Demethylation Inhibitor Fungicides Research Articles

Impact of Demethylation Inhibitor (DMI) Fungicides on Clipping Yield and Rooting Depth of Creeping Bentgrass

Demethylation inhibitor fungicides (DMIs) have been used to manage a wide range of turfgrass diseases for many decades. Unfortunately, this class of fungicide can have unwanted plant growth regulator (PGR) effects on amenity turfgrasses. Recently, a number of new DMI fungicides have been registered for turfgrasses and are seeing expanded use. To determine if differences exist in the PGR-related activity among products, a 2-year field study was conducted examining six different DMI fungicides. Fungicides were applied to creeping bentgrass maintained at 0.33 cm and applied five or six times every 14 days at equivalent active ingredient rates. The results demonstrated that although paclobutrazol statistically reduced clipping weights, there was very little effect from DMI fungicides. In the first study year, propiconazole did produce higher clipping weights on two rating dates but this was not observed in the second year. Rooting depth was unaffected by any of the chemicals in either year. Although phytotoxicity may be a concern of turfgrass managers when applying DMI fungicides during the summer, it seems unlikely that these materials applied without the additional use of labeled PGRs will have any noticeable negative effects on foliar and root growth of creeping bentgrass in New England.

Characterization of Monilinia fructicola Strains Resistant to Both Propiconazole and Boscalid.

In 2011 and 2012, significant brown rot disease caused by Monilinia fructicola was observed in a peach orchard in Spartanburg County, SC, despite preharvest fungicide applications of demethylation inhibitor (DMI), quinone outside inhibitor (QoI), and succinate dehydrogenase inhibitor (SDHI) fungicides. All 22 isolates obtained in 2011 from this orchard were sensitive to the QoI fungicide, azoxystrobin, and the methyl benzimidazole carbamate (MBC) fungicide, thiophanate-methyl. Five were resistant to the DMI fungicide, propiconazole, and were selected, together with five propiconazole-sensitive isolates, for further investigations. One of the 10 isolates was resistant to propiconazole but sensitive to the SDHI fungicide, boscalid (EC50 = 0.42 μg/ml), 3 were resistant to propiconazole with intermediate sensitivity to boscalid (EC50 0.72 to 2.1 μg/ml); 2 were sensitive to propiconazole with intermediate sensitivity to boscalid; 3 were sensitive to propiconazole but resistant to boscalid (EC50 ≥ 2.1 μg/ml); and 1 (isolate MD22) was resistant to both propiconazole and boscalid. Disease incidence on detached fruit treated with formulated propiconazole or boscalid was significantly higher for MD22 compared to a sensitive control isolate. Continued monitoring of fungicide resistance in the same orchard in 2012 revealed an increase of isolates resistant to propiconazole from 22.7% in 2011 to 34.7%, and an increase of isolates resistant to both propiconazole and boscalid from 4.5% in 2011 to 18.4%. Propiconazole resistance was always associated with the presence of the 'Mona' mobile element located upstream of the sterol 14α-demethylase (MfCYP51) gene. To investigate whether mutations in the subunits of the succinate dehydrogenase enzyme were involved in boscalid resistance, significant portions of the M. fructicola SdhA, SdhB, SdhC, and SdhD genes were cloned and analyzed for 2 sensitive, 2 boscalid-resistant, and 6 dual-resistant isolates. Although sequence variation was found among the isolates, no single change correlated with resistance. Interestingly, analysis of isolates collected from orchards in 2001 and 2002, prior to the registration of boscalid, revealed a range of sensitivities to boscalid (EC50 0.03 to 3.46 μg/ml) including boscalid-resistant isolates. The presence of boscalid-resistant isolates in the baseline population was unexpected and requires further investigation.

Induced overexpression of cytochrome P450 sterol 14α-demethylase gene (CYP51) correlates with sensitivity to demethylation inhibitors (DMIs) inSclerotinia homoeocarpa

The fungus Sclerotinia homoeocarpa causes dollar spot, the most important turfgrass disease worldwide. Demethylation inhibitor (DMI) fungicides have been relied upon heavily to manage this disease. Presently, populations of S. homoeocarpa with reduced sensitivity or resistance to DMIs are widespread in the United States. Cytochrome P450 sterol 14α-demethylase (ShCYP51) and its flanking regions were identified and sequenced in 29 isolates of S. homoeocarpa with a range of DMI sensitivities. No modifications were found in the gene coding and upstream regions that were consistently related to DMI sensitivity. In the absence of propiconazole, ShCYP51 was expressed at a similar low level among DMI baseline and resistant isolates. In the presence of propiconazole, DMI-resistant isolates were induced to express ShCYP51 at significantly higher levels than baseline isolates by propiconazole at 5 mg L(-1) for 5 h or at 0.5 mg L(-1) for 72 h. The ShCYP51 expression level after 72 h exposure to 0.5 mg L(-1) of propiconazole was linearly related to EC50 values and ΔRG (the change in relative growth rate over time), with R(2) values equal to 83.7 and 90.0% respectively. Induced overexpression of ShCYP51 in resistant isolates following DMI exposure is an important factor determining DMI sensitivity in S. homoeocarpa.

Evaluation of DMI fungicides against species of Diplodia and Neofusicoccum associated with Botryosphaeria canker of grapevine

C. Torres, B.A. Latorre, P. Undurraga, and X. Besoain. 2013. Evaluation of DMI fungicides against species of Diplodia and Neofusicoccum associated with Botryosphaeria canker of grapevine. Cien. Inv. Agr. 40(1):131-138. The Botryosphaeriaceae species have been widely studied and are considered a serious problem to the production of grapes worldwide. To aid in the development of control strategies for these fungi, in vitro experiments were conducted to evaluate the effectiveness of demethylation inhibiting (DMI) fungicides against Diplodia seriata, D. mutila, Neofusicoccum australe and N. parvum isolated from grapevine (Vitis vinifera). Fungicidal effectiveness was evaluated over a range of increasing concentrations using measurements of both the inhibition of conidial germination and the effects on mycelial growth. The effective concentrations (EC) necessary to inhibit conidial germination and mycelial growth by 50% (EC 50 ) and 90% (EC 90 ) were estimated by linear regression analysis between x = log 10 of the fungicidal concentration and y = the Probit value of the inhibition percentage. The results demonstrate that tebuconazole, myclobutanil, prochloraz and prochloraz plus epoxiconazole exhibit considerable in vitro activity against D. mutila, D. seriata, N. australe and N. parvum. These DMI compounds were able to arrest conidial germination and inhibit mycelial growth. The EC 50 values obtained for conidial germination were generally higher than the EC 50 values obtained for mycelial growth, suggesting that mycelia are more sensitive to the DMI compounds than are conidia. However, the effectiveness of these DMI compounds in the vineyard must be studied before any final conclusions can be reached.

Efficacy of Variable Tetraconazole Rates Against Cercospora beticola Isolates with Differing In Vitro Sensitivities to DMI Fungicides.

Cercospora leaf spot (CLS) of sugar beet is caused by the fungus Cercospora beticola. CLS management practices include the application of the sterol demethylation inhibitor (DMI) fungicides tetraconazole, difenoconazole, and prothioconazole. Evaluating resistance to DMIs is a major focus for CLS fungicide resistance management. Isolates were collected in 1997 and 1998 (baseline sensitivity to tetraconazole, prothioconazole, or difenoconazole) and 2007 through 2010 from the major sugar-beet-growing regions of Minnesota and North Dakota and assessed for in vitro sensitivity to two or three DMI fungicides. Most (47%) isolates collected in 1997-98 exhibited 50% effective concentration (EC50) values for tetraconazole of <0.01 μg ml-1, whereas no isolates could be found in this EC50 range in 2010. Since 2007, annual median and mean tetraconazole EC50 values have generally been increasing, and the frequency of isolates with EC50 values >0.11 μg ml-1 increased from 2008 to 2010. In contrast, the frequency of isolates with EC50 values for prothioconazole of >1.0 μg ml-1 has been decreasing since 2007. Annual median difenoconazole EC50 values appears to be stable, although annual mean EC50 values generally have been increasing for this fungicide. Although EC50 values are important for gauging fungicide sensitivity trends, a rigorous comparison of the relationship between in vitro EC50 values and loss of fungicide efficacy in planta has not been conducted for C. beticola. To explore this, 12 isolates exhibiting a wide range of tetraconazole EC50 values were inoculated to sugar beet but no tetraconazole was applied. No relationship was found between isolate EC50 value and disease severity. To assess whether EC50 values are related to fungicide efficacy in planta, sugar beet plants were sprayed with various dilutions of Eminent, the commercial formulation of tetraconazole, and subsequently inoculated with isolates that exhibited very low, medium, or high tetraconazole EC50 values. The high EC50 isolate caused significantly more disease than isolates with medium or very low EC50 values at the field application rate and most reduced rates. Because in vitro sensitivity testing is typically carried out with the active ingredient of the commercial fungicide, we investigated whether loss of disease control was the same for tetraconazole as for the commercial product Eminent. The high EC50 isolate caused more disease on plants treated with tetraconazole than Eminent but disease severity was not different between plants inoculated with the very low EC50 isolate.

In vivo sensitivity reduction of Puccinia triticina races, causal agent of wheat leaf rust, to DMI and QoI fungicides

Experiments were carried out to determine in vivo the IC50 and the IC90 for demethylation-inhibitor fungicides (DMIs, triazoles) and quinone outside inhibitors (QoIs, strobilurins) to the five most frequent races of Puccinia triticina in 2007 growing season in Southern Brazil. The tests were done in a greenhouse with wheat seedlings. DMI fungicides were tested at the concentrations, in mg/L, 0.0; 0.02; 0.2; 2.0; 20.0; 100.0 and 200.0, and QoIs at the concentrations 0.0; 0.0001; 0.001; 0.01; 0.1; 1 and 10.0 mg of active ingredient/L water. Fungicides were preventively applied at 24 hours before the inoculation of seedlings with the fungal spores. The effect of treatments was assessed based on the number of uredia/cm². The lowest IC50 (inhibitory concentration) for DMI fungicides determined for MCG-MN, sensitive race, ranged from 0.33 to 0.91 mg/L, while the highest values for MDP-MR, MDT-MR, MDK-MR, MFH-HT races, varied from 9.63 to 85.64 mg/L (suspected insensitivity). QoI fungicide presented an IC50 varying from 0.0018 to 0.14 mg/L. The sensitivity reduction factor for DMIs varied from 8.8 to 238.8, and for QoIs from 0.3 to 1.5 mg/L. Sensitivity reduction was confirmed for the races MDP-MR, MDT-MR, MDK-MR, MFH-HT to DMIs, as well as their sensitivity to QoI fungicides.

Overexpression of ShCYP51B and ShatrD in Sclerotinia homoeocarpa isolates exhibiting practical field resistance to a demethylation inhibitor fungicide.

We investigated genetic factors that govern the reduced propiconazole sensitivity of Sclerotinia homoeocarpa field isolates collected during a 2-year field efficacy study on dollar spot disease of turf in five New England sites. These isolates displayed a >50-fold range of in vitro sensitivity to a sterol demethylation inhibitor (DMI) fungicide, propiconazole, making them ideal for investigations of genetic mechanisms of reduced DMI sensitivity. The CYP51 gene homolog in S. homoeocarpa (ShCYP51B), encoding the enzyme target of DMIs, is likely a minor genetic factor for reduced propiconazole sensitivity, since there were no differences in constitutive relative expression (RE) values and only 2-fold-higher induced RE values for insensitive than for sensitive isolate groups. Next, we mined RNA-Seq transcriptome data for additional genetic factors and found evidence for the overexpression of a homolog of Botrytis cinerea atrD (BcatrD), ShatrD, a known efflux transporter of DMI fungicides. The ShatrD gene showed much higher constitutive and induced RE values for insensitive isolates. Several polymorphisms were found upstream of ShatrD but were not definitively linked to overexpression. The screening of constitutive RE values of ShCYP51B and ShatrD in isolates from two golf courses that exhibited practical field resistance to propiconazole uncovered evidence for significant population-specific overexpression of both genes. However, linear regression demonstrated that the RE of ShatrD displays a more significant relationship with propiconazole sensitivity than that of ShCYP51B. In summary, our results suggest that efflux is a major determinant of the reduced DMI sensitivity of S. homoeocarpa genotypes in New England, which may have implications for the emergence of practical field resistance in this important turfgrass pathogen.

Baseline Sensitivity and Cross-Resistance to Succinate-Dehydrogenase-Inhibiting and Demethylation-Inhibiting Fungicides in Didymella bryoniae.

Didymella bryoniae, which causes gummy stem blight (GSB) of watermelon, has a history of developing resistance to fungicides, most recently the succinate-dehydrogenase-inhibiting (SDHI) fungicide boscalid. To facilitate fungicide resistance monitoring, baseline sensitivity distributions were established for demethylation-inhibiting (DMI) fungicides tebuconazole and difenoconazole and the SDHI fungicide penthiopyrad, and reestablished for the SDHI fungicide boscalid. In all, 71 isolates with no known prior exposure to SDHIs or DMIs were used to determine the effective concentration at which mycelial growth was inhibited by 50% (EC50). EC50 values for boscalid, penthiopyrad, tebuconazole, and difenoconazole were 0.018 to 0.064, 0.015 to 0.057, 0.062 to 0.385, and 0.018 to 0.048 μg/ml, with median values of 0.032, 0.026, 0.118, and 0.031 μg/ml, respectively. Significant positive correlations between the sensitivity to penthiopyrad and boscalid (P < 0.0001, r = 0.75) and between tebuconazole and difenoconazole (P < 0.0001, r = 0.59) indicate a potential for cross-resistance between chemically related fungicides. In 2009, 103 isolates from fungicidetreated watermelon fields were tested for sensitivity to boscalid and penthiopyrad using a discriminatory concentration of 3.0 μg/ml. Of the isolates tested, 82 were insensitive and 14 were sensitive to both fungicides. Because of the significant potential for cross-resistance between closely related fungicides, growers will be advised not to use both SDHIs or both DMIs successively in the same fungicide spray program.

Orchard Factors Associated with Resistance and Cross Resistance to Sterol Demethylation Inhibitor Fungicides in Populations of Venturia inaequalis from Pennsylvania

Orchard management practices, such as destroying of overwintered inoculum and limiting the number of fungicide applications, are often recommended as tactics for slowing the development of resistance to sterol demethylation-inhibitor (DMI) fungicides in populations of Venturia inaequalis. However, there is little quantitative evidence relating the use of such practices to levels of resistance in orchards. The aim of this study was to evaluate the sensitivity of V. inaequalis isolates from Pennsylvania to DMI fungicides, and to identify orchard management factors related to the incidence of resistant isolates. In total, 644 single-spore V. inaequalis cultures obtained from 20 apple orchards in 2008 or 2009 were tested for sensitivity to myclobutanil, fenbuconazole, or difenoconazole. Growers provided management history of the sampled plots. Widespread shifts toward resistance to the three fungicides were noted, with mean effective concentration for 50% inhibition (EC(50)) values of 2.136, 0.786, and 0.187 μg/ml for myclobutanil, fenbuconazole, and difenoconazole, respectively. Cross resistance to the three fungicides was documented in high correlation (Spearman's r > 0.6) between mean EC(50) values for 14 orchards. Based on a 0.5-μg/ml threshold, 66 and 26% of isolates were resistant to myclobutanil and fenbuconazole, respectively, and 22% were cross resistant to the two fungicides. A significant between-year shift toward increased resistance was noted in two of three orchards surveyed in both years. Failure to use dormant copper sprays, older trees, larger orchards, orchards with ≤10 cultivars, and application of >4 DMI sprays were positively correlated (0.0001 < P < 0.05) with the incidence of resistant isolates. Isolates from orchards with >4 DMI sprays were four times as likely to be resistant to fenbuconazole (odds ratio = 4.57; P = 0.015). Isolates from orchards without dormant copper sprays were twice as likely to be cross-shifted toward resistance to all three fungicides (odds ratio = 1.76; P = 0.048). Results identify management practices that can reduce the risk of V. inaequalis developing resistance to DMI fungicides.

Characterization of CbCyp51 from Field Isolates of Cercospora beticola

The hemibiotrophic fungus Cercospora beticola causes leaf spot of sugar beet. Leaf spot control measures include the application of sterol demethylation inhibitor (DMI) fungicides. However, reduced sensitivity to DMIs has been reported recently in the Red River Valley sugar beet-growing region of North Dakota and Minnesota. Here, we report the cloning and molecular characterization of CbCyp51, which encodes the DMI target enzyme sterol P450 14α-demethylase in C. beticola. CbCyp51 is a 1,632-bp intron-free gene with obvious homology to other fungal Cyp51 genes and is present as a single copy in the C. beticola genome. Five nucleotide haplotypes were identified which encoded three amino acid sequences. Protein variant 1 composed 79% of the sequenced isolates, followed by protein variant 2 that composed 18% of the sequences and a single isolate representative of protein variant 3. Because resistance to DMIs can be related to polymorphism in promoter or coding sequences, sequence diversity was assessed by sequencing >2,440 nucleotides encompassing CbCyp51 coding and flanking regions from isolates with varying EC(50) values (effective concentration to reduce growth by 50%) to DMI fungicides. However, no mutations or haplotypes were associated with DMI resistance or sensitivity. No evidence for alternative splicing or differential methylation of CbCyp51 was found that might explain reduced sensitivity to DMIs. However, CbCyp51 was overexpressed in isolates with high EC(50) values compared with isolates with low EC(50) values. After exposure to tetraconazole, isolates with high EC(50) values responded with further induction of CbCyp51, with a positive correlation of CbCyp51 expression and tetraconazole concentration up to 2.5 μg ml(-1).

Sensitivity of Erysiphe necator to Demethylation Inhibitor Fungicides in Virginia.

Grape powdery mildew (Erysiphe necator) isolates were collected from 2005 to 2007 from vineyards mostly in Virginia but also some in Maryland, North Carolina, and Pennsylvania. Using a leaf disc assay, the isolates were tested against five demethylation inhibitor (DMI) fungicides. Most isolates exhibited reduced sensitivity to the five DMIs when compared with a sensitive group (n = 12) and compared with unexposed populations reported from other areas. The median resistance factor (RF) was highest for tebuconazole (RF = 399) and myclobutanil (RF = 378), followed by triflumizole (RF = 70), triadimefon (RF = 62), and fenarimol (RF = 44). The sensitive group used as the basis for comparison appears to have been more sensitive than unexposed isolates in New York and California. Our finding that the greatest resistance shift occurred with tebuconazole and myclobutanil contrasts with earlier reports from New York and California, where the greatest resistance shift was observed with triadimefon or triadimenol. Sensitivities to all five DMI fungicides were strongly correlated (pairwise r values of 0.70 to 0.87) but our data suggest that some may retain greater utility than others.

Toxicity and Resistance Potential of Selected Fungicides to Galactomyces and Penicillium spp. Causing Postharvest Fruit Decays of Citrus and Other Crops.

A diverse collection of isolates of Galactomyces citri-aurantii and G. geotrichum, the causal pathogens of sour rots of citrus and other fruit crops, respectively, was evaluated for sensitivity to demethylation-inhibiting (DMI) fungicides of the triazole group. Propiconazole was found to be highly effective in reducing mycelial growth of both species in vitro. For 139 isolates of G. citri-aurantii, a mean effective concentration for 50% reduction of mycelial growth (EC50 value) of 0.34 μg/ml was determined; whereas, for 33 isolates of G. geotrichum, this value was 0.14 μg/ml. In a comparison of additional DMI fungicides, mean EC50 values for 60 isolates of G. citri-aurantii and 20 isolates of G. geotrichum, were 0.27 and 0.17 μg/ml for cyproconazole, 0.25 and 0.14 μg/ml for metconazole, and 1.16 and 0.73 μg/ml for tebuconazole, respectively. Propiconazole was also highly active against mycelial growth of imazalil-sensitive isolates of Penicillium digitatum, the pathogen that causes green mold of citrus, with a mean EC50 value of 0.008 μg/ml for 63 isolates. Imazalil-resistant isolates of this fungus were cross-resistant to propiconazole. When G. citri-aurantii and P. digitatum were grown at selected pH values between 3 and 9, inhibition by propiconazole occurred over the entire pH range. The fungicide was most effective at pH 5 when compared with the non-fungicide-amended control grown at the same pH. In laboratory mass platings of single-spore isolates sensitive to propiconazole onto selective media, isolates with an up to 81.6-fold decrease in sensitivity to the fungicide were recovered for P. digitatum. For G. geotrichum, isolates with an approximately twofold decrease in sensitivity were obtained. No isolates with reduced sensitivity were recovered for G. citri-aurantii. Propiconazole is currently being registered for postharvest use on citrus and other crops, and the information provided will be valuable in monitoring of fungicide resistance and in designing effective fungicide application strategies.

Multiyear Evaluation of an Orange Peel Oil-based Spray Additive for Managing Insect Pests and Brown Rot of Nectarine

Adjuvants are often added to pesticide formulations to enhance their effectiveness. In this multiyear field study, a proprietary formulation of orange peel oil-based spray additive containing alcohol ethoxylate (OPO-AE) was used alone or in combination with the pyrethroid insecticide esfenvalerate or the demethylation inhibitor fungicide fenbuconazole for pest and disease control of nectarine. Field performance of fenbuconazole for preharvest and postharvest brown rot control was not improved when OPO-AE was added to the spray tank. However, OPO-AE alone reduced preharvest but not postharvest fruit rot compared to the nontreated control. Esfenvalerate was consistently the best treatment to control chewing insects but the insecticidal effect of the pyrethroid was negated when the adjuvant was added. Leaf damage likely due to insect feeding was significantly higher in the esfenvalerate + OPO-AE treatment compared to esfenvalerate alone. The OPO-AE treatment by itself had significantly greater leaf damage compared to the untreated control indicating that the orange peel oil-based formulation is an insect attractant likely due to the presence of d-limonene. In conclusion, our studies do not support claims that OPO-AE formulated as Vintre increases insecticidal or fungicidal efficacy of commercial products. Accepted for publication 14 May 2012. Published 17 June 2012.

Sensitivity of Mycosphaerella graminicola (anamorph: Septoria tritici) to DMI fungicides across Europe and impact on field performance

Since the occurrence and spread of resistance to quinone outside inhibitors (QoI) in Mycosphaerella graminicola in the early 2000s in Europe, demethylation inhibitors (DMIs) form the backbone for control of Septoria leaf blotch. European monitoring studies, carried out by various research institutes and DMI manufacturers, have shown a shift of the European M. graminicola population towards increased ED50 values for DMI fungicides. Populations of M. graminicola consist of very heterogeneous isolates within a region, and even within a field, in terms of DMI sensitivity. Sensitivity to DMIs is influenced by the haplotype of CYP51, the target of DMIs. New CYP51‐haplotypes have emerged and the frequency of less sensitive haplotypes in Europe has increased in recent years. Studies with efflux transporter inhibitors showed that not only CYP51, but also enhanced efflux, may play a role in the DMI sensitivity response. Sensitivity studies with 5 DMIs registered for Septoria leaf blotch control indicated that sensitivity of isolates to the 5 DMIs is heterogeneous and the overall correlation of sensitivity to the different DMIs is poor. A key requirement for sustainable control and resistance management of Septoria leaf blotch is therefore the continued availability of different DMIs.

Characterizing Fenbuconazole and Propiconazole Sensitivity and Prevalence of 'Mona' in Isolates of Monilinia fructicola from New York.

Demethylation inhibitor (DMI) resistant populations of Monilinia fructicola, the causal agent of brown rot of stone fruit, and the presence of the genetic DMI resistance determinant 'Mona' have been reported throughout the eastern United States. In this study, we endeavored to conduct a comprehensive investigation of DMI sensitivity, the prevalence of 'Mona', and implications of DMI use for M. fructicola populations from New York and Pennsylvania. Of the 18 orchards surveyed, only 9 were primarily composed of isolates with either resistance or reduced sensitivity to fenbuconazole and propiconazole. The DMI resistance determinant 'Mona' was only found in 5 orchards, present in isolates with a range of sensitivity phenotypes, and not always present in resistant isolates. These results suggested that 'Mona' only contributes to a portion of the quantitative resistance response to DMI fungicides. On detached blossoms and fruit, protective applications of fenbuconazole (Indar 2F) against isolates with resistance or reduced sensitivity resulted in significantly (P < 0.05) lower brown rot incidence compared to applications of propiconazole (Orbit 3.6EC) and water controls. By comparison, therapeutic applications of fenbuconazole and propiconazole against isolates with resistance or reduced sensitivity provided little to no reduction in brown rot incidence on blossoms and fruit.

DIFFERENTIAL SENSITIVITY TO TRIADIMENOL OF ERYSIPHE NECATOR ISOLATES BELONGING TO DIFFERENT GENETIC GROUPS

SUMMARY In populations of Erysiphe necator, the causal agent of grapevine powdery mildew, two genetic groups have been described, based on several molecular markers. In single vineyards, the two groups seem to occupy different temporal niches, with a temporal alternation that is clear-cut in vineyards intensively treated with chemical fungicides. To shed light on this behaviour, 80 isolates of E. necator from different vineyards and belonging to the two genetic groups were characterized for sensitivity to triadimenol, as representative of sterol demethylation inhibitor fungicides (DMIs). A leaf bioassay and an allele-specific PCR assay were used, based on the point mutation A495T in the CYP51 gene that causes the amino acid change Y136F, associated with high level of resistance to DMIs in E. necator. The CYP51 gene from isolates with different resistance levels was cloned and sequenced. The sensitivity to triadimenol (EC 50 ) of isolates of genetic group A was significantly higher than in group B isolates, to which all the moderately and highly resistant isolates belonged. The point mutation A495T was found in the CYP51 gene of isolates showing different levels of resistance, but additional point mutations were found in the same gene. The higher sensitivity to DMIs in group A isolates can be at the basis of their precocious disappearance in vineyards, and can have important implications for powdery mildew control strategies. The molecular basis of E. necator resistance to DMIs was confirmed to be complex and not always associated with the point mutation A495T.

In vitro Assessment of Sclerotinia homoeocarpa Resistance to Fungicides and Plant Growth Regulators.

Dollar spot (caused by Sclerotinia homoeocarpa) is the most economically important turfgrass disease in North America. This disease is primarily controlled by fungicide applications on golf courses; however, fungicide resistance has been confirmed in three of the four systemic fungicide classes commonly used to control dollar spot. The main objective of this study was to evaluate S. homoeocarpa sensitivity to multiple chemical classes and cross-resistance among active ingredients within the same class; in particular, the association between the fungistatic effect of demethylation inhibitors (DMIs) and plant growth regulators (PGRs). Fifty-eight isolates were selected arbitrarily from four locations in the United States and assayed for in vitro sensitivity to six DMI, two dicarboximide, one carboximide, and one benzimidazole fungicide as well as three type II PGRs. A series of concentrations for each active ingredient was used to determine the mean 50% effective concentration (EC50) values and correlation coefficients were calculated for all active ingredients. The EC50 values of all active ingredients from the DMI class were highly correlated (P < 0.0001) to each other as well as to the one dicarboximide (iprodione) and two PGRs (flurprimidol and paclobutrazol). Isolates resistant to thiophanatemethyl had significantly higher EC50 values than sensitive isolates for all active ingredients assayed except for boscalid. Findings showed that multiple and cross-resistance has developed in S. homoeocarpa and that the two PGRs have a fungistatic effect on this pathogen similar to that of DMI fungicides. The high correlation of in vitro sensitivities among PGRs and DMI fungicides further suggests that PGRs may contribute to the selection of DMI-resistant isolates or facilitate decreased sensitivity to DMI fungicides in the field.

Paralogous cyp51 genes in Fusarium graminearum mediate differential sensitivity to sterol demethylation inhibitors

Analysis of the genome sequence of Fusarium graminearum revealed three paralogous cyp51 genes (designated cyp51A, -B, and -C) encoding 14-α demethylases in this fungus. Targeted gene disruption showed that the cyp51A, -B or -C disruption mutants were morphologically indistinguishable from the parent isolate on potato dextrose agar medium, which indicates that none of these genes is essential for mycelial growth. The sensitivity of cyp51A deletion mutants to seven sterol demethylation inhibitor (DMI) fungicides increased significantly compared to the parent strain, while sensitivity of cyp51C deletion mutants increased to some but not all DMIs. No change in DMI sensitivity was observed for cyp51B deletion mutants. The parental phenotypes of cyp51A and cyp51C deletion mutants were completely restored by genetic complementation with the wild-type cyp51A and cyp51C genes, respectively. The sensitivity of F. graminearum isolates increased significantly when subjected in vitro to a mixture of DMI fungicides triadimefon and tebuconazole as compared to the individual components. These results indicate that different DMI fungicides target different CYP51 proteins in F. graminearum and that a mixture of DMI fungicides can result in synergistic effects. Our findings have directly implications on chemical management strategies of plant diseases caused by Fusarium species.

Synergism Between Demethylation Inhibitor Fungicides or Gibberellin Inhibitor Plant Growth Regulators and Bifenthrin in a Pyrethroid-Resistant Population of Listronotus maculicollis (Coleoptera: Curculionidae)

In 2007-2008, the "annual bluegrass weevil," Listronotus maculicollis Kirby (Coleoptera: Curculionidae), a serious pest of Poa annua L. (Poales: Poaceae) on U.S. golf courses, was shown to be resistant to two pyrethroids, bifenthrin and lambda-cyhalothrin. In 2008, we showed that bifenthrin resistance was principally mediated by oxidase detoxification (cytochrome P450 [P450]). P450s can be inhibited by demethylation inhibitor fungicides and gibberellin inhibitor plant growth regulators, both of which are commonly used on golf courses. We tested these compounds for synergistic activity with bifenthin against a pyrethroid-resistant population of L. maculicollis. The LD50 value for bifenthrin was significantly reduced from 87 ng per insect (without synergists) to 9.6-40 ng per insect after exposure to the fungicides fenarimol, fenpropimorph, prochloraz, propiconazole, and pyrifenox and the plant growth regulators flurprimidol, paclobutrazol, and trinexapac-ethyl. Simulated field exposure with formulated products registered for use on turf revealed enhanced mortality when adult weevils were exposed to bifenthrin (25% mortality, presented alone) combined with field dosages of propiconizole, fenarimol, flurprimidol, or trinexapac-ethyl (range, 49-70% mortality).

A sterol 14α-demethylase is required for conidiation, virulence and for mediating sensitivity to sterol demethylation inhibitors by the rice blast fungus Magnaporthe oryzae

The Magnaporthe oryzae genome contains two homologous CYP51 genes, MoCYP51A and MoCYP51B, that putatively encode sterol 14α-demethylase enzymes. Targeted gene deletion mutants of MoCYP51A were morphologically indistinguishable from the isogenic wild type M. oryzae strain Guy11 in vegetative culture, but were impaired in both conidiation and virulence. Deletion of MoCYP51B did not result in any obvious phenotypic changes compared with Guy11. The Δmocyp51A mutants were also highly sensitive to sterol demethylation inhibitor (DMI) fungicides, while Δmocyp51B mutants were unchanged in their sensitivity to these fungicides. Expression of both MoCYP51A and MoCYP51B was significantly induced by exposure to DMI fungicides. Analysis of intracellular localization of MoCyp51A showed that MoCyp51A was mainly localized to the cytoplasm of hyphae and conidia. Taken together, our results indicate that MoCYP51A is required for efficient conidiogenesis, full virulence and for mediating DMI sensitivity by the rice blast fungus.