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 and Characterization of QoI-Resistant Phytophthora cactorum and P. nicotianae Causing Leather Rot in Florida Strawberry

Phytophthora cactorum and P. nicotianae cause leather rot (LR) of fruit and Phytophthora crown rot (PhCR) in strawberry. LR occurs sporadically but can cause up to 70% fruit loss when weather is conducive. In Florida's annual strawberry winter production system, PhCR can be severe, resulting in plant stunting, mortality, and severe yield loss. Currently, azoxystrobin is labeled for control of LR but not for PhCR. The aims of this research were i) to determine the sensitivity of P. cactorum and P. nicotianae isolates from strawberry to azoxystrobin and ii) to investigate mechanisms of QoI-resistance present in P. cactorum and P. nicotianae based on the known point mutations within the cytb gene. Isolates of both Phytophthora spp. causing LR and PhCR were collected from multiple strawberry fields in Florida between 1997 and 2020. Isolates were tested for sensitivity to azoxystrobin at 0, 0.01, 0.1, 1.0, 10, and 50 µg/ml on potato dextrose agar (PDA) amended with SHAM (100 µg/ml). Isolates were separated into two groups, sensitive isolates, with the 50% effective concentration (EC50) values lower than 1.0 µg/ml, and resistant isolates having EC50 values higher than 50 µg/ml. P. cactorum and P. nicotianae resistance to azoxystrobin was found for isolates collected after 2010. The first 450 nucleotides of the mitochondrial cytochrome b (cytb) gene were sequenced from a selection of resistant and sensitive isolates of both species. The G143A mutation reported to confer resistance to azoxystrobin was found in all resistant P. cactorum isolates. However, in P. nicotianae, qualitative resistance was observed, but the isolates lacked all the known mutations in the cytb gene. This is the first report of resistance to azoxystrobin in P. cactorum and P. nicotianae.

Distribution of G143A mutations conferring fungicide resistance in Virginia populations of Parastagonospora nodorum infecting wheat

Stagonospora nodorum blotch (SNB) caused by Parastagonospora nodorum is an important leaf spot disease in the mid-Atlantic U.S. Disease management approaches include use of resistant varieties, cultural control, and foliar fungicides. Frequent use of foliar fungicides can select for fungicide resistance within pathogen populations. Recently, the first report of quinone outside inhibitor (QoI) fungicide resistance in the U.S. was made based on a relatively small collection of P. nodorum isolates from Virginia. The objective of this study was to conduct a state-wide, two-year survey of P. nodorum populations in Virginia wheat and quantify frequencies of the target-site mutation that confers QoI resistance. A total of 318 isolates of P. nodorum were obtained from wheat collected at seven locations distributed throughout the wheat-growing regions of Virginia in 2018 and 2019. A previously designed pyrosequencing assay that detects the G143A substitution in the cytochrome b gene of P. nodorum was used to screen isolates for the presence or absence of the target site mutation. The G143A substitution was detected in all sampled fields. Among locations and years, frequencies of the mutation in P. nodorum populations ranged from 5-32% (mean = 19%). Thus, the QoI-resistance conferring G143A mutation was widespread in P. nodorum populations in Virginia and it occurred at a relatively high frequency. Results suggest that fungicides containing QoI active ingredients may not be effective for controlling SNB in Virginia and the surrounding region, and application of stand-alone QoI fungicides for disease control in wheat is not recommended.

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.

Occurrence of quinone outside inhibitor (QoI) resistance in Virginia populations of Parastagonospora nodorum infecting wheat

Stagonospora nodorum blotch (SNB) of wheat, caused by Parastagonospora nodorum, is managed using cultural practices, resistant varieties, and foliar fungicides. Frequent fungicide use can select for fungicide resistance, making certain chemistries less effective; this may in part explain increasing severity of SNB in the mid-Atlantic U.S. Quinone outside inhibitor (QoI) resistance has been documented for a diversity of fungi, but it has not been reported for P. nodorum in the U.S. The objectives of this study were to 1) evaluate QoI sensitivity of P. nodorum from Virginia wheat fields, 2) screen P. nodorum for QoI target site mutations in the cytochrome b gene, and 3) develop a molecular assay to detect target site mutations associated with QoI resistance. Sensitivity of 16 isolates to pyraclostrobin and azoxystrobin was evaluated with radial growth assays, and the cytochrome b gene was sequenced. One isolate was insensitive to both fungicides, and it had the G143A mutation in the cytochrome b gene. For azoxystrobin, 10 isolates without target site mutations had reduced sensitivity. Additional isolates (N=74) were sequenced, and seven had the G143A mutation; all seven isolates with the mutation had reduced sensitivity to pyraclostrobin and azoxystrobin compared to a sensitive control isolate without the mutation. A pyrosequencing assay targeting G143A was developed as a rapid method to screen P. nodorum for the QoI resistance-conferring mutation. To our knowledge, this is the first report of QoI resistant P. nodorum in the U.S. Overall resistance frequency was low, but resistance management practices are needed to maintain the efficacy of fungicides for SNB control.

Widespread QoI Fungicide Resistance Revealed Among Corynespora cassiicola Tomato Isolates in Florida

Target spot of tomato caused by Corynespora cassiicola is one of the most economically destructive diseases of tomato in Florida. A collection of 123 isolates from eight counties in Florida were evaluated for sensitivity to azoxystrobin and fenamidone based on mycelial growth inhibition (MGI), spore germination (SG), detached leaflet assays (DLAs), and sequence-based analysis of the cytochrome b gene (cytb). Cleavage of cytb by restriction enzyme (Fnu4HI) revealed the presence of a mutation conferring a glycine (G) to alanine (A) mutation at amino acid position 143 (G143A) in approximately 90% of the population, correlating with quinone outside inhibitor (QoI) resistance based on MGI (<40% at 5 μg/ml), SG (<50% at 1 and 10 μg/ml), and DLA (<10% severity reduction). The mutation conferring a phenylalanine (F) to leucine (L) substitution at position 129 (F129L) was confirmed in moderately resistant isolates (#9, #19, and #74) based on MGI (40 to 50% at 5 μg/ml), SG (<50% at 1 μg/ml and >50% at 10 μg/ml), and DLA (>10% and <43% severity reduction) for both QoI fungicides, whereas sensitive isolates (#1, #4, #7, #28, #29, #46, #61, #74, #75, #76, #91, #95, and #118) based on MGI (>50% at 5 μg/ml), SG (>50% at 1 μg/ml and 10 μg/ml), and DLA (>50% severity reduction) correlated to non-mutation-containing isolates or those with a silent mutation. This study indicates that QoI resistance among C. cassiicola isolates from tomato is widespread in Florida and validates rapid screening methods using MGI or molecular assays to identify resistant isolates in future studies.

Cercospora nicotianae Isolates from Flue-Cured Tobacco in North Carolina Found with G143A and F129L Mutations in Cytochrome b Gene

Frogeye leaf spot of tobacco caused by Cercospora nicotianae (Ellis & Everhart) is a widespread disease of cultivated tobacco. Recently, flue-cured tobacco producers in North Carolina reported losses due to frogeye leaf spot disease despite the use of strobilurin fungicides. Isolates (n = 4) were obtained in 2018 from affected tobacco leaves from Cumberland, Lenoir, and Nash counties. In 2019, isolates (n = 28) were collected from a field in Wilson county. After sequencing the cytb region of 32 isolates, 30 contained a single point mutation conferring a G143A or F129L amino acid change that resulted in quinone outside inhibitor (QoI) fungicide resistance. Although these resistance mutations have been found in air-cured tobacco in Kentucky, to the best of our knowledge, the present study is the first to report QoI resistance mutations in C. nicotianae populations in flue-cured tobacco and a first report in North Carolina.