Biochemical Mechanisms Involved in Selective Fungitoxicity of Two Sterol 14α-Demethylation Inhibitors,Prochloraz and Quinconazole: Accumulation and Metabolism Studies

The selective fungitoxic actions of prochloraz (an imidazole) and a triazole fungicide, quinconazole (3-(2,4-dichlorophenyl)-2-(1 H- 1,2,4-triazol-1-yl)- 4(3H)-quinazolinone: II ), were studied with selected phytopathogenic fungi. With the exception of Ustilago maydis, all the fungi tested were more sensitive to prochloraz than to II. A number of DMI-resistant mutants of Penicillium digitatum and P. italicum showed positive cross-resistance to both DMIs, but except for P. italicum isolate H17, the levels of resistance to II were much higher than to prochloraz. The generally higher toxicity of prochloraz to the fungi investigated, as compared to II , could not be ascribed to the slightly higher accumulation of prochloraz. With regard to prochloraz, there was no general correlation between the sensitivity of the fungi tested and the amount of fungicide accumulated. A similar situation was evident for II. However, the DMI-resistant mutants of P. italicum did show a reduced accumulation of both azoles, which may account for a low level of acquired DMI-resistance in this fungus. Since accumulation levels of the test compounds in the isolates with different degrees of resistance were the same, additional mechanisms of resistance may be involved in isolates with relatively high degrees of DMI-resistance. No detectable amounts of fungicide metabolites were found in most fungi tested over a 16-hour incubation period. Therefore, fungal metabolism is not generally responsible for the differences in sensitivity between fungi to each azole tested. It also does not generally explain the differential toxicities of prochloraz and II to each individual species. The exception to this was Rhizoctonia solani which metabolized prochloraz to a non-fungitoxic compound. This correlated with its low prochloraz sensitivity.     

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

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

Effects of Infection by Mycosphaerella graminicola on Translocation of Fluquinconazole in Wheat Seedlings

ABSTRACT Translocation of (14)C-labeled fluquinconazole was measured using combustion analysis and radio thin-layer-chromatographic analysis in seedling wheat leaves uninfected and infected with Mycosphaerella graminicola. Two isolates were used with differing sensitivity to demethylation inhibitor fungicides. Fluquinconazole was translocated acropetally, but not basipetally. Fluquinconazole accumulated around infection sites within 6 days after treatment. Accumulation occurred before M. graminicola hyphae had colonized the host mesophyll further than one host cell around the invasion site. This suggested that the accumulation was caused by a host response to infection. Infrared gas analysis showed that rates of transpiration and stomatal conductance in inoculated leaves were significantly increased very soon after inoculation but net photosynthesis was decreased. The actual mechanism of fungicide accumulation was not determined.     

Overexpression of the sterol 14α-demethylase gene (MgCYP51) in Mycosphaerella graminicola isolates confers a novel azole fungicide sensitivity phenotype

BACKGROUND: The recent evolution towards resistance to azole fungicides in European populations of the wheat pathogen Mycosphaerella graminicola has been caused by the progressive accumulation of mutations in MgCYP51 gene, encoding the azole target sterol 14α‐demethylase. Particular combinations of mutations have been shown specifically to affect the interaction of the MgCYP51 protein with different members of the azole class. Although additional mechanisms, including increased MgCYP51 expression and enhanced active efflux, have been proposed, the genetic changes underlying these mechanisms are unknown. RESULTS: Analysis of the azole sensitivities of recent M. graminicola isolates identified a novel phenotype, seemingly independent of changes in MgCYP51 coding sequence. Characterised by a 7‐16‐fold reduction in in vitro sensitivity to all azoles tested and by growth on seedlings at higher doses of azoles in glasshouse tests compared with isolates carrying the same MgCYP51 variant (L50S, S188N, I381V, ΔY459/G460, N513K), isolates with this phenotype constitutively overexpress MgCYP51 by between 10‐ and 40‐fold compared with the wild type. Analysis of sequences upstream of the predicted MgCYP51 translation start codon identified a novel 120 bp indel, considered to be an insertion, in isolates overexpressing MgCYP51. CONCLUSIONS: The identification of an insertion in the predicted MgCYP51 promoter in azole‐resistant isolates overexpressing MgCYP51 is the first report of a genetic mechanism, other than changes in target‐site coding sequence, affecting sensitivity to multiple azoles in field isolates of M. graminicola. The identification of recent isolates overexpressing MgCYP51 confirms the ongoing evolution and diversification of resistance mechanisms in European populations of M. graminicola. Copyright © 2012 Society of Chemical Industry     

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.     

Genetic control of the resistance of Erysiphe graminis f.sp. hordei to five triazole fungicides

The genetics of resistance to ergosterol demethylation inhibitor (DMI) fungicides of the triazole (conazole) group was examined in a cross between two isolates of the barley powdery mildew fungus, Erysiphe graminis (=  Blumeria graminis ) f.sp. hordei . One isolate, E1, was previously identified as being resistant to the triazole fungicide triadimenol, while the other, HL3/5, was sensitive. The 56 progeny tested were classified into two distinct groups, either being resistant to triadimenol, like E1, or sensitive, like HL3/5. The segregation ratio was not significantly different from 1:1, consistent with responses to triadimenol being controlled by a single gene. In further tests with cyproconazole, epoxiconazole, propiconazole and tebuconazole, all the progeny classified as resistant to triadimenol were also more resistant to each of these other triazole fungicides than were any of the triadimenol-sensitive progeny. This is consistent with the triadimenol resistance allele also conferring cross-resistance to the other triazoles. The ratio between the responses of the resistant and sensitive progeny (the resistance factor, RF) was greatest for triadimenol, followed by tebuconazole, propiconazole, epoxiconazole and cyproconazole, in that order. The RF for triadimenol was much greater when the fungicide was applied as a seed treatment than when it was sprayed. Five isolates, covering the five levels of responses to triadimenol identified previously in the UK population of E. graminis f.sp. hordei , were used as standards; a triadimenol-sensitive isolate and one with the lowest level of resistance were sensitive to all four of the other fungicides, while three isolates with higher levels of triadimenol-resistance were also more resistant to the other chemicals.     

2011年我国主要麦区小麦白粉病菌群体对三唑酮和苯锈啶的敏感性

 小麦白粉病(Wheat Powdery Mildew)是我国小麦生产上常发性病害之一^[1]。小麦白粉病的防治主要采用抗病品种和化学药剂,辅之以栽培措施的综合防治技术。由于目前生产上抗病品种相对缺乏,药剂防治成为我国小麦白粉病防治的主要措施之一^[2]。自20世纪80年代以来三唑类杀菌剂一直是我国防治小麦白粉病的主要药剂,由于长期、大范围、单一的使用导致小麦白粉病菌对三唑类杀菌剂的抗药性大大提高。监测结果表明,2009年我国小麦白粉病菌群体对三唑酮的平均抗性水平已经达到56.58倍,抗性频率达到99.09%,其中高抗菌株占49.09%^[3]。目前小麦白粉病菌对三唑酮的抗性形势十分严峻,寻找三唑类杀菌剂的替代药剂成为控制该病害的迫切需求。     

Detection of Thiophanate-methyl-resistant Strains in Diplocarpon mail, Causal Fungus of Apple Blotch

Isolates of Diplocarpon mali, causal fungus of apple blotch, collected from four prefectures in Japan in 1997–1998 were tested for sensitivity to thiophanate-methyl. Results from mycelial growth tests showed that MIC values of the fungicide were 0.19 μg/ml against all isolates from Akita, Nagano and Saga prefectures but 100 or 200 μg/ml against all isolates from the Tokusa area in Yamaguchi prefecture. Detached apple leaves sprayed with the fungicide developed severe symptoms when inoculated with the isolate from Tokusa, but developed no symptoms with the isolate from Nagano. These results are the first confirmation of thiophanate-methyl-resistant strains in D. mali. Received 1 September 1999/ Accepted in revised form 15 October 1999     

Identification of isolate-specific and partial resistance to septoria tritici blotch in 238 European wheat cultivars and breeding lines

From a total of 238 European cultivars and breeding lines screened for isolate-specific resistance to septoria tritici blotch (STB) with eight Mycosphaerella graminicola isolates from five different countries, 142 lines were resistant to Ethiopian isolate IPO88004, and 43 lines were specifically resistant to IPO323, with little or no leaf area bearing pycnidia of M. graminicola . These lines probably all have the resistance gene Stb6 . Specific resistances to isolates CA30JI, IPO001, IPO89011, IPO92006 and ISR398 were less common. Seventy-three per cent of the lines were specifically resistant to at least one isolate and 36 lines were resistant to more than one isolate. The line with the greatest number of specific resistances was the spring cultivar Raffles, with five. The most resistant line in which no specific resistance was identified was the Italian landrace Rieti, an ancestor of many modern European wheat cultivars. There was also a wide range of partial resistance among the lines tested, expressed in detached seedling leaves. Information about the resistance of wheat lines to M. graminicola isolates will assist breeders to choose parents of crosses from which progeny with superior resistance to STB may be selected.     

Resistance to thiabendazole in isolates of Helminthosporium solani, the cause of silver scurf disease of potatoes

Isolates of Helminthosporium solani were obtained from potato tubers and cultured on agar containing thiabendazole. Some grew on agar containing 100 mg/l and these were classified as resistant, whereas sensitive isolates produced no mycelial growth on agar containing 5 mg/l. Isolates were also found that were intermediate in their sensitivity to the fungicide. All isolates from Scottish virus tested stem cutting stocks (VTSC) on two farms were resistant whereas those from another farm were sensitive to the fungicide. The proportion of resistant isolates from eight English once-grown seed stocks ranged from 0 to 96%. There has been no previous report of fungicide resistance in H. solani and the possible reasons for these results and their implications are discussed.     

Cloning and expression analysis of the ATP-binding cassette transporter gene MFABC1 and the alternative oxidase gene MfAOX1 from Monilinia fructicola

Brown rot, caused by Moniliniafructicola (G Wint) Honey, is a serious disease of peach in all commercial peach production areas in the USA, including South Carolina where it has been primarily controlled by pre-harvest application of 14-alpha demethylation (DMI) fungicides for more than 15 years. Recently, the Qo fungicide azoxystrobin was registered for brown rot control and is currently being investigated for its potential as a DMI fungicide rotation partner because of its different mode of action. In an effort to investigate molecular mechanisms of DMI and Qo fungicide resistance in M fructicola, the ABC transporter gene MfABC1 and the alternative oxidase gene MfAOX1 were cloned to study their potential role in conferring fungicide resistance. The MfABC1 gene was 4380 bp in length and contained one intron of 71 bp. The gene revealed high amino acid homologies with atrB from Aspergillus nidulans (Eidam) Winter, an ABC transporter conferring resistance to many fungicides, including DMI fungicides. MfABC1 gene expression was induced after myclobutanil and propiconazole treatment in isolates with low sensitivity to the same fungicides, and in an isolate with high sensitivity to propiconazole. The results suggest that the MfABC1 gene may be a DMI fungicide resistance determinant in M fructicola. The alternative oxidase gene MfAOX1 from M fructicola was cloned and gene expression was analyzed. The MfAOX1 gene was 1077 bp in length and contained two introns of 54 and 67 bp. The amino acid sequence was 63.8, 63.8 and 57.7% identical to alternative oxidases from Venturia inaequalis (Cooke) Winter, Aspergillus niger van Teighem and A nidulans, respectively. MfAOX1 expression in some but not all M fructicola isolates was induced in mycelia treated with azoxystrobin. Azoxystrobin at 2 microg ml(-1) significantly induced MfAOX1 expression in isolates with low MfAOX1 constitutive expression levels.     

Fungicide control of mushroom cobweb disease caused by Cladobotryum strains with different benzimidazole resistance profiles

The benzimidazole fungicides thiabendazole and carbendazim, and the imidazole fungicide prochloraz-Mn, were tested for their efficacy in controlling cobweb disease of mushrooms caused by two Cladobotryum isolates. Isolate 202A was benzimidazole-sensitive in vitro and cobweb growth on the casing was well controlled by both benzimidazole fungicides in cropping experiments. Carbendazim also controlled the development of spotting symptoms much more effectively than thiabendazole. A second isolate (192B1) was benzimidazole-resistant and was highly resistant to thiabendazole in vitro but it showed some sensitivity to carbendazim in vitro at moderate to high concentrations. Despite this, carbendazim did not control disease symptoms in cropping experiments, confirming that isolate 192B1 is cross-resistant to other benzimidazole fungicides. Both isolates showed some sensitivity to prochloraz-Mn in vitro. This fungicide gave between 45% and 65% control of cobweb growth on the casing caused by either 202A or 192B1 but gave no control of spotting symptoms. Reducing the fungicide application volume did not give enhanced disease control. The emergence of benzimidazole resistance reduces the value of benzimidazoles in the control of mushroom pathogens. However, the lack of effective alternatives means they continue to have utility in cases where pathogens are still sensitive but this requires regular monitoring of pathogen populations for resistance.     

Adaptation to fungicides in Monilinia fructicola isolates with different fungicide resistance phenotypes

The ability to develop fungicide resistance was assessed in Monilinia fructicola isolates with different fungicide sensitivity phenotypes by adapting mycelium and conidia to increasing concentrations of selective fungicides and UV mutagenesis. Results showed that adaptation to Quinone outside inhibitor (QoI) fungicide azoxystrobin and sterol demethylation inhibitor (DMI) fungicide propiconazole was more effective in conidial-transfer experiments compared to mycelial-transfer experiments. DMI-resistant (DMI-R) isolates adapted to significantly higher doses of azoxystrobin in both, mycelial- and conidial-transfer experiments compared to benzimidazole-resistant (BZI-R) and sensitive (S) isolates. Adaptation to propiconazole in conidial-transfer experiments was accelerated in BZI-R isolates when a stable, nonlethal dose of 50 microg/ml thiophanate-methyl was added to the selection medium. One of two azoxystrobin-resistant mutants from DMI-R isolates did not show any fitness penalties; the other isolate expired before further tests could be carried out. The viable mutant caused larger lesions on detached peach fruit sprayed with azoxystrobin compared to the parental isolate. The azoxystrobin sensitivity of the viable mutant returned to baseline levels after the mutant was transferred to unamended medium. However, azoxystrobin resistance recovered quicker in the mutant compared to the corresponding parental isolate after renewed subculturing on medium amended with 0.2 and 1 microg/ml azoxystrobin; only the mutant but not the parental isolate was able to adapt to 5 microg/ml azoxystrobin. In UV mutagenesis experiments, the DMI-R isolates produced significantly more mutants compared to S isolates. All of the UV-induced mutants showed stable fungicide resistance with little fitness penalty. This study indicates the potential for QoI fungicide resistance development in M. fructicola in the absence of a mutagen and provides evidence for increased mutability and predisposition to accelerated adaptation to azoxystrobin in M. fructicola isolates resistant to DMI fungicides.     

Genetic Variation for Virulence and Resistance in the Wheat-Mycosphaerella graminicola Pathosystem III. Comparative Seedling and Adult Plant Experiments

ABSTRACT Fourteen Dutch Mycosphaerella graminicola isolates were studied for their virulence to 22 wheat cultivars in the seedling stage in an experiment set up as a completely randomized block design with three repetitions. Isolate x cultivar interactions were highly significant. Cluster analyses were applied to select three isolates with significantly different virulence characteristics for both disease parameters. These were retested in the seedling stage and used to inoculate two field experiments that were planted according to a split plot design in 1992 and 1995. Overhead inoculations were conducted after flowering to avoid the effects of plant height; hence, these experiments were intended as monocyclic tests for virulence differences between the isolates. Significant isolate x cultivar interactions were detected in each experiment, demonstrating specificity in the wheat-M. graminicola pathosystem in the adult plant stage under field conditions. The reproducibility of the adult plant data was high. Genetic differences among the isolates were additionally demonstrated by randomly amplified polymorphic DNA (RAPD) patterns, which also showed that no significant contamination of the inoculated plots with the natural M. graminicola population had occurred. Rank correlations between seedling and adult plant data were significant for M. graminicola isolates IPO323 and IPO290, but not for isolate IPO001. Hence, evaluation of resistance and virulence may require seedling, as well as adult plant, tests.     

Triazole fungicides and the selection of resistance to medical triazoles in the opportunistic mould Aspergillus fumigatus

Azole resistance is an emerging problem in the opportunistic mould Aspergillus fumigatus. The triazoles are the most important agents for the management of Aspergillus diseases in humans. Selection for acquired resistance may occur in the hospital setting through exposure to high doses of azoles during azole therapy, but evidence is accumulating that A. fumigatus may become resistant to medical triazoles through environmental exposure to fungicides. The recovery of A. fumigatus isolates resistant to the medical triazoles from azole‐naive patients as well as from the environment strongly indicates an environmental route of resistance selection. Molecule alignment studies have identified five fungicides that share a very similar molecule structure with the medical triazoles, and thus may have selected for mechanisms that confer resistance to both groups of compounds. It is important to explore further the presumed fungicide‐driven route of resistance selection in order to implement effective preventive measures as the prevalence of azole resistance in A. fumigatus continues to increase and causes major challenges in the management of azole‐resistant Aspergillus diseases. Copyright © 2012 Society of Chemical Industry     

Aggressiveness of Mycosphaerella graminicola Isolates from Susceptible and Partially Resistant Wheat Cultivars

ABSTRACT The selective effect of quantitative host resistance on pathogen aggressiveness is poorly understood. Because two previous experiments with a small number of bread wheat cultivars and isolates of Mycosphaerella graminicola had indicated that more susceptible hosts selected for more aggressive isolates, we conducted a larger experiment to test that hypothesis. In each of 2 years, six cultivars differing in their levels of partial resistance were planted in field plots, and isolates were collected from each cultivar early and late in the growing season. The isolates were inoculated as populations bulked by cultivar of origin, field replicate, and collection date on seedlings of the same six cultivars in the greenhouse. The selective impact of a cultivar on aggressiveness was measured as the difference in aggressiveness between early and late isolates from that cultivar. Regression of those differences on disease severity in the field yielded significance values of 0.0531 and 0.0037 for the 2 years, with moderately resistant cultivars selecting for more aggressive isolates. In a related experiment, the protectant fungicide chlorothalonil was applied to plots of two susceptible cultivars to retard epidemic development. When tested in the greenhouse, isolates of M. graminicola from those plots were significantly more aggressive than isolates from the same cultivars unprotected by fungicide.     

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     

Resistance to metalaxyl in isolates of the sunflower pathogen Plasmopara halstedii

Plasmopara halstedii isolates showing an atypical reaction to metalaxyl were collected in France, in 1995 and 1996, and tested in the laboratory for their level of sensitivity to this fungicide. Primary and secondary infections caused by one of these isolates were not controlled by the metalaxyl concentration registered for seed treatment. The EC₅₀ of this isolate was 12 800 mg a.i. kg^-1 compared with 22 mg a.i. kg^-1 for sensitive isolate, indicating a 582-fold decrease in sensitivity to the compound. There was no reduction in the agressiveness of the resistant isolate. Using other anti-oomycete fungicides, it appeared that propamocarb, contact fungicides (fluazinam, folpet, mancozeb) and the mixed formulations dimethomorph + mancozeb, cymoxanil + mancozeb and ofurace + folpet were effective against primary infections made with metalaxyl resistant and sensitive isolates, but not against secondary infections. Metalaxyl mixed with fluazinam, folpet or mancozeb was more effective against primary infections with the resistant isolate than metalaxyl alone. The EC₅₀ of five other isolates ranged from 5 800 to 32 900 mg a.i. kg^-1, indicating a variability in metalaxyl sensitivity of resistant sunflower downy mildew isolates. This is the first report of physiological resistance to metalaxyl in Plasmopara halstedii.     

Specificity of incomplete resistance to Mycosphaerella graminicola in wheat

We examined interactions between wheat (Triticum aestivum) and Mycosphaerella graminicola, causal agent of Septoria tritici blotch, to determine whether specific interactions occur between host and pathogen genotypes that could be involved in eroding resistance. The moderate resistance of the wheat cultivar Madsen has eroded significantly in the Willamette Valley of Oregon since its release in 1990. Foote is a replacement cultivar expressing moderate resistance and was released in 2000. Isolates of M. graminicola were collected from Foote and Madsen in 2004 and 2005 and tested on each cultivar in growth chamber and greenhouse experiments. There was a significant (P 相似文献