QoI Resistance Research Articles

Fungicide Sensitivity and Non-Target Site Resistance in Rhizoctonia zeae Isolates Collected from Corn and Soybean Fields in Nebraska.

Rhizoctonia zeae was recently identified as the major Rhizoctonia species in corn and soybean fields in Nebraska and was shown to be pathogenic on corn and soybean seedlings. Fungicide seed treatments commonly used to manage seedling diseases include prothioconazole (demethylation inhibitor), fludioxonil (phenylpyrrole), sedaxane (succinate dehydrogenase inhibitor), and azoxystrobin (quinone outside inhibitor; QoI). To establish the sensitivity of R. zeae to these fungicides, we isolated this pathogen from corn and soybean fields in Nebraska during 2015 to 2017 and estimated the relative effective concentration for 50% inhibition (EC50) of a total of 91 R. zeae isolates from Nebraska and Illinois. Average EC50 for prothioconazole, fludioxonil, sedaxane, and azoxystrobin was 0.219, 0.099, 0.078, and > 100 µgml-1, respectively. In planta assays showed that azoxystrobin did not significantly reduce the disease severity on soybean (P > 0.05). The cytochrome b gene of R. zeae did not harbor any mutation known to confer QoI resistance and had a type-I intron directly after codon 143 suggesting that a G143A mutation is unlikely to evolve in this pathogen. For prothioconazole, fludioxonil, and sedaxane, EC50 of isolates did not differ significantly among years of collection (P > 0.05) and their single discriminatory concentrations were identified as 0.1 µgml-1. This is the first study to establish non-target site resistance of R. zeae to azoxystrobin and the sensitivity of R. zeae to commonly used seed treatment fungicides in Nebraska. This information will help to guide strategies for chemical control of R. zeae and monitor sensitivity shifts in future.

Comparative Fitness of Monilinia fructicola Isolates with Multiple Fungicide-Resistant Phenotypes.

This study characterized 52 isolates of Monilinia fructicola from peach and nectarine orchards for their multiresistance patterns to thiophanate-methyl (TF), tebuconazole (TEB), and azoxystrobin (AZO) using in vitro sensitivity assays and molecular analysis. The radial growth of M. fructicola isolates was measured on media amended with a single discriminatory dose of 1 μg/ml for TF and AZO and 0.3 μg/ml for TEB. Cyt b, CYP51, and β-tubulin were tested for point mutations that confer resistance to quinone outside inhibitors (QoIs), demethylation inhibitors (DMIs), and methyl benzimidazole carbamates (MBCs), respectively. Eight phenotypes were identified, including isolates with single, double, and triple in vitro resistance to QoI, MBC, and DMI fungicides. All resistant phenotypes to TF and TEB presented the H6Y mutation in β-tubulin and the G641S mutation in CYP51. None of the point mutations typically linked to QoI resistance were present in the Monilinia isolates examined. Moreover, fitness of the M. fructicola phenotypes was examined in vitro and in detached fruit assays. Phenotypes with single resistance displayed equal fitness in vitro and in fruit assays compared with the wild type. In contrast, the dual- and triple-resistance phenotypes suffered fitness penalties based on osmotic sensitivity and aggressiveness on peach fruit. In this study, multiple resistance to MBC, DMI, and QoI fungicide groups was confirmed in M. fructicola. Results suggest that Monilinia populations with multiple resistance phenotypes are likely to be less competitive in the field than those with single resistance, thereby impeding their establishment over time and facilitating disease management.

Revisiting the intron hypothesis of QoI resistance in Phyllosticta ampelicida, the causal agent of grape black rot, and other Phyllosticta species

AbstractChemical control of grape black rot, caused by Phyllosticta ampelicida, relies mainly on the use of demethylation inhibitors (DMIs) and quinone outside inhibitors (QoIs). The effectiveness of QoI fungicides is influenced by alternative respiration activity, and the exon/intron structure and point mutations in the target protein's gene, the cytochrome b (cytb) gene. Our study aims to investigate the QoI fungicide sensitivity of 48 P. ampelicida isolates in vitro by measuring EC50 and the molecular characteristics of the cytb gene and its mRNA in P. ampelicida and other Phyllosticta species. Mycelial growth tests revealed that the P. ampelicida isolates were sensitive to both azoxystrobin and trifloxystrobin; baseline EC50 values were 0.029 and 0.022 μg/mL, respectively. Addition of salicylhydroxamic acid (SHAM) resulted in lower EC50 values (0.024 and 0.017 μg/mL, respectively). None of the typical point mutations conferring resistance to QoIs in some fungi were detected. A group I intron was present right after the 143rd codon in the cytb gene in four of the six Phyllosticta species examined. The sequence and exon/intron structure of the cytb gene of P. ampelicida isolated from Vitis vinifera is studied in detail and published here. Our results indicate a low risk of QoI resistance development via the G143A mutation in P. ampelicida.

Development of a PNA-LNA-LAMP Assay to Detect an SNP Associated with QoI Resistance in Erysiphe necator.

The repetitive use of quinone outside inhibitor fungicides (QoIs, strobilurins; Fungicide Resistance Action Committee [FRAC] 11) to manage grape powdery mildew has led to development of resistance in Erysiphe necator. While several point mutations in the mitochondrial cytochrome b gene are associated with resistance to QoI fungicides, the substitution of glycine to alanine at codon 143 (G143A) has been the only mutation observed in QoI-resistant field populations. Allele-specific detection methods such as digital droplet PCR and TaqMan probe-based assays can be used to detect the G143A mutation. In this study, a peptide nucleic acid-locked nucleic acid mediated loop-mediated isothermal amplification (PNA-LNA-LAMP) assay consisting of an A-143 reaction and a G-143 reaction, was designed for rapidly detecting QoI resistance in E. necator. The A-143 reaction amplifies the mutant A-143 allele faster than the wild-type G-143 allele, while the G-143 reaction amplifies the G-143 allele faster than the A-143 allele. Identification of resistant or sensitive E. necator samples was determined by which reaction had the shorter time to amplification. Sixteen single-spore QoI-resistant and -sensitive E. necator isolates were tested using both assays. Assay specificity in distinguishing the single nucleotide polymorphism (SNP) approached 100% when tested using purified DNA of QoI-sensitive and -resistant E. necator isolates. This diagnostic tool was sensitive to one-conidium equivalent of extracted DNA with an R2 value of 0.82 and 0.87 for the G-143 and A-143 reactions, respectively. This diagnostic approach was also evaluated against a TaqMan probe-based assay using 92 E. necator samples collected from vineyards. The PNA-LNA-LAMP assay detected QoI resistance in ≤30 min and showed 100% agreement with the TaqMan probe-based assay (≤1.5 h) for the QoI-sensitive and -resistant isolates. There was 73.3% agreement with the TaqMan probe-based assay when samples had mixed populations with both G-143 and A-143 alleles present. Validation of the PNA-LNA-LAMP assay was conducted in three different laboratories with different equipment. The results showed 94.4% accuracy in one laboratory and 100% accuracy in two other laboratories. The PNA-LNA-LAMP diagnostic tool was faster and required less expensive equipment relative to the previously developed TaqMan probe-based assay, making it accessible to a broader range of diagnostic laboratories for detection of QoI resistance in E. necator. This research demonstrates the utility of the PNA-LANA-LAMP for discriminating SNPs from field samples and its utility for point-of-care monitoring of plant pathogen genotypes.

PCR–RFLP analysis for detecting potential QoI and CAA fungicide resistance in onion and lettuce downy mildews

PCR–RFLP analysis for detecting potential QoI and CAA fungicide resistance in onion and lettuce downy mildews

Fungicide sensitivity levels in the Lithuanian Zymoseptoria tritici population in 2021.

Zymoseptoria tritici causes the disease known as septoria leaf blotch in winter wheat and is a major factor in yield loss worldwide. Farmers are inclined to use fungicides to protect their crops; however, the efficacy of these measures is rapidly decreasing due to the natural mechanisms of mutation emergence in pathogen populations. Increasing fungicide resistance is being recorded worldwide, therefore, screening of the current situation in Lithuania is essential to determine the subsequent steps of crop protection strategies. In this study, in vitro fungicide sensitivity tests, mutation detection, and field experiments were carried out. The mean EC50 values for prothioconazole-desthio and mefentrifluconazole were 0.14 and 0.28 mg/l, respectively. Increased frequency of the mutation S524T, linked to DMIs resistance, was observed. Results revealed that the dominant point mutation in the gene CYP51 was I381V, and the most frequent CYP51 haplotype was D13 (V136C, I381V, Y461H, S524T). The mutation G143A, linked to QoI resistance, was detected in ¾ of the population. Mutations conferring resistance to SDHIs were not detected in single pycnidium isolates. Two-year field experiments likewise showed no decline in field efficacy of SDHI fungicide in Lithuania. Moreover, the baseline sensitivity of the Lithuanian Z. tritici population to QiI fungicide fenpicoxamid was established. The findings of this study provide an update on the current status of fungicide resistance in the Lithuanian Z. tritici population.

Evidence of Resistance to QoI Fungicides in Contemporary Populations of Mycosphaerella fijiensis, M. musicola and M. thailandica from Banana Plantations in Southeastern Brazil

Yellow and black Sigatoka, caused by Mycosphaerella fijiensis and M. musicola, respectively, are the most important worldwide foliar diseases of bananas. Disease control is heavily dependent on intensive fungicide sprays, which increase selection pressure for fungicide resistance in pathogen populations. The primary objective of this study was to assess the level and spread of resistance to quinone-outside inhibitors (QoI—strobilurin) fungicides in populations of both pathogens sampled from banana fields under different fungicide spray regimes in Southeastern Brazil. Secondly, we aimed to investigate when QoI resistance was confirmed if this was associated with the target-site alteration G143A caused by a mutation in the mitochondrial encoded cytochrome b gene. QoI resistance was detected in fungicide treated banana fields, while no resistance was detected in the organic banana field. A total of 18.5% of the isolates sampled from the pathogens’ populations were resistant to QoI. The newly described M. thailandica was also found. It was the second most abundant Mycosphaerella species associated with Sigatoka-like leaf spot symptoms in the Ribeira Valley and the highest level of QoI resistance was found for this pathogen. The G143A cytochrome b alteration was associated with the resistance to the QoI fungicides azoxystrobin and trifloxystrobin in M. fijiensis, M. musicola and M. thailandica strains. In order to reduce resistance development and maintain the efficacy of QoI fungicides, anti-resistance management strategies based on integrated disease management practices should be implemented to control the Sigatoka disease complex.

Resistance characterization of the natural population and resistance mechanism to pyraclostrobin in Lasiodiplodia theobromae

Lasiodiplodia theobromae is the main pathogen of mango stem-end rot disease, causing mango fruit decay and major economic loss. QoI resistance has been found in field populations of L. theobromae. The characterization and resistance mechanism of pyraclostrobin-resistant L. theobromae was investigated by using a combination of bioassays and biochemical and molecular methods. The pyraclostrobin resistance among the L. theobromae population samples from Hainan was 93.41%. The resistant isolates were stable after successive subculturing for 10 times on PDA. Cross-resistance was observed only between the Qols pyraclostrobin and azoxystrobin. The alternative oxidase (AOX) inhibitor SHAM notably decreased the EC50 values of pyraclostrobin for all tested L. theobromae isolates. Induction of AOX by pyraclostrobin was observed in mycelia cells of L. theobromae. After treatment with pyraclostrobin, the final ATP and AOX contents of all sensitive isolates were significantly lower than those of resistant isolates. The relevant mutation and high expression of the cytochrome b gene were not detected in resistant isolates. However, there were 4 mutations in the AOX gene, which were only observed in highly resistant isolates. Pretreatment with pyraclostrobin resulted in a significant upregulation of AOX gene expression, and the average expression level of the highly resistant isolates was 33-fold that of the control group. These results suggested that the AOX pathway is responsible for resistance to pyraclostrobin, and that the AOX-related resistance mechanism is common in field populations of L. theobromae in Hainan mango orchards.

Acquired QoI resistance in Pyrenophora teres through an interspecific partial gene transfer by Pyrenophora tritici-repentis?

Acquired QoI resistance in Pyrenophora teres through an interspecific partial gene transfer by Pyrenophora tritici-repentis?

Distribution of the F129L mutation conferring resistance to strobilurins in Alternaria solani populations in Serbia

AbstractAlternaria solani is a fungal pathogen that causes early blight, an economically important disease of potatoes worldwide. The application of fungicides differing in mode of action are the most important control measure for early blight management. However, frequent use of fungicides may cause fungal pathogen populations to be selected for mutations conferring fungicide resistance, resulting in significant losses of fungicide efficacy. Repeated use of QoI fungicides may generate a selection pressure on A. solani populations that promotes resistant genotypes, this quality in question being because of amino acid substitutions in the cytochrome b gene. During a four‐year survey, isolates of A. solani were collected from the primary potato production regions in Serbia. The cytochrome b gene region of each isolate was evaluated for the presence of the F129L substitution known to historically confer QoI resistance. Sequence analysis revealed two genotypes similar to those reported elsewhere in the world with a diverse geographic distribution in Serbia. Genotype I was dominant in the south of Serbia, genotype II was prevalent in north Serbia, and in central Serbia both genotypes were represented equally. The F129L mutation was detected in both genotypes, with a progressive shift from the wild type to mutated A. solani across years. A. solani isolates containing the F129L mutation were collected from the northern and the central Serbia in 2016 without any mutated isolates in the southern Serbia. In 2019, in all surveyed regions, a predominance of isolates with the F129L mutation was observed. The presence of two cytochrome b genotypes among A. solani isolates and significant presence of F129L mutations indicates a change in the genetic structure of this pathogens populations in potato fields nationwide, which could have a significant impact on control of early blight disease.

Fungicide resistance frequencies of Botrytis cinerea greenhouse isolates and molecular detection of a novel SDHI resistance mutation

Sensitivity of B. cinerea to commonly used fungicides against Gray mold with emphasis to the newer quinone outside inhibitor (QoIs), and succinate dehydrogenase inhibitors (SDHIs) was assessed during a monitoring survey from vegetable greenhouses in four representative regions of Crete. 42% from a total of 168 isolates were simultaneously resistant to boscalid, fluopyram, pyraclostrobin and fenhexamid but not to fludioxonil making this phenylpyrrole fungicide an excellent anti-resistance antifungal agent. Isolates with double resistance to SDHIs and QoIs were found in very high frequencies indicating a selection towards double resistance due to the use of pyraclostrobin-boscalid mixtures. A number of sdhB resistance mutations (H272R, N230I and P225F/H) were found in isolates also carrying the G143A cytb resistance mutation in the above isolates. A novel sdhB point mutation (I274V) was identified for the first time in B. cinerea isolates collected from greenhouses with a fluopyram spray history with specific resistance to SDHIs. A PCR-RFLP diagnostic assay was developed for the detection of this mutation in the sdhB gene. Mutations P225F/H and I274V were found to be associated with fitness penalties in terms of mycelial growth, sporulation or pathogenicity. Results suggest that, in order to retain effective control of gray mold in Crete, appropriate anti-resistance strategies should be implemented taking into account the high double SDHI and QoI resistance frequencies. Additional studies for monitoring the already known and the new SDHI-resistance mutations, are necessary in order to hinder the further spread and establishment of single or double resistant isolates of B. cinerea detected in greenhouses in Crete.

Assessment of QoI and CAA fungicide resistance of Plasmopara viticola populations in vineyards of the Great Lakes region in the United States of America

Assessment of QoI and CAA fungicide resistance of <i>Plasmopara viticola</i> populations in vineyards of the Great Lakes region in the United States of America

Detection and characterization of QoI resistance in Pyrenophora tritici‐repentis populations causing tan spot of wheat in Argentina

AbstractTan spot, caused by the fungus Pyrenophora tritici‐repentis (Ptr), is a disease that has become more prevalent and intense in wheat crops in Argentina in recent years. Failure to control the disease with strobilurin fungicides, which were once effective, has been observed in different zones where wheat is grown. However, whether or not true resistance is present in the pathogen population in the region is not scientifically confirmed. This study evaluated the sensitivity of numerous Ptr isolates to representative QoI fungicides used in Argentina through in vitro and in planta assays, as well as through molecular analysis. Eighty‐two monosporic isolates obtained in different locations in the north and south of Buenos Aires province in 2014, 2016, and 2018 were tested to determine sensitivity to selected QoI fungicides in conidial germination and mycelial inhibition assays, as well as in molecular analysis. Conidial germination was not inhibited at 1 µg/ml of azoxystrobin, trifloxystrobin, and pyraclostrobin. On the other hand, mycelial growth was inhibited by 59%, 56%, and 86% at 100 µg/ml of azoxystrobin, trifloxystrobin, and pyraclostrobin, respectively. The molecular analysis detected the G143A mutation in the cytb gene of all the 82 Ptr isolates, but the F129L and G137R substitutions were not present. This study documents the G143A mutation conferring QoI resistance in Ptr in South America. The findings of this study are key for future decisions regarding use of fungicide and rotation in the region.

First report of QoI resistance in Italian population of Pyricularia oryzae

First report of QoI resistance in Italian population of Pyricularia oryzae

Molecular and biochemical characterization of Colletotrichum gloeosporioides isolates resistant to azoxystrobin from grape in China

AbstractAnthracnose, caused by Colletotrichum gloeosporioides, is one of the most important diseases in grape‐growing regions worldwide. In Jiangsu Province of China, quinone‐outside inhibitor fungicides (QoIs) have been extensively sprayed as disease control for more than 10 years. A spore germination assay of 64 isolates obtained from 32 commercial vineyards was used to assess isolate sensitivity to azoxystrobin and 62 were found to be resistant to azoxystrobin. The biological fitness of QoI‐resistant (QoIR) isolates was significantly lower than the sensitive isolates (QoIS) in terms of mycelial growth and conidiation. Nucleotide sequence alignment of CgCytb genes from the QoIR and QoIS isolates revealed that two point mutations (F129L and G143A) are involved in the QoI resistance. Isolates with the G143A mutation expressed high resistance to azoxystrobin, whereas isolates carrying the F129L mutation exhibited moderate resistance. Positive cross‐resistance was observed between azoxystrobin and kersoxim‐methyl, pyraclostrobin, or benzothiostrobin, but not with fluazinam. This study provides important information for management of QoIR populations of C. gloeosporioides in the field.

Sensitivity of the Pyrenophora teres Population in Algeria to Quinone outside Inhibitors, Succinate Dehydrogenase Inhibitors and Demethylation Inhibitors.

Net blotch of barley caused by Pyrenophora teres (Died.) Drechsler, is one of the most destructive diseases on barley in Algeria. It occurs in two forms: P. teres f. teres and P. teres f. maculata. A total of 212 isolates, obtained from 58 fields sampled in several barley growing areas, were assessed for fungicide sensitivity by target gene analysis. F129L and G137R mitochondrial cytochrome b substitution associated with quinone outside inhibitors (QoIs) resistance, and succinate dehydrogenase inhibitors (SDHIs) related mutations (B-H277, C-N75S, C-G79R, C-H134R, and C-S135R), were analyzed by pyrosequencing. In vitro sensitivity of 45 isolates, towards six fungicides belonging to three chemical groups (QoI, demethylase inhibitor, and SDHI) was tested by microtiter technique. Additionally, sensitivity towards three fungicides (azoxystrobin, fluxapyroxad, and epoxiconazole) was assessed in planta under glasshouse conditions. All tested isolates were QoI-sensitive and SDHI-sensitive, no mutation that confers resistance was identified. EC50 values showed that pyraclostrobin and azoxystrobin are the most efficient fungicides in vitro, whereas fluxapyroxad displayed the best disease inhibition in planta (81% inhibition at 1/9 of the full dose). The EC50 values recorded for each form of net blotch showed no significant difference in efficiency of QoI treatments and propiconazole on each form. However, in the case of fluxapyroxad, epoxiconazole and tebuconazole treatments, analysis showed significant differences in their efficiency. To our knowledge, this study is the first investigation related to mutations associated to QoI and SDHI fungicide resistance in Algerian P. teres population, as well as it is the first evaluation of the sensitivity of P. teres population towards these six fungicides.

Discovery of metyltetraprole: Identification of tetrazolinone pharmacophore to overcome QoI resistance

Quinone outside inhibitors (QoIs) are one of the major agricultural fungicide groups used worldwide. However, the development of resistance by different pathogenic species associated with specific mutation at the target gene site is becoming a critical issue for the sustainable use of QoIs. The authors aimed to design a novel QoI molecule to overcome the aforementioned issue. A rational approach to avoid steric hindrance between the QoI molecule and the mutated target site was successfully employed. The resulting compound, metyltetraprole, is characterized by 3-substituted central ring with a tetrazolinone moiety, the key structure to retain potent activity against QoI-resistant mutants. Metyltetraprole is a promising new fungicide under commercial development, and its development in this study has paved the way to overcoming resistance to QoI fungicides.

First report of QoI resistance in Cercospora beticola in sugar beet in Japan

QoI fungicides have been an important tool for controlling Cercospora leaf spot (CLS) of sugar beet in Japan. In 2013, CLS control failed at an experimental plot in Okhotsk subprefecture of Hokkaido after the application of trifloxystrobin. Among isolates of Cercospora beticola, the causal agent of CLS, collected from this field were the first QoI-resistant strains found in Japan based on bioassays, culture assays and detection of G143A mutation in the cytochrome b gene. Additionally, our surveys of commercial fields in 2014–2015 revealed the widespread QoI resistance of C. beticola in Hokkaido.

Reduced sensitivity of azoxystrobin and thiophanate-methyl resistance in Lasiodiplodia theobromae from papaya

Stem-end rot caused by Lasiodiplodia theobromae is one of the most devastating diseases of papaya in northeastern Brazil. It is most effectively controlled by applications of fungicides, including site-specific fungicides at risk for resistance development. This study investigated the molecular mechanisms of reduced sensitivity to the QoI fungicide azoxystrobin and resistance to the MBC fungicide thiophanate-methyl in L. theobromae from Brazilian orchards. The EC50 values for azoxystrobin in sixty-four isolates ranged from 0.36 μg/ml to 364.24 μg/ml and the frequency distribution of EC50 values formed a multimodal curve, indicating reduced sensitivity to azoxystrobin. In detached fruit assays reduced sensitive isolates were not controlled as effectively as sensitive isolates at lowest label rate. Partial fragments were obtained from target genes β-tubulin (751 bp) and Cytb (687 bp) of isolates resistant to thiophanate-methyl and reduced sensitivity to azoxystrobin. Sequence analysis of the β-tubulin fragment revealed a mutation corresponding to E198K in all thiophanate-methyl-resistant isolates, while reduced sensitivity to axoxystrobin was not attributable to Cytb gene alterations. The target gene-based mechanism conferring resistance to thiophanate-methyl will likely be stable even if selection pressure subsides. However, the mechanism conferring reduced sensitivity to azoxystrobin is not based on target gene modifications and thus may not be as stable as other genotypes with mutations in Cytb gene.

Cloning of the Cytochrome b Gene From the Tomato Powdery Mildew Fungus Leveillula taurica Reveals High Levels of Allelic Variation and Heteroplasmy for the G143A Mutation.

Leveillula taurica is a major pathogen of tomato and several other crops that can cause substantial yield losses in favorable conditions for the fungus. Quinone outside inhibitor fungicides (QoIs) are routinely used for the control of the pathogen in tomato fields across California, but their recurrent use could lead to the emergence of resistance against these compounds. Here, we partially cloned the cytochrome b gene from L. taurica (Lt cytb) and searched within populations of the fungus collected from tomato fields across California for mutations that confer resistance to QoIs. A total of 21 single nucleotide polymorphisms (SNPs) were identified within a 704 bp fragment of the Lt cytb gene analyzed, of which five were non-synonymous substitutions. Among the most frequent SNPs encountered within field populations of the pathogen was the G143A substitution that confers high levels of resistance against QoIs in several fungi. The other four amino acid substitutions were novel mutations, whose effect on QoI resistance is currently unknown. Sequencing of the Lt cytb gene from individual single-cell conidia of the fungus further revealed that most SNPs, including the one leading to the G143A substitution, were present in a heteroplasmic state, indicating the co-existence of multiple mitotypes in single cells. Analysis of the field samples showed that the G143A substitution is predominantly heteroplasmic also within field populations of L. taurica in California, suggesting that QoI resistance in this fungus is likely to be quantitative rather than qualitative.