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

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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A Rapid Resazurin-Based Microtiter Assay to Evaluate QoI Sensitivity for Alternaria alternata Isolates and Their Molecular Characterization

Plant Disease ◽

10.1094/pdis-12-11-1037-re ◽

2012 ◽

Vol 96 (9) ◽

pp. 1262-1270 ◽

Cited By ~ 25

Author(s):

Byron Vega ◽

Daniele Liberti ◽

Philip F. Harmon ◽

Megan M. Dewdney

Keyword(s):

Alternaria Alternata ◽

Brown Spot ◽

Complete Medium ◽

Potato Dextrose Broth ◽

Qoi Fungicides ◽

Quinone Outside Inhibitor ◽

Yeast Peptone Dextrose ◽

Alternaria Brown Spot ◽

Microtiter Assay ◽

Qoi Resistance

Chemical management of Alternaria brown spot of citrus is based upon the timely application of site-specific fungicides, many of which are vulnerable to the development of fungicide resistance. A rapid microtiter bioassay based on the colorimetric changes of resazurin (RZ) dye was developed to evaluate the sensitivity of Alternaria alternata to quinone outside inhibitor (QoI) fungicides. Four liquid media (complete medium, minimal medium, potato dextrose broth, and yeast peptone dextrose broth), five conidia concentrations (from 101 to 105 conidia/ ml), and five RZ concentrations (10, 20, 30, 40, and 50 μM) were evaluated. Complete medium at 105 conidia/ml and 40 μM RZ were identified as optimal for measuring RZ reduction. The effective concentration of two QoI fungicides (azoxystrobin and pyraclostrobin) needed to reduce RZ by 50% (EC50) was calculated and compared with those obtained from conidia germination tests on fungicide-amended media. Concordant EC50 values were observed (R2 = 0.923; P < 0.0001) from both methods. Resistant phenotypes were further characterized by the partial sequencing of the cytochrome b gene. Genetic variability associated with the presence or absence of two introns was observed among isolates. The identified resistant isolates had the amino acid substitution G143A, typical of QoI resistance in other fungi.

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First Report on Quinone Outside Inhibitor Resistance of Alternaria alternata Causing Alternaria Brown Spot in Tangerines in São Paulo, Brazil

Plant Health Progress ◽

10.1094/php-12-18-0079-br ◽

2019 ◽

Vol 20 (2) ◽

pp. 94-94 ◽

Cited By ~ 1

Author(s):

Gabriel M. Chitolina ◽

Geraldo J. Silva-Junior ◽

Eduardo Feichtenberger ◽

Rosana G. Pereira ◽

Lilian Amorim

Keyword(s):

Alternaria Alternata ◽

Sao Paulo ◽

Field Resistance ◽

Brown Spot ◽

São Paulo ◽

Qoi Fungicides ◽

First Report ◽

Quinone Outside Inhibitor ◽

Alternaria Brown Spot ◽

Inhibitor Resistance

Field resistance to quinone outside inhibitor (QoI) fungicides in Alternaria alternata causing Alternaria brown spot of mandarins was reported by growers in Brazil in 2017. Conidial germination tests were performed, and isolates showed effective concentration to inhibit 50% of conidia germination (EC50) to be over 100 ppm. This is the first report of QoI resistance in A. alternata causing Alternaria brown spot in the state of São Paulo, Brazil.

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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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Molecular Identification of Mutations Conferring Resistance to Azoxystrobin in Cercospora nicotianae

Plant Disease ◽

10.1094/pdis-02-20-0441-re ◽

2020 ◽

Author(s):

Hua Li ◽

William Barlow ◽

Ed Dixon ◽

Bernadette F. Amsden ◽

Robert Hirsch ◽

...

Keyword(s):

Characteristic Curve ◽

Point Mutations ◽

Receiver Operator Characteristic Curve ◽

Resistance Mutations ◽

Pcr Assay ◽

Cytb Sequence ◽

Qoi Fungicides ◽

Quinone Outside Inhibitor ◽

Tobacco Production ◽

Moderately Resistant

Cercospora nicotianae, the causal agent of frogeye leaf spot (FLS) of tobacco, has been exposed to quinone outside inhibitor (QoI) fungicides for over a decade through azoxystrobin applications targeting other major foliar diseases. From 2016 to 2018, a total of 124 isolates were collected from tobacco fields throughout Kentucky. Sensitivity of these isolates to azoxystrobin was previously characterized by determining the effective concentration to inhibit 50% conidial germination (EC50). Based on azoxystrobin EC50, isolates were categorized into three discrete groups: high azoxystrobin sensitivity (< 0.08 µg/ml), moderate azoxystrobin sensitivity (0.14 to 0.64 µg/ml) and low azoxystrobin sensitivity (> 1.18 µg/ml). Variability in sensitivity in a limited number of C. nicotianae isolates was previously shown to be a result of resistance mutations in the fungicide target gene. To improve understanding of C. nicotianae cytochrome b (cytb) structure, the gene was cloned from three isolates representing each EC50group, and sequences were compared. Our analysis showed that cytb gene in C. nicotianae consists of 1161 nucleotides encoding 386 amino acids. Cytb sequence among the cloned isolates was identical with the exception of the F129L and G143A point mutations. To more rapidly determine the resistance status of C. nicotianae isolates to azoxystrobin, a PCR assay was developed to screen for mutations. Using this assay, 80% (n=99) of testedC. nicotianae isolates carried an F129L mutation and were moderately resistant to azoxystrobin, and 7% (n=9) carried the G143A mutation and were highly resistant. A receiver operator characteristic curve analysis suggested the PCR assay is a robust diagnostic tool to identify C. nicotianae isolates with different sensitivity to azoxystrobin in Kentucky tobacco production. The prevalence of both the F129L and G143A mutations in C. nicotianae from Kentucky differs from other pathosystems where resistance to QoI fungicides has been identified, in which the majority of sampled isolates of the pathogen species have a broadly-occurring cytb mutation.

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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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Prevalence of QoI resistance and mtDNA diversity in the Irish Zymoseptoria tritici population

Irish Journal of Agricultural and Food Research ◽

10.2478/ijafr-2019-0004 ◽

2019 ◽

Vol 58 (1) ◽

pp. 27-33

Author(s):

S. Kildea ◽

D.E. Bucar ◽

F. Hutton ◽

S. de la Rosa ◽

T.E. Welch ◽

...

Keyword(s):

Septoria Tritici Blotch ◽

Septoria Tritici ◽

Entire Group ◽

Zymoseptoria Tritici ◽

Qoi Fungicides ◽

Quinone Outside Inhibitor ◽

Strain Collection ◽

Increased Sensitivity ◽

Mtdna Diversity

Abstract The emergence and spread of Quinone outside Inhibitor (QoI) fungicide resistance in the Irish Zymoseptoria tritici population in the early 2000s had immediate impacts on the efficacy of the entire group of fungicides for the control of septoria tritici blotch. As a result, a dramatic reduction in the quantities applied to winter wheat occurred in the following seasons. Even in the absence of these fungicides, the frequency of the resistance allele, G143A in the pathogens mtDNA has remained exceptionally high (>97%), and as such, it can be anticipated that continued poor efficacy of current QoI fungicides will be observed. Amongst the isolates with G143A, differences in sensitivity to the QoI pyraclostrobin were observed in vitro. The addition of the alternative oxidase (AOX) inhibitor salicylhydroxamic acid increased sensitivity in these isolates, suggesting some continued impairment of respiration by the QoI fungicides, albeit weak. Interestingly, amongst those tested, the strains from a site with a high frequency of inserts in the MFS1 transporter gene known to enhance QoI efflux did not exhibit this increase in sensitivity. A total of 19 mtDNA haplotypes were detected amongst the 2017 strain collection. Phylogenetic analysis confirmed the suggestion of a common ancestry of all the haplotypes, even though three of the haplotypes contained at least one sensitive strain.

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

Plant Disease ◽

10.1094/pdis-03-19-0460-re ◽

2020 ◽

Vol 104 (3) ◽

pp. 893-903 ◽

Cited By ~ 2

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.

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