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

2021 ◽  
pp. PHP-04-21-0072-
Author(s):  
Tyler M. Smith ◽  
Heather Y. Kelly ◽  
Kurt H. Lamour
Keyword(s):  
United States ◽  
Cytochrome B ◽  
The United States ◽  
Corynespora Cassiicola ◽  
Cyt B ◽  
Qoi Fungicides ◽  
Quinone Outside Inhibitor ◽  
Target Spot ◽  
Causal Pathogen ◽  
Cyt B Gene

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.

scholarly journals Reduced Sensitivity to Azoxystrobin of Monilinia fructicola Isolates From Brazilian Stone Fruits is Not Associated With Previously Described Mutations in the Cytochrome b Gene

Plant Disease ◽  
2017 ◽  
Vol 101 (5) ◽  
pp. 766-773 ◽  
Author(s):  
Wagner V. Pereira ◽  
Isabela V. Primiano ◽  
Rafael G. F. Morales ◽  
Natalia A. Peres ◽  
Lilian Amorim ◽  
...  
Keyword(s):  
Cytochrome B ◽  
Ex Vivo ◽  
Brown Rot ◽  
Monilinia Fructicola ◽  
Cyt B ◽  
Qoi Fungicides ◽  
Quinone Outside Inhibitor ◽  
Low Sensitivity ◽  
Cyt B Gene

Quinone-outside inhibitor (QoI) fungicides are effective tools for preharvest control of brown rot of stone fruit. These fungicides have a very specific site of action so the risk of resistance selection is high. The sensitivity of Monilinia fructicola (G. Winter) Honey isolates to azoxystrobin (QoI) was investigated in 143 isolates collected between 2002 and 2011 from four Brazilian states in orchards with different frequencies of fungicide use (0 to 6 fungicides sprays/season). Sensitivity of the isolates to azoxystrobin was determined in vitro, by inhibition of mycelial growth and spore germination on fungicide-amended media or ex vivo by pathogen inoculation in untreated or treated fruit with azoxystrobin. Potential mutations in codons 143, 137, and 129 of the cytochrome b (Cyt b) gene and the occurrence of an intron immediately after codon 143 were analyzed in a subpopulation of the isolates. The M. fructicola population of São Paulo State was less sensitive to the fungicide than the population from the states of Paraná, Santa Catarina, and Rio Grande do Sul. The low sensitivity of the isolates was confirmed also by comparing to the sensitivity of the baseline isolates. Mutations in G143A, F129L, and G137R in Cyt b gene were not found. In addition, 58 isolates tested showed an intron after codon 143 in Cyt b gene. Our results indicate that other mechanisms of selection for low sensitivity to QoI fungicides should be investigated.

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

Horticulturae ◽  
2021 ◽  
Vol 7 (6) ◽  
pp. 155
Author(s):  
Xiuhuan Li ◽  
Chengcheng Li ◽  
Guixiang Li ◽  
Jiamei Zhu ◽  
Feng Liu ◽  
...  
Keyword(s):  
Point Mutation ◽  
Phytopathogenic Fungi ◽  
Shandong Province ◽  
Lamp Method ◽  
Corynespora Cassiicola ◽  
Cyt B ◽  
Qoi Fungicides ◽  
Quinone Outside Inhibitor ◽  
Target Spot ◽  
Qoi 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.

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 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.

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.

scholarly journals A Two-Step Molecular Detection Method for Pyrenophora tritici-repentis Isolates Insensitive to QoI Fungicides

Plant Disease ◽  
2011 ◽  
Vol 95 (12) ◽  
pp. 1558-1564 ◽  
Author(s):  
Jaimin S. Patel ◽  
Steven W. Meinhardt ◽  
Helge Sierotzki ◽  
Gerd Stammler ◽  
Neil C. Gudmestad ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Molecular Detection ◽  
Detection Method ◽  
The United States ◽  
Tan Spot ◽  
Complex Iii ◽  
Cyt B ◽  
Qoi Fungicides ◽  
Pyrenophora Tritici Repentis ◽  
Cyt B Gene

Tan spot, caused by Pyrenophora tritici-repentis, is an important disease of wheat worldwide. To manage tan spot, quinone outside inhibitor (QoI) fungicides such as azoxystrobin and pyraclostrobin have been applied in many countries. QoI fungicides target the cytochrome b (cyt b) site in complex III of mitochondria and, thus, pose a serious risk for resistance development. The resistance mechanism to QoI fungicides is mainly due to point mutations in the cyt b gene. The objective of this study was to develop a molecular detection method for the four currently known mutations responsible for shifts in sensitivity toward QoI fungicides in P. tritici-repentis. Twelve specific primers were designed based on sequences from the National Center for Biotechnology Information accessions AAXI01000704 and DQ919068 and used to generate a fragment of the cyt b gene which possesses four known single-nucleotide polymorphisms (SNPs). These mutant clones served as positive controls because QoI-insensitive and -reduced-sensitive isolates of P. tritici-repentis have not yet been reported in the United States. The partial cyt b gene clones were sequenced to identify the SNPs at sites G143A and F129L. Genomic DNA of the mutated partial cyt b gene clones and the European QoI-insensitive and -reduced-sensitive isolates of P. tritici-repentis possessing G143A (GCT) and F129L (TTA, TTG, and CTC) mutations were amplified by polymerase chain reaction (PCR) using two specific primer pairs and were further digested with three specific restriction enzymes (BsaJI, Fnu4HI, and MnlI). The results of the digested PCR product from genomic DNA of known QoI-insensitive and -reduced-sensitive isolates of P. tritici-repentis had DNA bands consistent with the mutation GCT at G143A and the mutations TTA, TTG, and CTC at F129L. The amplified region at the F129 site also had 99% sequence similarity with P. teres, the net blotch pathogen of barley. To validate mutations, we further tested two isolates of P. teres known to have reduced sensitivity to QoI fungicides possessing the mutations TTA and CTC at F129L. After PCR amplification and restriction digestion, DNA bands identical to those observed for the partial cyt b mutant clones were detected. These results suggest that this newly developed two-step molecular detection method is rapid, robust, and specific to monitor QoI-insensitive and -reduce-dsensitive isolates of P. tritici-repentis.

scholarly journals Heteroplasmy of the cytochrome b Gene in Venturia inaequalis and Its Involvement in Quantitative and Practical Resistance to Trifloxystrobin

2014 ◽  
Vol 104 (9) ◽  
pp. 945-953 ◽  
Author(s):  
Sara M. Villani ◽  
Kerik D. Cox
Keyword(s):  
Cytochrome B ◽  
Relative Abundance ◽  
Venturia Inaequalis ◽  
Apple Scab ◽  
Cyt B ◽  
Resistance Response ◽  
Relative Growth ◽  
Qoi Fungicides ◽  
Cyt B Gene ◽  
Moderate Resistance

Quantitative (partial) and qualitative (complete) resistance responses to quinone outside inhibitor (QoI) fungicides have been documented for the apple scab pathogen Venturia inaequalis. Resistance monitoring efforts have traditionally focused on the detection of qualitative resistance based on a single point mutation, G143A, within the cytochrome b (cyt b) gene. In order to better understand the role of heteroplasmy of the cyt b gene in the QoI resistance response for isolates and populations of V. inaequalis, an allele-specific quantitative polymerase chain reaction was developed to quantify the relative abundance of the A143 (resistant) allele in 45 isolates of V. inaequalis with differing in vitro resistance responses to the QoI fungicide trifloxystrobin. Although a high relative abundance of the A143 allele (>62%) was associated with isolates with high resistance responses (50 to 100% relative growth on trifloxystrobin-amended medium), heteroplasmy of the cyt b gene was not the primary factor involved in isolates with moderate resistance responses (29 to 49% relative growth). The relative abundance of the A143 allele in isolates with moderate resistance to trifloxystrobin rarely exceeded 8%, suggesting that other resistance mechanisms are involved in moderate resistance and, therefore, that the Qol resistance response is polygenic. In research orchards where QoI fungicides failed to control apple scab (practical resistance), field trials were conducted to demonstrate the link between practical resistance and the abundance of the A143 allele. Relative abundance of the A143 allele in these orchard populations exceeded 20% in 2011 and 2012. Similarly, of the eight additional commercial orchards screened in 2011, the relative abundance of the A143 allele always exceeded 20% in those with QoI practical resistance. Although heteroplasmy of the cyt b gene did not entirely explain the response of isolates with moderate resistance to QoIs, the relative abundance of A143 in orchard populations of V. inaequalis helps to explain the point of emergence for practical resistance to trifloxystrobin across several orchard populations with differing production histories.

scholarly journals Phylogenetic Diversity and Host Specialization of Corynespora cassiicola Responsible for Emerging Target Spot Disease of Cotton and Other Crops in the Southeastern United States

2018 ◽  
Vol 108 (7) ◽  
pp. 892-901 ◽  
Author(s):  
Leilani G. Sumabat ◽  
Robert C. Kemerait ◽  
Marin Talbot Brewer
Keyword(s):  
United States ◽  
Southeastern United States ◽  
Geographic Location ◽  
The United States ◽  
Plant Diseases ◽  
Host Specialization ◽  
Corynespora Cassiicola ◽  
Target Spot ◽  
Nuclear Loci

Corynespora cassiicola is a ubiquitous fungus causing emerging plant diseases worldwide, including target spot of cotton, soybean, and tomato, which have rapidly increased in incidence and severity throughout the southeastern United States. The objectives of this study were to understand the causes for the emerging target spot epidemics in the United States by comparing phylogenetic relationships of isolates from cotton, tomato, soybean, and other crop plants and ornamental hosts, and through the determination of the host range of isolates from emerging populations. Fifty-three isolates were sampled from plants in the southeastern United States and 1,380 nucleotides from four nuclear loci were sequenced. Additionally, sequences of the same loci from 23 isolates representing each of the distinct lineages of C. cassiicola described from previous studies were included. Isolates clustered based on host of origin, regardless of the geographic location of sampling. There was no genetic diversity detected among isolates from cotton, which were genetically distinct from isolates from other host species. Furthermore, pathogenicity and virulence assays of 40 isolates from various hosts onto cotton, soybean, tomato, and cucumber showed that isolates from cotton were more aggressive to cotton than those from other hosts. Soybean and tomato were most susceptible to isolates that originated from the same host, providing evidence of host specialization. These results suggest that emerging target spot epidemics in the United States are caused by either the introduction of host-specific isolates or the evolution of more aggressive strains on each host.

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

Plant Disease ◽  
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.

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.

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