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

2021 ◽  
Author(s):  
Danilo Neves ◽  
Richard W Webster ◽  
Damon L. Smith ◽  
Carl A Bradley
Keyword(s):  
Cultural Practices ◽  
Genetic Resistance ◽  
Integrated Approach ◽  
The United States ◽  
Management Tool ◽  
Cercospora Sojina ◽  
Qoi Fungicides ◽  
Molecular Assays ◽  
Quinone Outside Inhibitor ◽  
Qoi Resistance

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.

Resistance to Quinone Outside Inhibitor Fungicides Conferred by the G143A Mutation in Cercospora sojina (Causal Agent of Frogeye Leaf Spot) Isolates from Michigan, Minnesota, and Nebraska Soybean Fields

2020 ◽  
Vol 21 (4) ◽  
pp. 230-231 ◽  
Author(s):  
Danilo L. Neves ◽  
Martin I. Chilvers ◽  
Tamra A. Jackson-Ziems ◽  
Dean K. Malvick ◽  
Carl A. Bradley
Keyword(s):  
United States ◽  
Leaf Spot ◽  
Fungicide Resistance ◽  
The United States ◽  
Pcr Assay ◽  
Cercospora Sojina ◽  
Qoi Fungicides ◽  
Quinone Outside Inhibitor ◽  
Frogeye Leaf Spot ◽  
Important Disease

Frogeye leaf spot, caused by Cercospora sojina, is an important disease of soybean (Glycine max) in the United States. An important tactic to manage frogeye leaf spot is to apply foliar fungicides. Isolates of C. sojina were collected from soybean fields in one county in Michigan, three counties in Minnesota, and 10 counties in Nebraska in 2019, and they were tested for resistance to quinone outside inhibitor (QoI) fungicides using a discriminatory dose assay, a PCR assay, and DNA sequencing. Results of the testing indicated that QoI fungicide-resistant isolates were detected in isolates from all counties. Testing results also indicated that the G143A mutation was responsible for the QoI fungicide resistance. This is the first report of QoI fungicide-resistant C. sojina isolates in Michigan, Minnesota, and Nebraska and expands the geographical distribution of QoI fungicide-resistant C. sojina isolates to 18 states in total.

scholarly journals First Report of the Soybean Frogeye Leaf Spot Fungus (Cercospora sojina) Resistant to Quinone Outside Inhibitor Fungicides in North America

Plant Disease ◽  
2012 ◽  
Vol 96 (5) ◽  
pp. 767-767 ◽  
Author(s):  
G. R. Zhang ◽  
M. A. Newman ◽  
C. A. Bradley
Keyword(s):  
Leaf Spot ◽  
Management Practices ◽  
The United States ◽  
Conidial Germination ◽  
Cercospora Sojina ◽  
Soybean Field ◽  
Qoi Fungicides ◽  
First Report ◽  
Quinone Outside Inhibitor ◽  
Frogeye Leaf Spot

Quinone outside inhibitor (QoI; also known as strobilurin) fungicides sometimes are applied to soybean (Glycine max) fields to help manage frogeye leaf spot of soybean (caused by Cercospora sojina) in the United States. In August 2010, soybean leaflets exhibiting severe frogeye leaf spot symptoms were collected from a field in Lauderdale County, TN that had been treated twice with pyraclostrobin during that growing season. The field had been planted into soybean annually since at least 2008, and a QoI fungicide had been applied to the field in each of those years. Fifteen single-spore isolates of C. sojina were recovered from the affected soybean leaflets. These isolates were identified as C. sojina based on the observed symptoms on the soybean leaflets and the morphology and size of conidiophores and conidia (3). In addition, DNA was extracted from the cultures, PCR amplification of the small subunit rDNA and internal transcribed spacer (ITS) region was conducted (2), and the resulting PCR product was sequenced at the Keck Biotechnology Center at the University of Illinois, Urbana. The resulting nucleotide sequences were compared with sequences deposited in the nucleotide database ( http://www.ncbi.nlm.nih.gov ) and showed highest homology to sequences of C. sojina. The isolates were tested for their sensitivity to technical-grade formulations of the QoI fungicides azoxystrobin, pyraclostrobin, and trifloxystrobin with an in vitro conidial germination assay with fungicide + salicylhydroxamic acid (SHAM)-amended potato dextrose agar as described by Bradley and Pedersen (1). The effective concentration at which 50% conidial germination was inhibited (EC50) was determined for all 15 C. sojina isolates, with mean values of 3.1644 (2.7826 to 4.5409), 0.3297 (0.2818 to 0.6404), and 0.8573 (0.3665 to 2.5119) μg/ml for azoxystrobin, pyraclostrobin, and trifloxystrobin, respectively. When compared with previously established mean EC50 values of C. sojina baseline isolates (4), EC50 values of the C. sojina isolates collected from the Lauderdale County, TN soybean field were approximately 249- to 7,144-fold greater than the EC50 values of the baseline isolates. These results indicate that all isolates recovered from the Lauderdale County, TN soybean field were highly resistant to QoI fungicides. To our knowledge, this is the first report of QoI fungicide resistance occurring in C. sojina, and surveys for additional QoI fungicide-resistant C. sojina isolates are needed to determine their prevalence and geographic distribution. In light of these findings, soybean growers in Tennessee and adjacent states should consider utilizing alternative frogeye leaf spot management practices such as planting resistant cultivars, rotating to nonhost crops, and tilling affected soybean residue (3). References: (1) C. A. Bradley and D. K. Pedersen. Plant Dis. 95:189, 2011. (2) N. S. Lord et al. FEMS Microbiol. Ecol. 42:327, 2002. (3) D. V. Phillips. Page 20 in: Compendium of Soybean Diseases. 4th ed. G. L. Hartman et al., eds. The American Phytopathological Society, St. Paul, MN, 1999. (4) G. Zhang et al. Phytopathology (Abstr.) 100(suppl.):S145, 2010.

scholarly journals Characterization of Quinone Outside Inhibitor Fungicide Resistance in Cercospora sojina and Development of Diagnostic Tools for its Identification

Plant Disease ◽  
2015 ◽  
Vol 99 (4) ◽  
pp. 544-550 ◽  
Author(s):  
F. Zeng ◽  
E. Arnao ◽  
G. Zhang ◽  
G. Olaya ◽  
J. Wullschleger ◽  
...  
Keyword(s):  
Cytochrome B ◽  
Leaf Spot ◽  
Fungicide Resistance ◽  
Cytochrome B Gene ◽  
Diagnostic Tools ◽  
Polymerase Chain Reaction Primer ◽  
Cercospora Sojina ◽  
Qoi Fungicides ◽  
Quinone Outside Inhibitor ◽  
Frogeye Leaf Spot

Frogeye leaf spot of soybean, caused by the fungus Cercospora sojina, reduces soybean yields in most of the top-producing countries around the world. Control strategies for frogeye leaf spot can rely heavily on quinone outside inhibitor (QoI) fungicides. In 2010, QoI fungicide-resistant C. sojina isolates were identified in Tennessee for the first time. As the target of QoI fungicides, the cytochrome b gene present in fungal mitochondria has played a key role in the development of resistance to this fungicide class. The cytochrome b genes from three QoI-sensitive and three QoI-resistant C. sojina isolates were cloned and sequenced. The complete coding sequence of the cytochrome b gene was identified and found to encode 396 amino acids. The QoI-resistant C. sojina isolates contained the G143A mutation in the cytochrome b gene, a guanidine to cytosine transversion at the second position in codon 143 that causes an amino acid substitution of alanine for glycine. C. sojina-specific polymerase chain reaction primer sets and TaqMan probes were developed to efficiently discriminate QoI-resistant and -sensitive isolates. The molecular basis of QoI fungicide resistance in field isolates of C. sojina was identified as the G143A mutation, and specific molecular approaches were developed to discriminate and to track QoI-resistant and -sensitive isolates of C. sojina.

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

Plant Disease ◽  
2020 ◽  
Vol 104 (3) ◽  
pp. 893-903 ◽  
Author(s):  
Keevan J. MacKenzie ◽  
Katia V. Xavier ◽  
Aimin Wen ◽  
Sujan Timilsina ◽  
Heather M. Adkison ◽  
...  
Keyword(s):  
Amino Acid Position ◽  
Rapid Screening ◽  
Silent Mutation ◽  
Screening Methods ◽  
Corynespora Cassiicola ◽  
Qoi Fungicides ◽  
Quinone Outside Inhibitor ◽  
Target Spot ◽  
Moderately Resistant ◽  
Qoi Resistance

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.

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

Plant Disease ◽  
2020 ◽  
Author(s):  
Navjot Kaur ◽  
Chase Mullins ◽  
Nathan Michael Kleczewski ◽  
Hillary Laureen Mehl
Keyword(s):  
Cytochrome B ◽  
Management Practices ◽  
Cultural Practices ◽  
Resistance Management ◽  
Cytochrome B Gene ◽  
Target Site ◽  
Quinone Outside Inhibitor ◽  
Parastagonospora Nodorum ◽  
The U.S ◽  
Qoi Resistance

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.

scholarly journals Resistance to Quinone Outside Inhibitor Fungicides Conferred by the G143A Mutation in Cercospora sojina (Causal Agent of Frogeye Leaf Spot) Isolates from South Dakota Soybean Fields

2019 ◽  
Vol 20 (2) ◽  
pp. 104-105 ◽  
Author(s):  
Febina M. Mathew ◽  
Emmanuel Byamukama ◽  
Danilo L. Neves ◽  
Carl A. Bradley
Keyword(s):  
South Dakota ◽  
Leaf Spot ◽  
Causal Agent ◽  
Chain Reaction ◽  
Cercospora Sojina ◽  
Qoi Fungicides ◽  
Quinone Outside Inhibitor ◽  
Frogeye Leaf Spot ◽  
Polymerase Chain

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.

Assessment of quinone outside inhibitor sensitivity and frogeye leaf spot race of Cercospora sojina in Georgia soybean

Plant Disease ◽  
2021 ◽  
Author(s):  
Bennett Harrelson ◽  
Bikash Ghimire ◽  
Robert Kemerait ◽  
Albert Culbreath ◽  
Zenglu Li ◽  
...  
Keyword(s):  
Leaf Spot ◽  
Fungicide Resistance ◽  
Mycelial Growth ◽  
The United States ◽  
In Vitro Assays ◽  
Cercospora Sojina ◽  
Quinone Outside Inhibitor ◽  
Frogeye Leaf Spot ◽  
Differential Cultivars

Frogeye leaf spot (FLS), caused by the fungal pathogen Cercospora sojina K. Hara, is a foliar disease of soybean (Glycine max L. (Merr.)) responsible for yield reductions throughout the major soybean producing regions in the world. In the United States, management of FLS relies heavily on the use of resistant cultivars and in-season fungicide applications, specifically within the class of quinone outside inhibitors (QoIs), which has resulted in the development of fungicide resistance in many states. In 2018 and 2019, 80 isolates of C. sojina were collected from six counties in Georgia and screened for QoI fungicide resistance using molecular and in vitro assays, with resistant isolates being confirmed from three counties. Additionally, 50 isolates, including a “baseline isolate” with no prior fungicide exposure, were used to determine the percent reduction of mycelial growth to two fungicides, azoxystrobin and pyraclostrobin, at six concentrations: 0.0001, 0.001, 0.01, 0.1, 1, and 10 g ml-1. Mycelial growth observed for resistant isolates varied significantly from both the sensitive isolates and the baseline isolate for azoxystrobin concentrations of 10, 1, 0.1, and 0.01 g ml-1 and for pyraclostrobin concentrations of 10, 1, 0.1, 0.01 and 0.001 g ml-1. Moreover, 40 isolates were used to evaluate pathogen race on six soybean differential cultivars by assessing susceptible or resistant reactions. Isolate reactions suggested 12 races of C. sojina present in Georgia, four of which have not been previously described. Species richness indicators (rarefaction and abundance-based coverage estimator - ACE) indicated that within-county C. sojina race numbers were undersampled in the present study, suggesting the potential for the presence of either additional undescribed races or known but unaccounted for races in Georgia. However, no isolates were pathogenic on differential cultivar ‘Davis’, carrying the Rcs3 resistance allele, suggesting the gene is still an effective source of resistance in Georgia.

scholarly journals Baseline Sensitivity of Cercospora zeae-maydis to Quinone Outside Inhibitor Fungicides

Plant Disease ◽  
2011 ◽  
Vol 95 (2) ◽  
pp. 189-194 ◽  
Author(s):  
C. A. Bradley ◽  
D. K. Pedersen
Keyword(s):  
United States ◽  
Leaf Spot ◽  
The United States ◽  
Gray Leaf Spot ◽  
Alternative Respiration ◽  
Baseline Sensitivity ◽  
Qoi Fungicides ◽  
Quinone Outside Inhibitor ◽  
Cercospora Zeae Maydis

Cercospora zeae-maydis, the causal agent of gray leaf spot on corn (Zea mays), can cause severe yield loss in the United States. Quinone outside inhibitor (QoI) fungicides are effective tools that can be used to manage gray leaf spot, and their use has increased in corn production in the United States. In total, 61 C. zeae-maydis isolates collected from fields in which QoI fungicides had never been applied were tested in vitro using azoxystrobin-, pyraclostrobin-, or trifloxystrobin-amended medium to determine the effective fungicide concentration at which 50% of the conidial germination was inhibited (EC50). The effect of salicylhydroxamic acid (SHAM) also was evaluated for seven isolates to determine whether C. zeae-maydis is capable of using alternative respiration in azoxystrobin-amended medium. All seven C. zeae-maydis isolates tested had significantly greater (P < 0.02) EC50 values when SHAM was not included in medium amended with azoxystrobin, indicating that C. zeae-maydis has the potential to utilize alternative respiration to overcome QoI fungicide inhibition in vitro. Baseline EC50 values of azoxystrobin ranged from 0.003 to 0.031 μg/ml, with mean and median values of 0.018 and 0.019 μg/ml, respectively. Baseline EC50 values of pyraclostrobin ranged from 0.0003 to 0.0025 μg/ml, with mean and median values of 0.0010 and 0.0010 μg/ml, respectively. Baseline EC50 values of trifloxystrobin ranged from 0.0004 to 0.0034 μg/ml, with mean and median values of 0.0023 and 0.0024 μg/ml, respectively. These baseline sensitivity values will be used in a fungicide resistance monitoring program to determine whether shifts in sensitivity to QoI fungicides are occurring in C. zeae-maydis populations.

Widespread Occurrence of Quinone Outside Inhibitor Fungicide-Resistant Isolates of Cercospora sojina, Causal Agent of Frogeye Leaf Spot of Soybean, in the United States

2018 ◽  
Vol 19 (4) ◽  
pp. 295-302 ◽  
Author(s):  
Guirong Zhang ◽  
Tom W. Allen ◽  
Jason P. Bond ◽  
Ahmad M. Fakhoury ◽  
Anne E. Dorrance ◽  
...  
Keyword(s):  
United States ◽  
Leaf Spot ◽  
Management Practices ◽  
Integrated Management ◽  
The United States ◽  
Causal Agent ◽  
Cercospora Sojina ◽  
Widespread Occurrence ◽  
Quinone Outside Inhibitor ◽  
Frogeye Leaf Spot

Isolates of Cercospora sojina, causal agent of frogeye leaf spot of soybean (Glycine max), were collected across Alabama, Arkansas, Delaware, Illinois, Indiana, Iowa, Kentucky, Louisiana, Mississippi, Missouri, North Carolina, Ohio, Tennessee, and Virginia and were evaluated for quinone outside inhibitor (QoI) fungicide resistance. Collection of these isolates from these 14 states occurred between 2010 and 2017. QoI fungicide-resistant C. sojina isolates were detected in all 14 states surveyed and represent a total of 240 counties or parishes. In 2017, these 240 counties and parishes represented approximately 13% of the harvested soybean hectares in the United States. In light of this widespread occurrence of QoI fungicide-resistant C. sojina isolates, management of frogeye leaf spot should focus on integrated management practices such as planting resistant soybean cultivars, rotating with nonhost crops, and tilling to speed up decomposition of infested soybean residue. When foliar fungicide application is warranted, fungicide products that contain active ingredients from chemistry classes other than the QoI class should be applied for frogeye leaf spot management.

scholarly journals Distribution of QoI Resistance in Populations of Tangerine-Infecting Alternaria alternata in Florida

Plant Disease ◽  
2014 ◽  
Vol 98 (1) ◽  
pp. 67-76 ◽  
Author(s):  
Byron Vega ◽  
Megan M. Dewdney
Keyword(s):  
Alternaria Alternata ◽  
Brown Spot ◽  
Cross Resistance ◽  
Qoi Fungicides ◽  
Quinone Outside Inhibitor ◽  
Citrus Orchards ◽  
Application Frequency ◽  
Polymerase Chain ◽  
Highly Correlated ◽  
Qoi Resistance

Chemical control, based on copper and quinone outside inhibitor (QoI) fungicides, has been essential for the management of brown spot of citrus, caused by Alternaria alternata. However, QoI control failures were detected recently in Florida. From 2008 to 2012, 817 monoconidial isolates of A. alternata from 46 citrus orchards were examined for sensitivity to azoxystrobin (AZ) and pyraclostrobin (PYR). Of the isolates, 57.6% were resistant to both fungicides, with effective concentration to inhibit 50% growth (EC50) values greater than 5 μg/ml for AZ and 1 μg/ml for PYR. The mean EC50 values for sensitive isolates were 0.139 and 0.020 μg/ml for AZ and PYR, respectively. The EC50 values of both fungicides were highly correlated (P < 0.0001), indicating cross resistance. The proportion of resistant isolates differed significantly (P < 0.0001) among cultivars and with QoI application frequency (P < 0.0001). However, resistance was not significantly related (P = 0.364) to disease severity in the field (low, moderate, and high) or isolate virulence (P = 0.397). The molecular basis for QoI resistance was determined for a subset of 235 isolates using polymerase chain reaction restriction fragment length polymorphism of the cytochrome b gene. All resistant isolates showed the point mutation G143A. Based on the presence of one or two introns, isolates were classified as profile I and profile II, respectively. The resistance frequency was significantly higher (P < 0.0001) in isolate profile II, suggesting a higher selection pressure for resistant population profile II.

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