Fungicide Resistance in Cercospora kikuchii, a Soybean Pathogen

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.

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

Plant Disease ◽

10.1094/pdis-04-21-0693-re ◽

2021 ◽

Author(s):

Hafiz Muhammad Usman ◽

Qin Tan ◽

Mohammad Mazharul Karim ◽

Muhammad Adnan ◽

Weixiao Yin ◽

...

Keyword(s):

High Resistance ◽

Mycelial Growth ◽

Management Strategies ◽

Cross Resistance ◽

Qoi Fungicides ◽

Quinone Outside Inhibitor ◽

Dmi Fungicides ◽

Colletotrichum Gloeosporioides Species Complex ◽

Cyt B Gene

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.

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The G143A Mutation is Responsible for Strobilurin Fungicide Resistance in Cercospora cf. flagellaris, a Leaf Blight and Purple Seed Stain Pathogen of Louisiana Soybean

Plant Health Progress ◽

10.1094/php-br-16-0043 ◽

2016 ◽

Vol 17 (3) ◽

pp. 197-197 ◽

Cited By ~ 4

Author(s):

Sebastian Albu ◽

Trey Price ◽

Vinson Doyle ◽

Boyd Padgett ◽

Ray Schneider

Keyword(s):

Cytochrome B ◽

Mississippi River ◽

Fungicide Resistance ◽

Radial Growth ◽

Leaf Blight ◽

Cytochrome B Gene ◽

Pathogen Population ◽

Lower Mississippi River ◽

Strobilurin Fungicide ◽

Purple Seed Stain

Cercospora leaf blight and purple seed stain are economically important diseases of soybean throughout the Lower Mississippi River Valley and Gulf South USA. Fungicide resistance was documented in Louisiana in the pathogen population using radial growth assays. Using some of the same isolates, a portion of the cytochrome b gene was sequenced. The G143A mutation was found in resistant isolates, and results correlated with radial growth assays 97% of the time. Accepted for publication 1 September 2016.

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

Plant Disease ◽

10.1094/pdis-05-14-0460-re ◽

2015 ◽

Vol 99 (4) ◽

pp. 544-550 ◽

Cited By ~ 21

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.

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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

Plant Health Progress ◽

10.1094/php-06-20-0052-br ◽

2020 ◽

Vol 21 (4) ◽

pp. 230-231 ◽

Cited By ~ 1

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.

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Point Mutations in the β-Tubulin of Phytophthora sojae Confer Resistance to Ethaboxam

Phytopathology ◽

10.1094/phyto-01-19-0032-r ◽

2019 ◽

Vol 109 (12) ◽

pp. 2096-2106 ◽

Cited By ~ 3

Author(s):

Qin Peng ◽

Zhiwen Wang ◽

Yuan Fang ◽

Weizhen Wang ◽

Xingkai Cheng ◽

...

Keyword(s):

Fungicide Resistance ◽

Germination Rate ◽

Management Strategies ◽

Resistance Management ◽

Point Mutations ◽

Phytophthora Sojae ◽

Cross Resistance ◽

Baseline Sensitivity ◽

Resistant Mutants ◽

Β Tubulin

Ethaboxam is a β-tubulin inhibitor registered for the control of oomycete pathogens. The current study was established to determine the ethaboxam sensitivity of the plant pathogen Phytophthora sojae and investigate the potential for the emergence of fungicide resistance. The effective concentration for 50% inhibition (EC50) of 112 Phytophthora sojae isolates exhibited a unimodal distribution with a mean EC50 for ethaboxam of 0.033 µg/ml. Establishing this baseline sensitivity provided critical data for monitoring changes in ethaboxam-sensitivity in field populations. The potential for fungicide resistance was investigated using adaptation on ethaboxam-amended V8 agar, which resulted in the isolation of 20 resistant mutants. An assessment of the biological characteristics of the mutants including mycelial growth, sporulation, germination rate and pathogenicity indicated that the resistance risk in Phytophthora sojae was low to medium with no cross-resistance between ethaboxam and cymoxanil, metalaxyl, flumorph, and oxathiapiprolin being detected. However, positive cross-resistance was found between ethaboxam and zoxamide for Q8L and I258V but negative cross-resistance for C165Y. Further investigation revealed that the ethaboxam-resistant mutants had point mutations at amino acids Q8L, C165Y, or I258V of their β-tubulin protein sequences. CRISPR/Cas9-mediated transformation experiments confirmed that the Q8L, C165Y, or I258V mutations could confer ethaboxam resistance in Phytophthora sojae and that the C165Y mutation induces high levels of resistance. Taken together, the results of the study provide essential data for monitoring the emergence of resistance and resistance management strategies for ethaboxam, as well as for improving the design of novel β-tubulin inhibitors for future development.

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Sensitivity of Bipolaris oryzae Isolates Pathogenic on Cultivated Wild Rice to the Quinone Outside Inhibitor Azoxystrobin

Plant Disease ◽

10.1094/pdis-12-18-2267-re ◽

2019 ◽

Vol 103 (8) ◽

pp. 1910-1917 ◽

Cited By ~ 2

Author(s):

Claudia V. Castell-Miller ◽

Deborah A. Samac

Keyword(s):

Cytochrome B ◽

Fungicide Resistance ◽

Wild Rice ◽

Brown Spot ◽

Bipolaris Oryzae ◽

Qoi Fungicides ◽

Quinone Outside Inhibitor ◽

Group I ◽

Mitochondrial Cytochrome B

The occurrence of fungal brown spot, caused by Bipolaris oryzae, has increased in cultivated wild rice (Zizania palustris) paddies in spite of the use of azoxystrobin-based fungicides. The active ingredient blocks electron transfer at the quinone outside inhibitor (QoI) site in the mitochondrial cytochrome b within the bc1 complex, thus obstructing respiration. The in vitro averaged EC50 of baseline isolates collected in 2007 before widespread fungicide use was estimated to be 0.394 µg/ml with PROBIT and 0.427 µg/ml with linear regression analyses. Isolates collected during 2008, 2015, and 2016 had a range of sensitivity as measured by relative spore germination (RG) at a discriminatory dose of 0.4 µg/ml azoxystrobin. Isolates with a higher (≥80%) and lower RG (≤40%) had the wild type nucleotides at amino acid positions F129, G137, and G143 of cytochrome b, sites known to be associated with QoI fungicide resistance. Two Group I introns were found in the QoI target area. The splicing site for the second intron was found immediately after the codon for G143. A mutation for fungicide resistance at this location would hinder splicing and severely reduce fitness. B. oryzae expresses an alternative oxidase in vitro, which allows the fungus to survive inhibition of respiration by azoxystrobin. This research indicates that B. oryzae has not developed resistance to QoI fungicides, although monitoring for changes in sensitivity should be continued. Judicious use of QoI fungicides within an integrated disease management system will promote an effective and environmentally sound control of the pathogen in wild rice paddies.

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Fungicide Resistance in the Cercospora Leaf Blight and Purple Seed Stain Pathogen of Soybean

Grow: Plant Health Exchange ◽

10.1094/grow-soy-07-15-118 ◽

2015 ◽

Keyword(s):

Fungicide Resistance ◽

Leaf Blight ◽

Purple Seed Stain

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Growth Potential of Different Fungicide Resistant Alternaria solani Isolates of Tomato Leaf Blight Pathogen, under In Vitro, in the Presence of Different Fungicide, Growth Temperature and pH Ion Concentration

European Journal of Agriculture and Food Sciences ◽

10.24018/ejfood.2020.2.5.110 ◽

2020 ◽

Vol 2 (5) ◽

Author(s):

V. A. Chavan ◽

S. G. Borkar ◽

Nivedita A Kadam

Keyword(s):

Growth Rate ◽

Fungicide Resistance ◽

Plant Disease ◽

Plant Pathogens ◽

Alternaria Solani ◽

Leaf Blight ◽

Growth Potential ◽

Resistant Isolate ◽

Cross Resistance

Evolution of fungicide resistance in plant pathogens and cross resistance to other fungicides in such pathogens is one of major concerns in sustainable plant disease management. Frequent and unwarranted use of fungicides to control plant diseases not only pollute the earth soil and environment, but also causes the development of fungicide resistance in the plant pathogens, which makes it difficult to manage the plant disease. In India, particularly in western Maharashtra, Alternaria leaf blight pathogen of tomato developed resistance and cross resistance to various fungicides. How these fungicide resistant isolates grow in the different fungicidal environment is reported in the present investigation. Eight different fungicide resistant isolates of Alternaria solani responsible for causing leaf blight in tomato crop were tested for their growth potential, under in vitro condition, on the potato-dextrose-agar(PDA) growth media amended with different fungicides viz. Dithane M-45, Blitox, Kavach, Ridomil, Nativo, Bavistin, Captaf and Score. Different fungicide resistant isolates showed different pattern of growth i.e. complete inhibition of growth, reduced growth or enhancement of growth on different fungicide amended PDA media. The growth of Dithane M-45 resistant isolate was enhanced by fungicide Kavach and Bavistin while reduced by fungicide Blitox. The growth of this fungicide resistant isolate was completely inhibited by fungicide Ridomil, Nativo, Captaf and Score. The maximum enhancement of growth was for Nativo resistant isolate to Bavistin amended PDA media. Bavistin resistant isolate had decreased growth on all fungicides amended PDA media. The minimum reduction in growth was recorded for Kavach resistant isolate on Dithane M-45 fungicide amended PDA media. The growth rate (cm/day) of these fungicide resistant isolates was maximum at 220C temperature as compared to 25 and 300C temperature on the routine PDA growth medium. Captaf resistant isolate produced more growth followed by Nativo, Dithane M-45 and score resistant isolate. The minimum growth was observed for Blitox resistant isolate. As the temperature increases, the growth rate decreased. The growth of fungicide resistant isolates was favored by neutral pH of 7.0 and decreased toward the acidic and alkaline condition. Captaf resistant isolate produced more growth followed by Nativo and score resistant isolates. The minimum growth was produced by Blitox resistant isolate. More growth of fungicide resistant Alternaria isolate means production of more inoculum for disease initiation and spread which is favored at 22 °C temperature and further indicate which fungicide should be used to restrict/manage the growth of particular Alternaria fungicide resistant isolate.

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

Plant Disease ◽

10.1094/pdis-04-13-0449-re ◽

2014 ◽

Vol 98 (1) ◽

pp. 67-76 ◽

Cited By ~ 15

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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