NCR13 peptide protects soybean against Cercospora sojina using multi-faceted modes of action and additive interaction with chemical fungicides.

Reduced sensitivity to demethylation inhibitor (DMI) and quinone outside inhibitor (QoI) fungicides in Nothopassalora personata, the cause of late leaf spot of peanut (Arachis hypogaea), complicates management of this disease in the Southeastern United States. Mixtures with protectant fungicides may help preserve the use of members of both DMI and QoI fungicide groups for leaf spot management. Field experiments were conducted in Tifton, GA, from 2019 to 2021 and in Plains, GA, during 2019 and 2020. The primary objective was to determine the effects of mixtures of DMI fungicides tebuconazole and mefentrifluconazole and QoI fungicides azoxystrobin and pyraclostrobin with micronized elemental sulfur on late leaf spot in fields with populations of N. personata with suspected reduced sensitivity to DMI and QoI fungicides. In four of the experiments, the efficacies of elemental sulfur and chlorothalonil as mixing partners were also compared. In most cases, standardized area under the disease progress curve (sAUDPC) and final percentage defoliation were less for all DMI and QoI fungicides mixed with sulfur or chlorothalonil than for the respective fungicides alone. In most cases, sAUDPC and final percentage defoliation were similar for sulfur and chlorothalonil when mixed with the respective DMI or QoI fungicide. These results indicate that mixtures of DMI or QoI fungicides with either micronized sulfur or chlorothalonil can improve control of late leaf spot compared with the DMI or QoI fungicide alone. These results also indicate that elemental sulfur has potential as an alternative to chlorothalonil in tank mixes where that protectant fungicide is currently being used as a mixing partner to improve leaf spot control.

Sensitivity of Cercospora sojina isolates to quinone outside inhibitor fungicides

The effect of quinone outside inhibitor fungicides on powdery mildew in a grape vineyard in Hungary

Isolates of Cercospora kikuchii, the causal agent of Cercospora leaf blight (CLB) and purple seed stain (PSS), were used to determine baseline sensitivities to selected quinone outside inhibitor (QoI) and demethylation inhibitor (DMI) fungicides by conducting radial growth assays on fungicide-amended media. The effective concentration to inhibit 50% radial growth (EC50) for each isolate was calculated by linear interpolation of the dose-response relationship. All baseline distributions were non-normal with outliers towards the less sensitive ends of the spectra, and median EC50 values for azoxystrobin, pyraclostrobin, trifloxystrobin, flutriafol, propiconazole, and tetraconazole were 0.081, 0.013, 0.012, 0.273, 0.143, 1.47 µg/ml, respectively. When compared to baseline sensitivities, median EC50 values for isolates exposed to azoxystrobin, pyraclostrobin, and trifloxystrobin in 2011/2012 were significantly higher at 37.2/57.6, 10.1/12.2, and 20.1/29.1 µg/ml, respectively. Cross-resistance to all three QoI fungicides was observed in the 2011 and 2012 populations. Discriminatory doses of 10 µg/ml were developed for all three QoI fungicides to distinguish between sensitive and resistant isolates. Approximately 83% of all isolates screened in 2011 and 2012 were resistant to QoI fungicides, and isolates from 21 of 27 parishes tested positive for resistance. Median EC50 values for isolates exposed to flutriafol, propiconazole, and tetraconazole in 2011/2012 were 0.41/0.54, 0.33/0.24, and 0.75/0.73 µg/ml. Significant shifts from the baseline towards less sensitivity were detected in isolates exposed to flutriafol and propiconazole. Additionally, outliers towards less DMI sensitivity were detected for all three DMI fungicides 2012. Strong, positive, and significant cross-sensitivity was observed among all three DMI fungicides. At a discriminatory dose of 5 µg/ml thiophanate methyl, methyl benzimidazole carbamate (MBC) resistance was detected in the 2000, 2011, and 2012 populations at 23.3, 44.8, and 35.7%, respectively, with resistant isolates in 19 of 27 parishes. Isolates exhibiting multiple resistance to QoI and MBC fungicides also were detected in 15 of 27 parishes. Ninety-eight percent of MBC-resistant isolates also were resistant to QoI fungicides. Based on results from this research, CLB/PSS management strategies with QoI and MBC fungicides should be reconsidered in areas where resistance has been confirmed, and C. kikuchii populations should be further monitored for shifts in DMI sensitivity.

Downy mildew (Plasmopara viticola) is a significant problem in grape vineyards throughout the growing season. Control of downy mildew is carried out with a combination of host tolerance and chemical applications. Even in vineyards planted with very tolerant varieties (e.g., Concord), control is important in years with ideal pathogen conditions. Fungicides with a single mode of action possess a very high potential for the development of resistance. Resistance has been observed often in the Quinone outside inhibitor (QoI) fungicides, such as strobilurins. We ascertained the levels of QoI resistance in downy mildew colonies on diseased leaves using CAPS-PCR to detect the glycine to alanine mutation (G143A) known to confer a qualitative level of resistance in fungal pathogens. Our data uncovered a small percentage of samples that contain G143A, suggesting an overall low level of QoI resistance. The low prevalence of the resistant single nucleotide polymorphism (SNP) suggests that QoI fungicides should remain a viable control mechanism in Lake Erie vineyards. Additionally, it appears that a viticultural region where tolerant hosts predominant and QoI use is minimal, resistance buildup in the pathogen population will be minimal. Accepted for publication 15 January 2013. Published 22 April 2013.

Botrytis cinerea, the causal agent of gray mold disease, is one of the most important plant-pathogenic fungi affecting strawberry. During the last decade, control of gray mold disease in the southeastern United States has largely been dependent on captan and the use of at-risk fungicides with single-site modes of action, including a combination of the quinone outside inhibitor (QoI) fungicide pyraclostrobin and succinate dehydrogenase inhibitor (SDHI) fungicide boscalid formulated as Pristine 38WG. Reports about loss of efficacy of Pristine in experimental fields in North Carolina prompted us to collect and examine 216 single-spore isolates from 10 conventional fields and 1 organic field in North Carolina and South Carolina in early summer 2011. Sensitivity to pyraclostrobin or boscalid was determined using a conidial germination assay with previously published discriminatory doses. Pyraclostrobin- and pyraclostrobin+boscalid-resistant isolates were found in all conventional fields (with some populations revealing no sensitive isolates) and in the organic field. Among the isolates collected, 66.7% were resistant to pyraclostrobin and 61.5% were resistant to both pyraclostrobin and boscalid. No isolates were identified that were resistant to boscalid but sensitive to pyraclostrobin, indicating that dual resistance may have derived from a QoI-resistant population. The molecular basis of QoI and SDHI fungicide resistance was determined in a subset of isolates. Polymerase chain reaction-restriction fragment length polymorphism analysis of the partial cytochrome b (CYTB) gene showed that pyraclostrobin-resistant isolates possessed the G143A mutation known to confer high levels of QoI fungicide resistance in fungi. Boscalid-resistant isolates revealed point mutations at codon 272 leading to the substitution of histidine to arginine (H272R) or tyrosine (H272Y), affecting the third Fe-S cluster region of the iron-sulfur protein (SdhB) target of SDHIs. The results of the study show that resistance to QoI fungicides and dual resistance to QoI and SDHI fungicides is common in B. cinerea from strawberry fields in the Carolinas. Resistant strains were more frequent in locations heavily sprayed with QoI and SDHI fungicides. However, resistance to both fungicides was also found in the unsprayed, organic field, indicating that some resistant strains may have been introduced from the nursery.

Early blight of potato (Solanum tuberosum L.) caused by Alternaria solani Sorauer is a frequent concern for potato growers in Canada. Management of early blight has relied on foliar fungicides that often include quinone outside inhibitor (QoI) fungicides such as azoxystrobin. In recent years, isolates of A. solani with reduced sensitivity to QoI fungicides, conferred by the presence of the F129L mutation (in the cytochrome b gene causing amino acid substitution of phenylalanine with leucine at position 129), have become widespread in potato-production areas of the United States, leading to a reduced efficacy of these products (3). Observations of reduced fungicide efficacy, following application of QoI fungicides to commercial fields in Manitoba, Canada in 2007, prompted an examination of the fungicide sensitivity of isolates of A. solani collected from fields in this province. Nine isolates of A. solani were obtained from potato foliage with typical early blight symptoms from four fields in Manitoba using standard protocols (2). Isolates were maintained on clarified V8 agar (1) and identified to species level based on conidial morphology (4). The sensitivity of each isolate to azoxystrobin was determined by assessing conidial germination on water agar plates amended with 0, 0.001, 0.01, 0.1, 1.0, or 10.0 mg/liter of azoxystrobin with protocols described previously (1). Two reference isolates of A. solani from North Dakota with known sensitivities to azoxystrobin and one isolate from Prince Edward Island (PEI), Canada, (a province yielding only isolates sensitive to azoxystrobin in previous surveys; R. D. Peters, unpublished data) were included in the assays. Calculated effective concentration (EC50) values (azoxystrobin concentration inhibiting conidial germination by 50%) were determined for each isolate response from two replications of the assays. The reference isolates of A. solani from North Dakota were sensitive or had reduced sensitivity to azoxystrobin with mean EC50 values of 0.02 and 0.2 mg/liter, respectively. The isolate from PEI was sensitive to azoxystrobin with a mean EC50 value of 0.04 mg/liter. By contrast, isolates of A. solani from Manitoba had reduced sensitivity to azoxystrobin with mean EC50 values from 0.2 to 0.8 mg/liter. Real-time PCR analysis of each isolate was performed (2) and confirmed the presence of the F129L mutation in the Manitoba isolates and the isolate with reduced sensitivity to azoxystrobin from North Dakota. The F129L mutation was absent in the azoxystrobin-sensitive wild-type isolates from PEI and North Dakota. To our knowledge, this is the first report of isolates of A. solani with reduced sensitivity to azoxystrobin in Canada. Since cross resistance among QoI fungicides has been demonstrated in A. solani isolates with the F129L mutation (3), adoption of resistance management strategies, including alternating QoI fungicides with fungicides having different modes of action and further monitoring pathogen populations for QoI sensitivity in Canadian production areas, is recommended.

This chapter discusses the history, current situation and molecular mechanism of resistance to quinone outside inhibitor (QoI) fungicides, which are inhibitors of mitochondrial respiration at Qo site of cytochrome bc1 enzyme complex, in some plant pathogenic fungi in Japan. The molecular diagnostic methods, in vitro methods and bioassay for identifying QoI resistance are described. Strategies against QoI resistance on rice and the development of novel QoI fungicides are presented. The resistance to succinate dehydrogenase inhibitors (SDHI) fungicides, so-called complex II inhibitors in the mitochondrial respiration chain, is also discussed, as well as the molecular mechanism of and strategies against resistance to these fungicides.

Target spot, caused by Corynespora cassiicola, is a soybean disease of increasing importance in the southern United States. Recently, isolates of C. cassiicola with resistance to quinone outside inhibitor (QoI) fungicides have been confirmed with the G143A mutation in multiple southern states, including Alabama, Arkansas, Mississippi, and Tennessee. From 2017 to 2019, a total of 84 isolates of C. cassiicola were recovered from soybean field in 12 counties in Kentucky. DNA sequencing of the cytochrome b gene of these isolates revealed that 15.5% of the isolates had the G143A mutation that confers resistance to QoI fungicides and were in 50% of the counties in which isolates originated. This represents the first report of QoI fungicide resistance in C. cassiicola isolates from Kentucky soybean fields. Considering these findings, Kentucky soybean growers should adopt target spot management practices which include rotating to non-host crops, planting resistant soybean cultivars, and applying fungicides from different fungicide classes.

Frogeye leaf spot, caused by Cercospora sojina, is an important foliar disease of soybean (Glycine max) in the United States. Application of quinone outside inhibitor (QoI) fungicides has been an important management tool available to farmers to help manage this disease, but in 2010, C. sojina isolates with resistance to QoI fungicides were first discovered in Tennessee and then additional states in the years to follow. During the 2020 growing season, C. sojina isolates collected from Wisconsin soybean fields were tested for QoI resistance using laboratory and molecular assays. The results of these assays showed that QoI fungicide-resistant C. sojina isolates are present in Wisconsin. Similar to previous findings in other states, these QoI-resistant C. sojina isolates contain the G143A mutation. Soybean farmers in Wisconsin will need to use an integrated approach of cultural practices, genetic resistance, and using fungicides with multiple modes of action to manage this disease in light of QoI-resistant C. sojina isolates being present in the state.

Distribution of Alternaria alternata isolates with resistance to quinone outside inhibitor (QoI) fungicides in Brazilian orchards of tangerines and their hybrids

Isolates of Cercospora kikuchii, a soybean (Glycine max) pathogen causing Cercospora leaf blight and purple seed stain, were tested to determine baseline sensitivities (n = 50) to selected quinone outside inhibitor (QoI) fungicides by conducting radial growth assays on fungicide-amended media. Baseline effective fungicide concentration to inhibit 50% of fungal radial growth (EC50) values were compared with EC50 values for isolates collected in 2011 (n = 50), 2012 (n = 50), and 2013 (n = 36) throughout soybean-producing areas in Louisiana. Median EC50 values for isolates subjected to QoI fungicides were significantly (P = 0.05) higher across all 3 years. Cross-resistance to QoI fungicides was observed in resistant isolates collected in 2011 to 2013. Discriminatory doses were developed for QoI fungicides to distinguish between sensitive and resistant isolates. On average, 89% of all isolates screened in 2011 to 2013 were resistant to QoI fungicides. At a discriminatory dose of thiophanate methyl (TM), a methyl benzimidazole carbamate (MBC) fungicide, at 5 μg/ml, resistance was detected in the 2000, 2011, 2012, and 2013 collections at 23, 38, 29, and 36%, respectively. Isolates exhibiting multiple resistance to QoI fungicides and TM also were detected in 2011, 2012, and 2013 at frequencies of 34, 26, and 31%, respectively. Based on these results, Cercospora leaf blight management strategies in Louisiana using solo applications of QoI or MBC fungicides in soybean should be reconsidered.

Frogeye leaf spot, caused by Cercospora sojina, is an important foliar disease of soybean (Glycine max) in the United States. Application of quinone outside inhibitor (QoI) fungicides has been an important tool available to farmers to help manage this disease, but in 2010, C. sojina isolates with resistance to QoI fungicides were first discovered in Tennessee and then additional states in the years to follow. During the 2020 growing season, C. sojina isolates collected from North Dakota soybean fields were tested for QoI resistance using laboratory and molecular assays. The results of these assays showed that QoI fungicide-resistant C. sojina isolates are present in North Dakota. Similar to previous findings in other states, these QoI-resistant C. sojina isolates contain the G143A mutation. Soybean farmers in North Dakota will need to use an integrated approach of cultural practices, genetic resistance, and fungicides with multiple modes of action to manage this disease in light of QoI-resistant C. sojina isolates being present in the state.

Resistance to quinone outside inhibitor (QoI) fungicides was detected in Cercospora sojina (causal agent of frogeye leaf spot) isolates collected from soybean (Glycine max) fields in four South Dakota counties during the 2018 growing season. A discriminatory dose assay was used to detect QoI-resistant isolates, and a follow-up polymerase chain reaction assay was used to determine the presence of the G143A mutation in QoI-resistant isolates. This is the first report of resistance to QoI fungicides in C. sojina isolates from South Dakota.

It is a common practice to add salicylhydroxamic acid (SHAM) into artificial medium in the in vitro sensitivity assay of fungal phytopathogens to the quinone outside inhibitor (QoI) fungicides. The rationale for adding SHAM is to inhibit fungal alternative oxidase, which is presumed to be inhibited by secondary metabolites of plants. Therefore, the ideal characteristics of SHAM should be almost nontoxic to phytopathogens and have no significant effect on control efficacy of fungicides. However, this study showed that the average effective concentration for 50% inhibition (EC50) of mycelial growth values of SHAM were 97.5 and 401.4 μg/ml for Sclerotinia sclerotiorum and Botrytis cinerea, respectively. EC50 values of the three QoI fungicides azoxystrobin, kresoxim-methyl, and trifloxystrobin in the presence of SHAM at 20 and 80 μg/ml for S. sclerotiorum and B. cinerea, respectively, declined by 52.7 to 78.1% compared with those without SHAM. For the dicarboximide fungicide dimethachlone, the average EC50 values in the presence of SHAM declined by 18.2% (P = 0.008) for S. sclerotiorum and 35.9% (P = 0.012) for B. cinerea. Pot experiments showed that SHAM increased control efficacy of the three QoI fungicides against the two pathogens by 43 to 83%. For dimethachlone, SHAM increased control efficacy by 134% for S. sclerotiorum and 86% for B. cinerea. Biochemical studies showed that SHAM significantly inhibited peroxidase activity (P = 0.024) of B. cinerea and esterase activity (P = 0.015) of S. sclerotiorum. The strong inhibitions of SHAM per se on mycelial growth of B. cinerea and S. sclerotiorum and significant influences on the sensitivity of the two pathogens to both the QoI fungicides and dimethachlone as well as inhibitions on peroxidase and esterase indicate that SHAM should not be added in the in vitro assay of sensitivity to the QoI fungicides.