The G143A Mutation in the Cytochrome b Gene is Associated with Quinone Outside Inhibitor Fungicide Resistance in Cercospora sojina from Soybean Fields in Wisconsin

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 use fungicides with multiple modes of action to manage this disease in light of QoI-resistant C. sojina isolates being present in the state.

Detection of the G143A Mutation in the Cytochrome b Gene of Corynespora cassiicola Isolates from Soybean in Tennessee

Corynespora cassiicola is the causal pathogen of target spot in soybean and cotton grown in the United States. With target spot increasing in importance, fungicides are becoming an important tool for control of this disease. Unfortunately, there are reports of C. cassiicola isolates in other crops being resistant to some fungicide classes. The objective of this study was to identify if resistance to quinone outside inhibitor (QoI) fungicides is present in Tennessee soybean and cotton isolates of C. cassiicola. Four isolates of C. cassiicola were evaluated at a range of doses for the fungicide pyraclostrobin. Isolates were also sequenced to determine if the G143A mutation was present in the cyt b gene. Two isolates previously reported to be resistant to QoIs were also used as positive checks. Two isolates of C. cassiicola from Tennessee soybean were found to have the G143A mutation. EC50 values for the two isolates ranged from 15.7 to 121 μg/ml. As a result of this study, C. cassiicola isolates have exhibited resistance to QoI fungicides in Tennessee soybean.

Sensitivity of the U.S. wheat powdery mildew population to QoI fungicides and determination of the complete Blumeria graminis f. sp. tritici cytochrome b gene

Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici, is managed primarily with cultivar resistance and foliar fungicides. Quinone outside inhibitors (QoIs), which target the mitochondrial cytochrome b (cytb) gene, are one of the two main fungicide classes used on wheat. While European populations of B. graminis f. sp. tritici are widely insensitive to QoIs, largely due to the cytb mutation G143A, the QoI sensitivity of the U.S. B. graminis f. sp. tritici population had never been evaluated despite years of QoI use on U.S. wheat. A total of 381 B. graminis f. sp. tritici isolates from 15 central and eastern U.S. states were screened for sensitivity to QoI fungicides pyraclostrobin and picoxystrobin. A modest range of sensitivities was observed, with maximum resistance factors of 11.2 for pyraclostrobin and 5.3 for picoxystrobin. The F129L, G137R, and G143A cytb mutations were not detected in the U.S. B. graminis f. sp. tritici population, nor were mutations identified in the PEWY loop, a key part of the Qo site. Thus, no genetic basis for the observed quantitative variation in QoI sensitivity of U.S. B. graminis f. sp. tritici was identified. Isolate sporulation was weakly negatively associated with reduced QoI sensitivity, suggesting a fitness cost. In the course of the study, the complete B. graminis f. sp. tritici cytb gene sequence was determined for the first time in the isolate 96224 v. 3.16 reference genome. Contrary to previous reports, the gene has an intron that appears to belong to intron group II, which is unusual in fungi. The study was the first QoI sensitivity screening of a large, geographically diverse set of U.S. B. graminis f. sp. tritici isolates, and while the population as a whole remains relatively sensitive, some quantitative loss of efficacy was observed.

Detection of a Point Mutation (G143A) in Cyt b of Corynespora cassiicola That Confers Pyraclostrobin Resistance

Point mutation G143A in the cytochrome b (Cyt b) protein commonly confers resistance to quinone outside inhibitor (QoI) fungicides in phytopathogenic fungi, including Corynespora cassiicola, which causes cucumber target spot disease. However, the effect of G143A on the binding between the QoI fungicide and the Cyt b protein, and the use of LAMP (loop-mediated isothermal amplification) to detect this point mutation had not been reported previously in C. cassiicola. In this study, the sensitivity of 131 C. cassiicola isolates—collected from Shandong province, China in 2019 and 2020—to pyraclostrobin was determined. The EC50 values ranged from 1.67 to 8.82 μg/mL, and sequencing results showed that all C. cassiicola isolates contained the G143A mutation. Molecular docking results suggested that G143A significantly alters the affinity of pyraclostrobin to the Cyt b protein. Following development of three LAMP primer pairs, the best reaction condition for LAMP analysis was 65 °C for 60 min, and the detection limit was 0.01 ng/μL of DNA containing the point mutation. In conclusion, the G143A mutation conferring pyraclostrobin resistance is widespread in C. cassiicola from Shandong province, and the LAMP method can be used to monitor QoI resistance in C. cassiicola caused by the G143A mutation in the field.

Sensitivity of C. fructicola and C. siamense of peach in China to multiple classes of fungicides and characterization of pyraclostrobin-resistant isolates

Anthracnose, mainly caused by Colletotrichum gloeosporioides species complex including C. fructicola and C. siamense, is a devastating disease of peach. The chemical control has been widely used for years and management failures have increased towards commonly used fungicides. Therefore, screening of sensitivity of Colletotrichum spp. to fungicides with different modes of action is needed to make proper management strategies for peach anthracnose. In this study, sensitivity of 80 isolates of C. fructicola and C. siamense was screened for pyraclostrobin, procymidone, prochloraz and fludioxonil based on mycelial growth inhibition at discriminatory doses. Results showed that C. fructicola and C. siamense isolates were highly resistant to procymidone and fludioxonil with 100% resistance frequencies to both fungicides, but sensitive to prochloraz, i.e., no resistant isolates were found. For pyraclostrobin, 74% of C. fructicola isolates showed high resistance and 26 % were low resistant, all of the C. siamense isolates were low resistant. No positive cross-resistance was observed between pyraclostrobin and azoxystrobin, even they are members of the same quinone outside inhibitor (QoI) fungicide group, and between pyraclostrobin and non-QoIs. Resistant isolates to QoI fungicides were evaluated for the fitness penalty. Results showed that no significant differences except for mycelial growth rates were detected between highly resistant and low-resistant isolates of C. fructicola. Molecular characterization of Cyt b gene revealed that the G143A point mutation was the determinant of the high resistance in C. fructicola. This study demonstrated the current resistance status of C. fructicola and C. siamense to different fungicides and their future perspectives. Demethylation inhibitor (DMI) fungicides are the best option among different chemicals to control peach anthracnose in China.

Sensitivity of Rhizoctonia solani AG 2-2 isolates from soybean and sugar beet to selected SDHI and QoI fungicides

Rhizoctonia solani causes root and stem diseases on soybean and sugar beet, and fungicides are commonly used to manage these diseases. Quinone outside inhibitor (QoI) fungicides (pyraclostrobin and azoxystrobin) have been used for in-furrow and post-emergence application since 2000. Succinate dehydrogenase inhibitor (SDHI) fungicides (sedaxane, penthiopyrad, and fluxapyroxad) became popular seed treatments following their registration in Minnesota and North Dakota between 2012 and 2016. Periodic monitoring of sensitivity to these fungicides in R. solani anastomosis group (AG) 2-2 is important to detect potential shifts in sensitivity over time. R. solani AG 2-2 isolates (n=35) collected from soybean and sugar beet in Minnesota and North Dakota were evaluated in vitro for sensitivity. Isolates were considered as baseline or non-baseline for the above mentioned fungicides based on previous potential exposure. The effective concentration (EC50) required to suppress radial fungal growth by 50% was determined. The mean EC50 values for sedaxane, penthiopyrad, fluxapyroxad and pyraclostrobin were 0.1, 0.15, 0.16, and 0.25 µg ml-1, respectively. The mean EC50 value for azoxystrobin for 22 isolates was 0.76 to 1.56 µg ml-1; and EC50 could not be determined for 13 isolates due to < 50% inhibition at the highest concentrations used. The EC50 values for the QoI fungicides did not differ significantly between baseline and non-baseline isolates. EC50 values for SDHI fungicides were significantly higher for isolates collected from soybean than from sugar beet, and isolates collected from both crops had similar EC50 values for pyraclostrobin. All SDHI fungicides and pyraclostrobin effectively suppressed R. solani isolates from soybean and sugar beet at low concentrations in vitro.

Delayed Development of Resistance to QoI Fungicide in Venturia inaequalis in Israeli Apple Orchards and Improved Apple Scab Management Using Fungicide Mixtures

Quinone outside inhibitors (QoI) fungicides group were introduced for commercial use against apple scab (Venturia inaequalis) in Israel in 1997. Unlike other regions in the world, in which resistance of V. inaequalis to QoI fungicides was observed within 3–5 years of use, in Israel it only occurred after 14 years of use. Field trials conducted between 2007 and 2017 showed a significant reduction in susceptibility to QoIs in northern Israel only since 2011. The delay in the development of resistance is related to limited fungicidal sprays resulting from unfavorable conditions for the pathogen. Of the 28 isolates collected from infected leaves or fruits of commercial orchards in northern Israel, 27 were resistant to the QoI fungicide Kresoxim-methyl. Amplification of the CYTB gene and sequencing of the G143A mutation region confirmed the resistance of all 27 isolates to QoIs. Resistance is demonstrated in the orchard, in vitro and molecular-based study, which forced the growers to avoid using QoIs against apple scab. We show that foliar applications of tank mixtures of systemic fungicides plus captan or prepacked fungicidal mixtures improved efficacy and can be used as a strategic approach in fungicide resistance management, including in orchards in which resistance to QoIs has been detected.

Are DMI+QoI Fungicide Premixes During flowering Worthwhile for Fusarium head blight Control in Wheat? A Meta-analysis

Fusarium head blight (FHB), caused mainly by Fusarium graminearum, is best controlled with demethylation inhibitor (DMI) fungicides during flowering. However, the use of premixes of DMI and quinone outside inhibitor (QoI) fungicides to control FHB has increased in Brazil. Data on FHB severity and wheat yields measured in field experiments conducted in Brazil were gathered from both peer- and non-peer-reviewed sources published from 2000 to 2018. After applying selection criteria, 73 field trials from 35 bibliographic sources were identified, among which 50% of the data were obtained from cooperative network trials conducted after 2011. To be included in the analysis, a DMI+QoI premixes or tebuconazole (TEB) were tested in at least 14 trials and three years. Four premixes met the criteria. Estimates of percent control (and respective 95% confidence interval) by a network model fitted to the log of the treatment means ranged from 44.1% (pyraclostrobin + metconazole applied once; 32.4 to 53.7) to 64.3% (pyraclostrobin + metconazole; 58.4 to 69.3); the latter not differing from TEB (59.9%, 53.6 to 65.3). Yield response was statistically similar for pyraclostrobin + metconazole (532.1 kg/ha, 441 to 623) and trifloxystrobin + prothioconazole (494.9 kg/ha, 385 to 551), and both differed statistically from a group composed of TEB (448.2 kg/ha, 342 to 554), trifloxystrobin + TEB (468.2 kg/ha, 385 to 551), azoxystrobin + TEB (462.4 kg/ha, 366 to 558) and pyraclostrobin + metconazole applied once (413.7 kg/ha, 308 to 518). The two categories of FHB index (7% cut off) and yield (3,000 kg/ha cut off), both in the non-treated check, did not explain the heterogeneity in the estimates. Two sequential sprays of TEB or one spray of pyraclostrobin + metconazole as management choices are likely more profitable than DIM+QoI premixes sprayed twice during flowering considering only the fungicide effects on yield.