in the field) to this fungicide which were widely reported from the mid 1980s onward. Prochloraz sensitivity was more complex, with the emergence of a higher resistance category of isolates in the late 1980s and early 1990s which then decreased in frequency towards the end of the survey. This may be partly explained by the introduction and increased use of cyprodinil in the mid 1990s. Although all trends were similar across Europe, differences were observed between the two eyespot species. A higher frequency of O. yallundae isolates showed decreased sensitivity to MBC, whereas decreased sensitivity to prochloraz was at a higher frequency in O. acuformis populations. The relative abundance of the two eyespot species was influenced by their differential levels of fungicide sensitivity, with the ratio increasing toward the species with the highest level of resistance to the prevailing fungicide.
... In the early 1980s, because of the selection of resistant strains, the benzimidazoles were replaced by C-14 demethylation inhibitors (DMIs) such as prochloraz (imidazole group) or flusilazole (triazole group). In the early 1990s the efficiency of these DMIs was also compromised by fungicide resistance in some regions (Leroux and Gredt 1997). The most-used current fungicide is cyprodinil, but decreased sensitivity from repeated applications has also been observed with this fungicide (Babij et al., 2000). ...
... The accuracy of early diagnosis of SBD causal agents could have potentially significant repercussions where they differ in sensitivity to chemical control agents or where host resistances vary. For example, the eyespot species T. yallundae has been found to be more sensitive to flusilazole and other triazole fungicides than T. acuformis (Cavalier et al., 1987: Bateman, 1990: Leroux & Gredt, 1997. Also, over 90% of M. nivale isolates obtained from brown foot rot lesions on wheat in the UK in 1986 were found to be resistant to the methyl benzimidazole carbamate (MBC) fungicide benomyl while all isolates of F. culmorum and F. avenaceum were sensitive (Locke et al., 1987). ...
Stem base disease (eyespot, sharp eyespot and brown foot rot) was assessed visually and by the polymerase chain reaction (PCR) technique on single plants sampled at four-week intervals in two crops of winter wheat grown in the UK in 1992–3. PCR assays were conducted for Fusarium avenaceum, F. culmorum, both varieties of Microdochium nivale, both eyespot-causing species of Tapesia and Rhizoctonia cerealis. PCR diagnoses were compared with visual diagnoses at each time point. Eyespot was caused principally by T. acuformis (R-type) and developed rapidly late in the season. Visual diagnoses of eyespot were largely confirmed by PCR but T. acuformis was detected in many plants lacking eyespot symptoms. R. cerealis was detected at relatively low incidences on both sites, and sharp eyespot visual diagnoses did not correlate with the incidence of any of the pathogens assayed by PCR. Brown foot rot, caused principally by Microdochium nivale var. majus, accumulated earlier in the season than eyespot. Overall, visual diagnoses of stem base disease coincided poorly with PCR data until after growth stage (GS) 30.
... Field isolates of the cereal eyespot pathogen Tapesia yallundae with reduced sensitivity to the imidazole DMI fungicide prochloraz (1-[N-propyl-N-[2-(2,4,6-trichlorophenoxy)ethyl]carbamoyl]-imidazole) have recently been detected in France and New Zealand [3,4]. Understanding the molecular genetic basis of resistance should aid in developing improved strategies for managing eyespot disease and predicting models of resistance to prochloraz in other pathogens [5]. ...
Resistance to sterol 14α-demethylase inhibitor (DMI) fungicides has been correlated with mutations in the CYP51 gene encoding the target enzyme eburicol 14α-demethylase. CYP51 was isolated from the eyespot pathogen Tapesia yallundae revealing a predicted 526-amino acid product exhibiting homology to other fungal CYP51s. CYP51 was sequenced from four field isolates sensitive or resistant to the DMI fungicide prochloraz and partially sequenced from two further isolates and eight progeny from a cross between prochloraz-sensitive and -resistant parents. Two alleles of the gene were detected termed CYP51-1 and CYP51-2. No correlation was found between sequence change and fungicide sensitivity. Therefore prochloraz resistance involved a mechanism other than mutation in the target site gene.
... The evolution of resistance to pesticides is a common phenomenon in insects, weeds, and plant pathogens (Jasieniuk and Maxwell, 1994;Leroux and Gredt, 1997;Rosenheim et al., 1996). Pest population sizes are typi-cally quite large, which increases the chance that a mutation inXuencing resistance will occur, and selection in the form of the application of pesticides is quite strong, driving mutations or standing genetic variation that enables survival and reproduction towards Wxation quickly. ...
Microevolution may determine both the safety and efficacy of classical biological control. Despite a growing body of literature, there are several key unanswered questions regarding the role of evolution in biological control: (1) How common is local adaptation of natural enemies to their hosts or the environment in the native range? How critical is it for success of biological control to find locally adapted agents for importation? (2) Does adaptive evolution following introductions play an important role in biological control? (3) Do introductions of biological control agents impose bottlenecks in population size that reduce genetic variation, and is reduced genetic variation associated with low fitness and poor performance? (4) How great is the risk of evolution of host range of biological control agents? (5) What is the risk of target pests evolving resistance to biological control agents? If pests evolve increased resistance, will biological control agents evolve mechanisms to overcome that resistance? Here, we review the four fundamental processes of microevolution, and discuss how they interact in the context of biological control. We discuss our current state of knowledge regarding the outstanding questions, highlight the types of experiments that can address them, and suggest ways to use microevolution to define risks, and enhance efficacy and safety of biological control.
... Chemical fungicides are commonly used to control plant pathogens and reduce the economical losses from disease. However, the development of resistance to chemical fungicides is a common phenomenon among plant pathogens (Gisi et al., 2002;Hufbauer and Roderick, 2005;Leroux and Gredt, 1997). The likelihood for pathogens to develop resistance is dependent on pathogen population size which often is large enough for mutations to occur against the fungicide that is being used. ...
The necrotrophic pathogen Heterobasidion annosum sensu lato (Fr.) Bref. causes severe root rot on coniferous trees in the boreal and temperate forests. The annual economic losses caused by this fungus in Europe are estimated to at least 790 million €. In managed forests, the major route of infection is via stump surfaces from which the H. annosum s.l. grows through the roots and attacks adjacent healthy trees. A biocontrol method to reduce H. annosum s.l. infection is to apply the wood degrading fungus Phlebiopsis gigantea in a spore solution (Rotstop) directly on the freshly cut stumps immediately after cutting. We investigated the potential risk for a build-up in the capacity of H. annosum s.l. to resist overgrowth by P. gigantea. Wood blocks of Picea abies, precolonized with the two fungal species, were juxtaposed on top of agar and the overgrowth of the P. gigantea strain (Rotstop) on the H. annosum s.l. was measured periodically. We found a natural variation in Heterobasidion parviporum to resist overgrowth by P. gigantea. There was no difference between homo- and heterokaryotic strains. In a mapping population of 91 progenies from a H. annosum hybrid strain we were able to identify one quantitative trait locus (QTL) which controls the examined resistance capacity. We estimated the broad sense heritability to 0.336 for the capacity to resist the P. gigantea overgrowth. We conclude that there exists a theoretical risk for resistance build-up in the H. annosum s.l. population towards its biological control agent P. gigantea.
... There are now new fungicides that are effective against eyespot, such as prochloraz of the chemical class azole and cyprodinil of the chemical class pyrimidine. The latter is more effective but fungicide resistance may also evolve for both of these types of fungicides, and resistant varieties are preferred to fungicides in many ways (Leroux & Gredt, 1997; Babij et al., 2000; Bateman et al., 2000; Ray et al., 2004). Cappelle-Desprez was used extensively in the UK for more than 20 years, probably due to its resistance against eyespot (Hollins et al., 1988). ...
The relationships between plant diseases, winter wheat characteristics, air temperature and precipitation, site factors and agricultural practices were investigated. Regression analyses revealed that control of LBDs (Leaf Blotch Diseases, including septoria tritici blotch, stagonospora nodorum blotch and tan spot) explained 74% of the yield increase achieved by fungicide treatment at GS 45-61, followed by powdery mildew (20%), brown rust (5%) and yellow rust (1%). Yield of both untreated and fungicide-treated plots increased from 6000 to 12000 kg/ha over the period 1983-2005. Single eyespot treatment improved mean yield by ~320 kg/ha/yr during the period 1977-2002, mainly due to occasional years with severe eyespot. A fungicide treatment at GS 45-61 increased mean yield by 10.3% or 810 kg/ha/yr (9.9% or 660 kg/ha/yr for 1983-1994 and 10.7% or 970 kg/ha/yr for 1995-2005) due to increased TGW and grain numbers, especially in high yielding stands. Air temperature and precipitation as monthly means explained more than 50% of the variation between years regarding yield increase, TGW, LBDs, brown rust, yellow rust and eyespot, but less than 50% of the variation in yield and powdery mildew. Precipitation in May was the factor most consistently related to LBD disease intensity, and adding another two weather factors further improved the degree of explanation. Weather factors in the preceding growing season influenced growth stage, powdery mildew and brown rust. Mild winters and springs favoured the biotrophs, i.e. powdery mildew, brown rust and yellow rust. The mean net return from fungicide use was negative in 10 years and less than 50% of the entries were profitable to treat in 11 years. Fungicide use was in fact more profitable (mean net return 21 compared with 3 €/ha) during the latter part of the period (1995-2007) than in the earlier part (1983-1994). The role of site factors and agricultural factors is complex but some factors, such as pre-crop and dose of nitrogen, can probably be used in plant disease warning and prediction models. Wheat as pre-crop to wheat gave 1.6 tons/ha lower yield than rape as pre-crop. The results confirm the potential and limits of fungicides and the need for supervised control strategies that include factors affecting disease, yield, interactions and overall profitability.
... This is crucial in order to prevent the pathogen from developing into strains that are more tolerant to the control method and thereby creating future complications in the field of use. Resistance to chemical fungicides is a common phenomenon among different fungal pathogens (Leroux & Gredt 1997; Gisi et al. 2002; Hufbauer & Roderick 2005). But so far, resistance to biological control methods is not known to be as pronounced as the resistance to chemical compounds (Holt & Hochberg 1997). ...
This thesis summarises the author's work on the effectiveness of two control methods against the necrotrophic fungal pathogen Heterobasidion annosum s.l. (Fr.) Bref. The studied control methods were stump treatments through the application of a fungal biocontrol agent (Phlebiopsis gigantea) and application of urea solution. An additional component to this thesis was to develop a useful Agrobacterium tumefaciens-mediated transformation system for the H. annosum s.l. The potential risk for a build-up in the capacity of H. annosum s.l. to resist overgrowth by P. gigantea was investigated by utilising a P. gigantea strain used in the commercial preparation Rotstop and different strains of H. annosum s.l. The impact on intraspecific genetic diversity from the P. gigantea used in Rotstop four years after stump treatment was estimated using both microsatellite markers and AP-PCR. The long-term effect of urea treatment during first rotation thinning of Picea abies stands was evaluated 15 years post-treatment. An A. tumefaciens-mediated transformation system was developed using two different transformation plasmids and a North American H. annosum strain.
The results revealed differences between H. annosum s.l. strains in their ability to resist overgrowth by P. gigantea. This trait was found to be heritable which suggests that H. annosum s.l. has the capacity to develop more tolerant strains against the biocontrol agent if the conditions are met. Also, one QTL effect associated with this trait was identified. Genetic diversity was lower within Rotstop treated plots and the resident population was more genetically similar to the applied P. gigantea strain than other populations sampled in Sweden. This effect on genetic structure was not detected 100-200 m from the treated plot which suggests that stump treatments with P. gigantea only has a local effect on genetic diversity. Urea treated plots exhibited less rot than untreated plots and the urea treatments also significantly reduced the occurrence of windthrown trees. This shows that urea treatment during thinning operations is an efficient method to reduce root rot in P. abies stands. A total of 18 H. annosum s.l. transformants were obtained from the A. tumefaciens-mediated transformation system. The transformants expressed GFP throughout the mycelia and had a normal growth rate. This system will be very useful for genomic work and interaction studies.
... Resistance to DMIs in Tapesia spp. has not been reported from the PNW, but has been reported in Europe (18). Leroux and Gredt (18) found that T. acuformis is intrinsically less sensitive toward most DMIs compared with T. yallundae, which could have facilitated the increase of T. acuformis in Europe. ...
ABSTRACT Eyespot of wheat is caused by the discomycete fungi Tapesia yallundae and T. acuformis. T. yallundae is considered the most important causal agent of the disease in this region but no apothecia of either species have been found in the U.S. Pacific Northwest (PNW). Two compatible isolates of T. yallundae from the PNW were used to inoculate a field plot in the fall of 1998 and apothecia developed in the spring and fall of 2000 on standing wheat stubble. In the spring of 2000, wheat stubble from eight naturally infected fields was examined for the presence of apothecia of T. yallundae and T. acuformis. Apothecia of T. acuformis were found in two fields but no apothecia of T. yallundae were found. This is the first report of apothecia of the eyespot pathogens occurring in the PNW. Species and mating-type distribution of T. yallundae and T. acuformis in the PNW were determined from 817 isolates collected from diseased wheat over 3 years at spatial scales ranging from within fields to across states. In all, 460 isolates were identified as T. yallundae and 357 isolates were identified as T. acuformis with MAT1-1/MAT1-2 ratios not significantly different from 1:1 based on chi(2) tests at most scales tested. The apparent increase in frequency of T. acuformis from previous surveys may indicate a shift in the predominant species causing eyespot. The occurrence of apothecia under field conditions, along with the widespread distribution of mating types of both species, suggests that sexual reproduction may be occurring in both species.
... However, the extensive use of synthetic fungicides has raised concerns regarding decreases in the fungicide sensitivity of pathogens. A number of studies have reported the emergency of fungal resistance to prochloraz [Leroux and Credt, 1997; Gea et al., 2005]. ...
Laboratory studies on the formation of bound residues and on the degradation of the triazole fungicide propiconazole were conducted in two different soils. Soils treated with ¹⁴ C‐propiconazole were incubated at 22 °C and extracted exhaustively with a solvent at each sampling date until no further propiconazole was extracted. The solvent‐extractable residues were used to measure propiconazole remaining in the soil, and the extracted soils were used to investigate bound residues of propiconazole. Mineralization of propiconazole was investigated by measuring [ ¹⁴ C]carbon dioxide evolved from the soil samples. Formation of bound residues of propiconazole was higher in silty clay loam soil than in sandy loam soil, giving approximately 38 and 23% of the applied ¹⁴ C, respectively. In contrast, the rates of degradation and mineralization of propiconazole were lower in silty clay loam soil than in sandy loam soil. Decreased extractability of the ¹⁴ C residues with incubation time was observed with increased formation of bound residues. When the propiconazole remaining in the solvent‐extractable residues was quantitatively measured by high‐pressure liquid chromatographic analysis, the half‐life value in sandy loam soil was about 315 days, while the half‐life in silty clay loam soil exceeded the duration of the 1 year experimental period. Increased formation of bound residues was observed as propiconazole degraded with incubation time, suggesting that degradation products are involved in the formation of bound residues. Our study suggests that the formation of bound residues of propiconazole contributes to the persistence of this fungicide in soil.
© 2003 Society of Chemical Industry
... In some of them there was not always cross-resistance between all tested DMIs (Tri R1, Tri R2, Tri R3 and Tri R7), as some were sensitive to the imidazole derivatives triflumizole and prochloraz, as was previously recorded in Oculimacula sp., the causal agents of wheat eyespot. 17 This lack of cross-resistance extended to the triazoles fluquinconazole and tebuconazole. On the other hand, according to the RF values, it was possible to classify the DMI-resistant phenotypes into two categories: Tri LR (low RFs) and Tri MR (medium to high RFs). ...
In France, as in many other European countries, Mycosphaerella graminicola (Fuckel) Schröter 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.
Eyespot, caused by the related fungal pathogens Oculimacula acuformis (OA) and O. yallundae (OY), is an important cereal stem-base disease in temperate parts of the world. Both species are dispersed mainly by splash-dispersed conidia but are also known to undergo sexual reproduction yielding apothecia containing ascospores. Field diagnosis of eyespot can be challenging with other pathogens causing similar symptoms, which complicates eyespot management strategies. Differences between OA and OY (e.g. host pathogenicity and fungicide sensitivity) require that both be targeted for effective disease management. Here, we develop and apply two molecular methods for species-specific and mating-type (MAT1-1 or MAT1-2) discrimination of OA and OY isolates. First, a multiplex PCR-based diagnostic assay targeting the MAT idiomorph region was developed allowing simultaneous determination of both species and mating type. This multiplex-PCR assay was successfully applied to type a global collection of isolates. Second, the development of loop-mediated isothermal amplification (LAMP) assays targeting beta-tubulin sequences is described, which allow fast (<9 min) species-specific discrimination of global OA and OY isolates. The LAMP assay can detect very small amounts of target DNA (1 pg) and was successfully applied in planta. In addition, mating-type specific LAMP assays were also developed for rapid (<12 min) genotyping of OA and OY isolates. Finally, the multiplex PCR-based diagnostic was applied, in conjunction with spore trapping in field experiments, to provide evidence of the wind dispersal of ascospores from a diseased crop. The results indicate an important role of the sexual cycle in the dispersal of eyespot.
Fungal pathogens pose an increasing threat to global food security through devastating effects on staple crops and contamination of food supplies with carcinogenic toxins. Widespread deployment of agricultural fungicides has increased crop yields but is driving increasingly frequent resistance to available agents and creating environmental reservoirs of drug-resistant fungi that can also infect susceptible human populations. To uncover non-cross-resistant modes of antifungal action, we leveraged the unique chemical properties of boron chemistry to synthesize novel 6-thiocarbamate benzoxaboroles with broad spectrum activity against diverse fungal plant pathogens. Through whole genome sequencing of Saccharomyces cerevisiae isolates selected for stable resistance to these compounds, we identified mutations in the protein prenylation-related genes, CDC43 and ERG20. Allele-swapping experiments confirmed that point mutations in CDC43, which encodes an essential catalytic subunit within geranylgeranyl transferase I (GGTase I) complex, were sufficient to confer resistance to the benzoxaboroles. Mutations in ERG20, which encodes an upstream farnesyl pyrophosphate synthase in the geranylgeranylation pathway, also conferred resistance. Consistent with impairment of protein prenylation, the compounds disrupted membrane localization of the classical geranylgeranylation substrate Cdc42. Guided by molecular docking predictions, which favored Cdc43 as the most likely direct target, we overexpressed and purified functional GGTase I complex to demonstrate direct binding of benzoxaboroles to it and concentration-dependent inhibition of its transferase activity. Further development of the boron-containing scaffold described here offers a promising path to the development of GGTase I inhibitors as a mechanistically distinct broad spectrum fungicide class with reduced potential for cross-resistance to antifungals in current use.
We collected 1017 and 1931 isolates of wheat eyespot pathogen from 555 fields in various regions of Hokkaido in 1989 and 1992, respectively. Of these, 52.3% in 1989 and 43.7% in 1992 were identified as Oculimacula acuformis based on mycelial growth and culture morphology. The others (47.7% in 1989 and 56.7% in 1992) were O. yallundae. The 2-year data showed that O. acuformis was widely distributed in Hokkaido and was the dominant species in the northern and eastern regions. The susceptibility of O. yallundae and O. acuformis to demethylation inhibitor (DMI) fungicides was tested. The EC50 values for propiconazole ranged from 0.16 to 0.32 ppm and 1.0 to 4.5 ppm, and those for prochloraz ranged from 0.03 to 0.09 ppm and 0.04 to 0.18 ppm in O. yallundae and O. acuformis, respectively. The 2-year results indicate that the susceptibility of O. acuformis to propiconazole was relatively low. Efficacy of propiconazole and prochloraz on wheat eyespot was investigated in the fields where the proportion of O. acuformis was different from 5.0 to 100%. Propiconazole was effective in the fields dominated by O. yallundae, whereas the effect was not observed in those dominated by O. acuformis. Prochloraz had a stable effect in all the fields tested.
The targeted use of fungicides can help minimise the yield losses caused to grain cereals by plant pathogenic fungi. However, the long-term use of fungicide classes based on a single mode of action has imposed consistent selection pressures on certain pathogens. Over time, this has resulted in many cases of fungicide resistance, leading to a reduction or the total loss of field performance of a range of active ingredients. There is a pressing need to develop anti-resistance management strategies. This chapter briefly reviews the development of resistance in cereal pathogens, before presenting and evaluating a variety of anti-resistance strategies. We include case studies of how data from field trials have helped to illustrate the impact of different control strategies.
Cereal stem base diseases occur in the Czech Republic every year. In Europe today, there is a trend towards more comprehensive strategies for growing and protecting crops with the aim of reducing risks to the environment and to health associated with pesticide residues. Precise knowledge as to the dynamics of causative agents of stem base disease may constitute an important part of the decision support system for cereal-growing strategies. Over 17 years (1999–2015), 482,219 plants were collected within the Czech Republic and visually inspected for stem base disease symptoms. An in vitro culture test was then conducted to determine the causative agents. During 2012–2015, the response of Oculimacula spp. and Microdochium spp. populations to prochloraz was tested. The most plants infected with Oculimacula spp. (>6 %) were detected in 2004, 2008, 2014 and 2015. The lowest infection rates were recorded in 2005, 2006, 2011 and 2012, when the level did not exceed 2 %. Microdochium spp. occurred most frequently in 2003, 2006 and 2013 (29–37 % incidence). Its lowest occurrence was recorded in 2014, when only 13 % of plants were infected. All isolates of Microdochium spp. were sensitive to prochloraz. Some isolates of Oculimacula spp. showed low to medium resistance (12 and 5, respectively, from 86 total isolates through the entire testing period). In all years, ED50 for the Oculimacula spp. population was ≤0.1 μg ml⁻¹. ED50 for Microdochium spp. ranged between 0.01 and 0.06 μg ml⁻¹.
To investigate the changes of the resistance to prochloraz of Fusarium species causing bakanae disease, Fusarium isolates were collected from various regions in Korea, and pathogenicity tests were performed using rice seeds. Minimum inhibitory concentration (MIC) and effective concentration of 50% () values of isolates were determined using the agar dilution method. High frequency distribution of MIC values of prochloraz against isolates collected in 2006~2007 and 2013~2014 years were and , respectively. The mean value of isolates increased from in 2006~2007 to in 2013~2014. Based on the value of isolates collected in 2006~2007, the resistant baseline of prochloraz was determined as . Compared with the ratio of resistant isolates in 2006~2007, the ratio of resistant isolates in 2013~2014 increased from 6.5% to 41.6%.
New N-(heterocyclo)phthalimides (III), (V) and (VII) are synthesized from corresponding phthalic anhydride derivatives and tested for their antifungal activities.
A total of 25 isolates of Fusarium fujikuroi were obtained from diseased rice plants in Korea from 2006 to 2007 to assess their resistance against fungicides prochloraz and benomyl + thiram. Minimal inhibitory concentration (MIC) values of F. fujikuroi isolates were examined by agar dilution method. Most of the isolates were sensitive to the fungicides. Out of 25 isolates, six were resistant to prochloraz and three to benomyl + thiram. In addition, the isolates CF245, CF249 and CF337 showed resistant to both fungicides. The progenies ( isolates) obtained through two different crosses between sensitive parental isolates(CF202, CF232 and CF179) and resistant parental isolate (CF337) were evaluated for their mycelial growth at different temperatures and resistance against fungicides. Mycelial growth rate of isolates originated from CF202 CF232 was similar to the parental isolates. However mycelial growth rate of isolates originated from CF179 CF337 was faster than their parent isolates. In case of prochloraz, distribution ratio of sensitivity(S) to resistance(R) against to the fungicide of isolates originated from CF202 CF232 and CF179 CF337 was 86 : 14 and 78 : 22, respectively. In case of benomyl+thiram, all the isolates originated from CF202 CF232 were sensitive to the fungicide, however ratio of sensitivity(S) to resistance(R) against to the fungicide of isolates originated from CF179 CF337 was 35 : 65.
Sexual crosses were used to determine the genetic basis of resistance to the sterol 14 α-demethylase inhibitor fungicide prochloraz
in the cereal eyespot pathogen Tapesia yallundae. Three different crosses between sensitive parental strains (22-432 and 22-433 [the concentration required to inhibit growth
by 50% {IG50} for each was ≤0.03 mg/liter]) and field isolates from France and New Zealand with differing levels of resistance (PR11 [IG50 = 0.5 mg/liter], PR1 [IG50 = 1.0 mg/liter], and 11-3-18 [IG50 = 2.4 mg/liter]) yielded progeny showing a bimodal distribution, with an even number of sensitive and resistant progeny.
This indicated the segregation of a single major gene for resistance in each cross, which was confirmed by the use of backcrosses,
crosses between F1 progeny, and control crosses between sensitive parents. However, there was also evidence of additional quantitative genetic
components responsible for the increased IG50s of the more resistant isolates. A further cross was made between isolate PR11 and an F1 progeny arising from isolate 11-3-18, and this also yielded progeny which were entirely prochloraz resistant. This suggested
that resistance genes were allelic in these two isolates, with resistance conferred by a gene at the same locus (or closely
linked loci), despite the fact that the isolates (PR11 and 11-3-18) originated from different continents.
Eyespot, caused by Oculimacula acuformis and Oculimacula yallundae, is the major foot disease of winter wheat in several European countries, including France. It can be controlled by chemical treatment between tillering and the second node stage. The fungicides used include antimicrotubule toxicants (benzimidazoles), inhibitors of sterol 14α-demethylation (DMIs) or of succinate dehydrogenase (SDHIs), the anilinopyrimidines cyprodinil and the benzophenone metrafenone. Since the early 1980s, a long-term survey has been set up in France to monitor changes in the sensitivity of eyespot populations to fungicides. Resistance to benzimidazoles has become generalised since the early 1990s, in spite of the withdrawal of this class of fungicides. In the DMI group, resistance to triazoles is generalised, whereas no resistance to the triazolinethione prothioconazole has yet developed. Resistance to the imidazole prochloraz evolved successively in O. acuformis and O. yallundae and is now well established. Specific resistance to cyprodinil has also been detected, but its frequency has generally remained low. Finally, since the early 2000s, a few strains of O. yallundae displaying multidrug resistance (MDR) have been detected. These strains display low levels of resistance to prothioconazole and SDHIs, such as boscalid. Knowledge of the spatiotemporal distribution in France of O. acuformis and O. yallundae field strains resistant to fungicides allows resistance management strategies for eyespot fungi in winter wheat to be proposed.© 2012 Society of Chemical Industry.
Eyespot disease caused by the soil-borne facultative fungi Oculimacula yallundae and O. acuformis is the major component of the stem-base disease complex of wheat in temperate regions of the world with a cool and wet climate. In this review, we focus on results of genetic studies concerning both partners of the host–pathogen interaction. This comprises analyses of genetic diversity of the pathogen and identification of particular genes within it, evaluation and screening methods for host resistance, resistance sources and genetics of these resistances, breeding of resistant cultivars in wheat, and application of genetic markers in tagging and tracking of eyespot resistance genes. We also attempt to foresee some of the key issues and developments that may occur in future. The identification of markers tightly linked to eyespot resistance genes is the important research focus opening the door to marker-assisted selection of resistant varieties.
Cereal eyespot fungi Tapesia acuformis and Tapesia yallundae are closely related species which show different behaviours upon treatment with sterol 14-demethylase inhibitors (DMIs). T. acuformis is naturally resistant to DMIs belonging to the triazole family and susceptible to the imidazole ones, whilst T. yallundae is sensitive to both inhibitors. Cloning of the target enzyme gene, CYP51, from the two species revealed an important polymorphism between them. Further sequencing of CYP51 from sixteen T. acuformis and eleven T. yallundae strains with different phenotypes with regards to resistance to DMIs confirmed that at least eleven variations are species related. Among them, a conserved phenylalanine residue at position 180, found both in T. yallundae and in all known CYP51 proteins from filamentous fungi and yeast, was replaced in T. acuformis by a leucine. Therefore, a leucine at 180 could be possibly involved in natural resistance of T. acuformis to triazoles. Other mutations were observed in some resistant strains, sometimes simultaneously, but in contrast to what was reported for other filamentous fungi, where a mutation at the 136 position of the CYP51 gene product seemed to correlate with resistance to DMIs, we did not find a clear relationship between a given mutation and a particular phenotype. This result suggests that resistance to DMIs could have a polygenic nature in Tapesia. We took advantage of species-related variations to develop a PCR-based assay allowing rapid and easy discrimination between field strains of the two species.
Eyespot pathogens, Tapesia yallundae and Tapesia acuformis, were isolated from two trial sites in the UK over several years. Both sites were treated with 2 applications per year of cyprodinil (a new anilinopyrimidine fungicide), prochloraz and a mixture of cyprodinil with prochloraz. One trial site was exposed to cyprodinil for 3 years, and the second for a total of 11 years, including 5 years before the trial was initiated. Control of eyespot and sensitivity to cyprodinil were monitored. During the first 3 years of the trial, disease control with all fungicide treatments ranged from 43% to 82%. At the site, where the trial was extended for a further 3 years, control then began to decline but no practical resistance was detected. The decline in control by both fungicides suggests that factors other than reduced sensitivity might be involved. Field isolates of both T. yallundae and T. acuformis with reduced sensitivity to cyprodinil were found predominantly in plots treated with cyprodinil. A reduction in sensitivity to cyprodinil was identified in the population from cyprodinil-treated plots in two years out of six, and in the population from mixture plots in the final year. No obvious trends could be identified and in-vivo studies showed control of most isolates with reduced sensitivity could be regained by increasing the dose to one tenth of the recommended field rate. Analysis of progeny from sexual crosses involving a sensitive isolate and a field isolate with an ED50 value higher than the baseline sensitivity range indicated that a single gene controlled the reduction in sensitivity to cyprodinil in one T. yallundae isolate. There is clearly a resistance risk in eyespot to cyprodinil. The reduction in sensitivity is monogenic in inheritance and at a significant level in some isolates, but any shift in sensitivity in field populations has so far been gradual.
Seven phenotypes were identified among field isolates of Tapesia yallundae and Tapesia acuformis when tested for susceptibility to the benzimidazole fungicides carbendazim and thiabendazole and the N-phenylcarbamates diethofencarb, MDPC, and swep. PCR was used to amplify and sequence 627-bp fragments of the β-tubulin gene from 32 Tapesia spp. strains representing the seven field phenotypes and from six T. yallundae laboratory mutants. All benzimidazole-resistant field isolates analyzed had a punctual allelic mutation at codon 198, 200, or 240 of the β-tubulin gene fragment. Codon 198, which encodes glutamic acid in benzimidazole-sensitive strains (resistant to N-phenylcarbamates), was converted to a codon for alanine, glycine, lysine, or glutamine in benzimidazole-resistant strains exhibiting increased sensitivity toward the N-chlorophenylcarbamates MDPC and swep; the first two allelic mutations (alanine and glycine) also conferred susceptibility to diethofencarb. In T. yallundae, benzimidazole-resistant phenotypes, which remained resistant to all the tested N-phenylcarbamates, had a tyrosine instead of a phenylalanine at codon 200 or a phenylalanine instead of a leucine at codon 240. In T. acuformis, however, the change of a phenylalanine at codon 200 for a tyrosine conferred a weaker susceptibility to MDPC and swep as well as a reduced resistance to benzimidazoles compared to their T. yallundae counterparts. The same molecular analysis was performed with T. yallundae laboratory mutants obtained after UV mutagenesis and selection on carbendazim or diethofencarb of a former benzimidazole-sensitive or benzimidazole-resistant field strain. We found in two mutants a punctual change at codon 198, replacing the glutamic acid by a glycine or an aspartic acid, but multiple mutations were observed in the four other mutant strains: a double mutation in codon 198 resulting in the substitution of the glutamic acid by a threonine; a mutation at codon 198 (an alanine instead of a glutamic acid) and a mutation at codon 200 (a serine instead of a phenylalanine); a mutation at codon 198 (an alanine instead of a glutamic acid) and a mutation at codon 250 (a phenylalanine instead of a leucine); and one mutant had four codon changes: at codon 179 (a glycine substituting a valine), at codon 185 (a serine replacing an alanine), at codon 190 (an asparagine replacing a histidine), and at codon 198 (an alanine instead of a glutamic acid). We show here that each different phenotype could be correlated with particular mutations at the β-tubulin gene level.
Eyespot is an important stem-base disease of cereal crops in temperate regions. Cultural, genetic and molecular criteria have been used to separate the fungi responsible into two species, Tapesia yallundae (previously W-type) and T. acuformis (previously R-type). Discovery of the apothecial sexual stage (teleomorph) of Tapesia yallundae on straw stubble was a key factor in this change for a pathogen previously believed to be asexual (anamorph: Pseudocercosporella herpotrichoides). Sexual reproduction is controlled by a two-allele heterothallic system in both species, although mating appears to be rare in T. acuformis. Infection of cereal hosts is achieved by formation of multicellular plaques, and the colonization process is described. The host range includes wild grass species as well as small-grain cereals, and new genetic sources of resistance to the disease have been identified in Triticum species and wild relatives. Recent advances in the molecular genetics of the pathogens will aid analysis of pathogenic variation in eyespot.
Sterol 14α-demethylation inhibitors (DMIs) have been widely used in many European countries to control septoria leaf blotch, which is caused by Mycosphaerella graminicola (Fückel) J Schrot (anamorph Septoria tritici Berk & MA Curtis). However, treatment efficacy has declined, and significant shifts in population susceptibility have occurred in recent years, with the isolation of particularly highly resistant strains from French, English and Irish populations. The present aim was to determine the phenotypic characteristics of these field isolates and to identify the possible resistance mechanisms.
Target alteration, linked to 11 possible changes in the gene encoding 14α-demethylase (Cyp51), was the basic resistance mechanism in weakly, moderately and highly resistant strains. Changes in Cyp51 combined with the overexpression of drug efflux transporters probably result in multidrug resistance in some of the most resistant phenotypes. Finally, some moderately or highly resistant isolates were found to harbour an insertion in the Cyp51 promoter and/or new combinations of known mutations in the target gene.
An updated overview of M. graminicola field strains displaying low to high resistance to DMIs is provided here. The management of field resistance and efficacy should be adapted to take these findings into account.
This review considers factors affecting the coexistence of closely related pathogen species on arable crops, with particular reference to data available at Rothamsted for Septoria tritici/Stagonospora nodorum (Mycosphaerella graminicola/Phaeosphaeria nodorum) (septoria leaf blotch diseases on winter wheat), Oculimacula yallundae/O. acuformis (eyespot disease of winter cereals), and Leptosphaeria maculans/L. biglobosa (phoma stem canker on winter oilseed rape). Factors affecting the short-term, medium-term, and long-term coexistence of such related pathogen species are reviewed, and their evolution from common ancestors considered. Small niche differences between the related pathogen species enable them to coexist on the same host. The niche differences result from small differences in their biology/epidemiology, leading to separation in space, time, or resource use. Changes in both natural (e.g., fluctuating temperature) and man-made (e.g., agronomic practices, pollution) factors influence the coexistence. Such factors may result in coexistence between the related species in some parts of the world, whereas in other parts only one species occurs. These principles illustrated with pathogens of arable crops are generic to other host-pathogen systems.
Control of diseases caused by fungi in both medicine and agriculture is heavily dependent on the use of triazoles. As a consequence, resistance to triazoles is a threat to both human health and the sustainability of agricultural production systems. In human pathogens, particularly Candida albicans, mutations encoding alterations in the target cytochrome P450 sterol 14alpha-demethylase (CYP51; where CYP is cytochrome P450) enzyme are the primary determinants of triazole resistance. In fungal pathogens of cereals, CYP51A1 modifications, some at positions known to contribute to a resistant phenotype in human pathogens, have also been identified in isolates with altered triazole-sensitivity. However, unlike medicine where resistance to triazoles is a major clinical problem, failures of triazoles to control crop diseases in the field are rare with mean population sensitivities generally remaining low, perhaps due to differences in the selection pressures imposed on human and cereal pathogen populations. Nonetheless, the biological potential for resistance exists, and the question remains as to whether widespread triazole resistance can develop in an important cereal pathogen.
Pseudocercosporella herpotrichoides, l'agent du piétin-verse des céréales, présente une variabilité intraspécifique mise en évidence par plusieurs critères : pouvoir pathogène, morphologie et physiologie in vitro, profils protéiques et enzymatiques, sensibilité aux fongicides. Une synthèse des données est réalisée concernant ces différents critères. L'ensemble de ces caractères permet la différentiation de 2 groupes de souches au sein de l'espèce: souches à croissance normale ou type W ou P h var herpotrichoides d'une part, souches à croissance lente ou type R ou P h var acuformis d'autre part. La variabilité à l'intérieur de ces 2 populations est également importante si l'on se base sur ces mêmes critères d'étude. Les divers aspects de la variabilité de l'agent pathogène sont développés de manière à montrer leur intérêt dans la lutte contre cette maladie, tant au niveau de l'utilisation raisonnée de fongicides qu'à celui de la recherche de sources de résistance. Variability of Pseudocercosporella herpotrichoides (Fron) Deighton, the cause of eyespot in cereals: a review. Pseudocercosporella herpotrichoides, the cause of eyespot in cereal crops, shows variability characterized on the basis of pathogenicity, cultural morphology, physiology, protein and isozyme patterns and sensitivity to fungicides. The species herpotrichoides comprises 2 main pathotypes distinguished by these criteria: fast growing colonies (I or N) correlated with W-type and P h var herpotrichoides; slow growing colonies (II or L) correlated with R-type and P h var acuformis. Growth rate (on agar medium), colony morphology and pigmentation, conidial form allow differentiation into 2 groups. Nevertheless, there are considerable differences between individual isolates in both groups and these criteria do not fully confirm the distinction made on the basis of pathogenicity. The first virulence group is characterised by the W type isolates: their pathogenicity is high to wheat and barley, and is low to rye. The second group is characterized by the R type isolates, which are equally pathogenic to wheat, barley and rye. A type is also described which is pathogenic to couch grass. Apart from these types, 1 type was observed which is pathogenic only to wheat and Aegilops squarrosa. All types are pathogenic to Aegilops ventricosa. There are considerable differences in pathogenicity between individual isolates in both the W and the R type groups. Differences in controlled environment conditions may explain the varying results in the studies on pathogenicity of both types. The penetration of the stem by the W type (or N, or var herpotrichoides) occurs earlier than that by the R type (or L, or var acuformis), which are isolated more frequently at the end of the season. Both types can exist in the same plant or even on the same lesion; interactions between both types can influence the development of eyespot. Electrophoretic studies show that specific patterns (proteins, isozymes) exist for the W type, N type and var herpotrichoides, and also for the R type, L type and var acuformis. Contrary to the former characteristics, high homogeneity is observed within the same type. P herpotrichoides isolates can be clearly and more objectively differentiated by biochemical markers. The development of resistance to fungicides has induced different phenotypes which react in a very specific way to 1 type of compound. Sensitive or resistant isolates to benzimidazole, thiophanate and phenylcarbamate compounds are common in both types. Three types are distinguished according to their sensitivity to ergosterol biosynthesis inhibitors: la or Na which are sensitive to DMI, Ib or Nb and II or L which are less sensitive to DMI. Recently, several prochloraz resistant strains (II or L type) have been isolated on winter wheat in France. These different aspects of the variability of the fungus are discussed to show their importance in eyespot control both for the optimal use of fungicides and for research into host resistance sources. The perfect state in which P herpotrichoides is found, Tapesia yallundae, has only recently been described and its importance is not yet known. It provides a new means for the study of the relationships between isolates types.
This review considers the effect of various fungicides, especially synthetic strobilurins, on fungal respiration, discusses recent contributions of biochemistry to the anilinopyrimidines and the phenylpyrroles and also discusses the future development of chemical inducers of systemic acquired resistance in host plants.
Strains of Pseudocercosporella herpotrichoides collected in France on winter wheat give either fast‐growing mycelial colonies with regular margins or slow‐growing mycelial colonies with irregular margins. Most of the fastgrowing isolates were sensitive to triadimenol (EC 50 below 2mg litre ⁻¹ ), but some of them were resistant to this inhibitor of sterol C‐14 demethylation. In contrast, all the slow‐growing strains were highly resistant to triadimenol (EC 50 greater than 100 mg litre ⁻¹ ). This resistance was also expressed in inhibition of germ‐tube elongation. Positive cross‐resistance was observed between most of the inhibitors of sterol C‐14 demethylation, with the exception of some imidazole derivatives (clotrimazole, prochloraz). All the fast‐growing strains were tolerant to fenpropimorph and fenpropidin whereas the slow‐growing ones were susceptible; the reverse was true with piperalin and tridemorph. All the field isolates were inhibited to the same extent by the inhibitors of squalene‐epoxidase, nafifine and terbinafine.
Two types of mutant resistant to triadimenol have been induced under laboratory conditions from sensitive fast‐growing strains. The most common mutants were resistant to all the inhibitors of sterol C–14 demethylation and also in some conditions to fenpropimorph, tridemorph and the inhibitors of squalene‐epoxidase. The other mutants were characterised by a reduced spectrum of cross‐resistance between triadimenol and the other inhibitors of sterol biosynthesis.
The field isolates and laboratory mutants resistant to triadimenol and propiconazole were also resistant to each of the four enantiomers of these two fungicides.
Cyprodinil and fludioxonil are two new active ingredients for the control of Botrytis cinerea, representing the two novel fungicide classes anilinopyrimidines and phenylpyrroles, respectively, with two new and different modes of action. While cyprodinil seems to interfere with the synthesis of certain amino acids, the mode of action of fludioxonil is less well understood. To help design appropriate use recommendations, the risk for resistance in B. cinerea against these two agents was assessed by establishing baseline sensitivities and by monitoring sensitivity of Botrytis to these two fungicides at various trial sites over a number of years. Monitoring was carried out at three sites in France and at one site in Switzerland where phenylpyrroles and anilinopyrimidines were applied over five years in longterm strategy trials, either twice per season in mixture or once each as a single product in alternation. An additional site in Switzerland was monitored where anilinoyrimidines and phenylpyrroles had been applied three to four times per year in development trials starting in 1986. Monitoring with fludioxonil at all five trials sites over five years included 279 isolates from untreated and 702 isolates from treated plots. This monitoring did not show any changes in sensitivity to fludioxonil. Monitoring with cyprodinil over four years in the four long-term strategy trials included 173 isolates from untreated and 482 isolates from treated plots. It revealed two isolates from France with reduced sensitivity in vitro and in vivo, without however affecting performance of the products. At the site in Switzerland where the development trials were carried out, a higher proportion of isolates with reduced sensitivity to anilinopyrimidines began to appear in 1993, which led to a reduced performance of anilinopyrimidines applied alone in one trial. However, Botrytis control by a mixture of cyprodinil with fludioxonil remained excellent and superior to the standards also in 1994. No cross-resistance exists between cyprodinil and fludioxonil. Also, strains with reduced sensitivity to the market products carbendazim, diethofencarb or vinclozolin showed no cross-resistance to fludioxonil. Other studies had already shown, that cross-resistance exists between cyprodinil and the other two anilinopyrimidines, mepanipyrim and pyrimethanil, but not between anilinopyrimidines and the market products mentioned above. The results of these comprehensive studies form the basis for the use strategy proposed by CIBA for the use of cyprodinil and fludioxonil against Botrytis. A prepacked mixture of the two new active ingredients is being developed and will be introduced worldwide for the control of Botrytis in grapes and vegetables.
CGA 219417 (proposed common name: cyprodinil) is a pyrimidinamine currently being developed as a foliar fungicide for cereals, grapes, vegetable crops and deciduous fruit. It has a new mode of action and shows no cross-resistance to any current market product. On cereals, CGA 219417 exhibits a broad fungicidal spectrum with good activity against Pseudocercosporella herpotrichoides, Erysiphe graminis, Pyrenophora teres, Rhynchosporium secalis and Septoria nodorum on ears. The strength of CGA 219417 on grapes, vegetable crops and strawberries is its excellent activity against Botrytis spp. On deciduous fruit the key target pathogen genera are Venturia, Alternaria and Monilinia spp. The fungicide has excellent safety for users, consumers and environment. In addition, its other characteristics, such as not being harmful to beneficial organisms, make it a promising product for flexible integrated disease-control practices.
A partir des isolats de P. herpotrichoides collectes en France, sur ble d'hiver de 1984 a 1987, il a ete possible de caracteriser 3 types de souches en fonction de leur sensibilite a des inhibiteurs de la biosynthese des sterols. Les differences s'observent notamment en presence de fenpropimorphe et d'inhibiteurs de la C14-demethylation des sterols comportant l'heterocycle triazole (cyproconazole, flusilazol, hexaconazole, penconazole, tebuconazole, triadimenol, …). Quant au prochloraze, il inhibe fortement tous les isolats de P. herpotrichoides. […]
Des souches de P herpotrichoides résistantes au prochloraze ont été isolées pour la première fois en France au cours de l'année 1990 sur des tiges de blé prélevées dans 3 parcelles ayant reçu au moins un traitement à base de prochloraze. Les niveaux de résistance de ces nouveaux phénotypes qui sont tous caractérisés par une croissance mycélienne lente sur milieu nutritif gélosé, sont de l'ordre de 20-30. Ces nouvelles souches peuvent présenter une sensibilité réduite (ex: imazalil, flusilazole, flutriafol, propiconazole, tébuconazole) ou accrue (ex : triflumizole, fénarimol, cyproconazole, hexaconazole, triadiménol) vis-à-vis d'autres inhibiteurs de la C-14 déméthylation des stérols, ceci comparativement aux souches à croissance lente isolées antérieurement. À la différence de mutants induits en laboratoire et résistants au prochloraze, ces souches naturelles ne présentent pas une sensibilité réduite vis-à-vis de la terbinafine, un inhibiteur de la squalène-époxydase. Les conséquences du développement au champ de tels phénotypes de P herpotrichoides résistants au prochloraze sont discutées. Resistance to prochloraz in strains of the cereal eyespot fungus Pseudocercosporella herpotrichoides isolated from winter wheat in France. Several prochloraz-resistant strains of P herpotrichoides were isolated for the first time in France during the year 1990 on winter wheat stems taken from 3 separate plots which had been previously treated with prochloraz. At sites 1 and 2 in the North of France the efficiency of prochloraz was found to be reduced. At site 3 where a long-term trial was taking place, prochloraz was found to be highly effective (table II). The resistance levels of these new phenotypes (Ilp-type), which all produced slow-growing colonies (II-or R-type), were = 20-30 in tests on mycelial growth or germ tube elongation (tables I and II; figs 1 and 2). Compared to the slow-growing strains isolated previously (Ils-type), the new strains were more tolerant towards imazalil, flusilazol, flutriafol, propiconazole and tebuconazole, more sensitive towards triflumizol, fenarimol, cyproconazole, hexaconazole and triadimenol, and equally sensitive towards clotrimazole, dichlobutrazol and diniconazole (table II). These fungicides, like prochloraz, are inhibitors of sterol C-14 demethylation. The prochloraz-resistant strains did not exhibit cross-resistance towards terbinafine and fenpropimorph which respectively inhibit the squalene-epoxidase and the sterol Δ14 reductase or Δ8 -> Δ7 isomerase (tables IV and V). The practical consequences of the development of such prochloraz-resistant strains of P herpotrichoides in winter wheat are discussed.
Agent du pie tin-verse des ce re ales : e volution des populations et re sultats de huit anne es dÏessais multilocaux
- P A Lardier
- N Cavelier
Lardier, P. A. & Cavelier, N., Agent du pie tin-verse des ce re ales : e volution des populations et re sultats de huit anne es dÏessais multilocaux. Phytoma, 491 (1997) 16È22.
Immu-nodiagnostic assay for cereal eyespot : novel technology for disease detection
- C M Smith
- D W Saunders
- D A Allison
- L E B Johson
- B Labit
- S J Kendall
- D W Hollomon
Smith, C. M., Saunders, D. W., Allison, D. A., Johson, L. E. B., Labit, B., Kendall, S. J. & Hollomon, D. W., Immu-nodiagnostic assay for cereal eyespot : novel technology for disease detection. Proc. Brighton Crop Prot. Conf.È Pests and Diseases, 2 (1990) 763È70.
Me thode dÏessais dÏefficacite au champ de produits fongicides destine s a` combattre lÏagent du pie tin-verse des ce re ales : Pseudocercosporella herpotrichoides : Me thode C.E.B no. 64. Association Nationale pour la Protection des Plantes
- Anon
Anon., Me thode dÏessais dÏefficacite au champ de produits fongicides destine s a` combattre lÏagent du pie tin-verse des ce re ales : Pseudocercosporella herpotrichoides : Me thode C.E.B no. 64. Association Nationale pour la Protection des Plantes, Paris, 1986, 13 pp.
Pie tin-verse des ce re alesÈcartographie 1993 : De Ðni-tion dÏun risque re gional enmatiè re de type de souches
- J L Migeon
- M P Mathon
- A M Chudzicki
- J P Leroy
Migeon, J. L., Mathon, M. P., Chudzicki, A. M. & Leroy, J. P., Pie tin-verse des ce re alesÈcartographie 1993 : De Ðni-tion dÏun risque re gional enmatiè re de type de souches. Phytoma, 485 (1994) 24È6.
Interaction of the anilinopyrimidine fungicide pyrimethanil with amino acids and sulfur-containing metabolites in Botrytis cinerea
- P Leroux
- V Colas
- R Fritz
- C Lanen
Leroux, P., Colas, V., Fritz, R. & Lanen, C., Interaction of the anilinopyrimidine fungicide pyrimethanil with amino acids and sulfur-containing metabolites in Botrytis cinerea. T hird International symposium ReinhardsbrunnÈ Modern fungicides and Antifungal compounds, ed. H. Lyr, P. E. Russel & H. D. Sisler. Intercept, Andover, U.S.A. 1996, pp. 61È7.