Demethylation Inhibitor Resistance Research Articles

Transcriptome Analysis Reveals Potential Regulators of DMI Fungicide Resistance in the Citrus Postharvest Pathogen Penicillium digitatum.

Green mold, caused by Penicillium digitatum, is the major cause of citrus postharvest decay. Currently, the application of sterol demethylation inhibitor (DMI) fungicide is one of the main control measures to prevent green mold. However, the fungicide-resistance problem in the pathogen P. digitatum is growing. The regulatory mechanism of DMI fungicide resistance in P. digitatum is poorly understood. Here, we first performed transcriptomic analysis of the P. digitatum strain Pdw03 treated with imazalil (IMZ) for 2 and 12 h. A total of 1338 genes were up-regulated and 1635 were down-regulated under IMZ treatment for 2 h compared to control while 1700 were up-regulated and 1661 down-regulated under IMZ treatment for 12 h. The expression of about half of the genes in the ergosterol biosynthesis pathway was affected during IMZ stress. Further analysis identified that 84 of 320 transcription factors (TFs) were differentially expressed at both conditions, making them potential regulators in DMI resistance. To confirm their roles, three differentially expressed TFs were selected to generate disruption mutants using the CRISPR/Cas9 technology. The results showed that two of them had no response to IMZ stress while ∆PdflbC was more sensitive compared with the wild type. However, disruption of PdflbC did not affect the ergosterol content. The defect in IMZ sensitivity of ∆PdflbC was restored by genetic complementation of the mutant with a functional copy of PdflbC. Taken together, our results offer a rich source of information to identify novel regulators in DMI resistance.

Optimizing Use of DMI Fungicides for Management of Apple Powdery Mildew Caused by Podosphaera leucotricha in New York State.

Powdery mildew, caused by the ascomycete Podosphaera leucotricha, is an endemic disease found wherever apples are grown that reduces both tree vigor and fresh market yield. In the absence of durable host resistance, chemical management is the primary means of disease control. Demethylation inhibitor (DMI) fungicides are widely used to manage apple powdery mildew, but members within this fungicide class have been observed to differ in efficacy with respect to disease control. Moreover, debate exists as to the optimal timing of DMI fungicide applications for management of apple powdery mildew. In this regard, the goal of this study was to determine the best-use practices for DMI fungicides to manage apple powdery mildew in New York State. Multiyear trials were conducted to evaluate the potential differential efficacy performance of four common DMI fungicides, and additional trials were conducted to assess optimal application timing. In all years, we observed that treatments of flutriafol and myclobutanil consistently had the lowest incidences of powdery mildew compared with difenoconazole and fenbuconazole. In the 2018 and 2021 trials, the newly registered mefentrifluconazole was more comparable to the difenoconazole program with respect to powdery mildew disease incidence. We hypothesize that differences in DMI efficacy may result from each fungicide's water solubility and lipophilicity characteristics and thus their ability to move systemically in the host or more easily penetrate the surface of germinating conidia. Applications timed between petal fall and first cover resulted in the lowest incidence of powdery mildew on terminal leaves of apple shoots compared with applications timed before petal fall. These observations are contrary to previous studies conducted in regions with differing climates. We also found that the incidence of secondary powdery mildew observed 2 weeks after petal fall was influenced by applications of DMI fungicides during the previous season. For example, management programs consisting of applications of flutriafol or myclobutanil in the previous season tended to have lower incidence of apple powdery in the next spring, presumably because of reductions in overwintering inoculum. Despite reports of DMI resistance in other apple pathosystems, the DMI fungicide class is still relevant for the successful management of apple powdery mildew in New York State.

Characterization of the Molecular Mechanisms of Resistance against DMI Fungicides in Cercospora beticola Populations from the Czech Republic.

Cercospora leaf spot (CLS), caused by the fungal pathogen Cercospora beticola, is the most important foliar pathogen of sugar beet worldwide. Extensive reliance on fungicides to manage CLS has resulted in the evolution of fungicide resistance in C. beticola worldwide, including populations in the Czech Republic. One important class of fungicides used to manage CLS is the sterol demethylation inhibitors (DMI). The aim of our study was to assess DMI resistance in C. beticola from the Czech Republic and elucidate the molecular basis of DMI resistance in this population. A total of 50 isolates were collected in 2018 and 2019 from the major sugar beet growing regions of the Czech Republic and assessed for in vitro sensitivity to the DMI fungicides propiconazole, prochloraz, and epoxiconazole. These analyses identified three strains that exhibited 50% effective concentration (EC50) values > 1.0 μg mL–1 against respective fungicides, which were therefore considered resistant. In contrast, strains that exhibited lowest EC50 values were considered sensitive. To explore the molecular basis of resistance in these three strains, the cytochrome P450-dependent sterol 14α-demethylase (Cyp51) gene was sequenced. Sequence analysis identified a Y464S mutation in all three resistant strains. To assess whether Cyp51 gene expression may play a role in DMI resistance, selected strains were grown in vitro with and without fungicide treatment. These analyses indicated that Cyp51 gene expression was significantly induced after fungicide treatment. Thus, we conclude that Y464S point mutation along with induced Cyp51 gene overexpression is likely responsible for resistance against DMI fungicides in C. beticola from the Czech Republic.

Lack of an Intron in Cytochrome b and Overexpression of Sterol 14α-Demethylase Indicate a Potential Risk for QoI and DMI Resistance Development in Neophysopella spp. on Grapes.

Asian grapevine leaf rust, caused by Neophysopella meliosmae-myrianthae and N. tropicalis, is often controlled by quinone outside inhibitor (QoI) and demethylation inhibitor (DMI) fungicides in Brazil. Here, we evaluated the sensitivity of 55 Neophysopella spp. isolates to pyraclostrobin (QoI) and tebuconazole (DMI). To elucidate the resistance mechanisms, we analyzed the sequences of the cytochrome b (CYTB) and cytochrome P450 sterol 14α-demethylase (CYP51) target proteins of QoI and DMI fungicides, respectively. The CYP51 expression levels were also determined in a selection of isolates. In leaf disc assays, the mean 50% effective concentration (EC50) value for pyraclostrobin was about 0.040 µg/ml for both species. CYTB sequences were identical among all 55 isolates, which did not contain an intron immediately after codon 143. No amino acid substitution was identified at codons 129, 137, and 143. The mean EC50 value for tebuconazole was 0.62 µg/ml for N. tropicalis and 0.46 µg/ml for N. meliosmae-myrianthae, and no CYP51 sequence variation was identified among isolates of the same species. However, five N. meliosmae-myrianthae isolates grew on leaf discs treated at 10 µg/ml tebuconazole, and these were further exposed to tebuconazole selection pressure. Tebuconazole-adapted laboratory isolates of N. meliosmae-myrianthae showed an eight- to 25-fold increase in resistance after four rounds of selection that was not associated with CYP51 targetalterations. In comparison with sensitive isolates, CYP51 expression was induced in the presence of tebuconazole in three out of four tebuconazole-adapted isolates tested. These results suggest a potential risk for QoI and DMI resistance development in Neophysopella spp.

Spatio-temporal distribution of DMI and SDHI fungicide resistance of Zymoseptoria tritici throughout Europe based on frequencies of key target-site alterations.

Over the past decade, demethylation inhibitor (DMI) and succinate dehydrogenase inhibitor (SDHI) fungicides have been extensively used to control to septoria tritici blotch, caused by Zymoseptoria tritici on wheat. This has led to the development and selection of alterations in the target-site enzymes (CYP51 and SDH, respectively). Taking advantage of newly and previously developed qPCR assays, the frequency of key alterations associated with DMI (CYP51-S524T) and SDHI (SDHC-T79N/I, C-N86S and C-H152R) resistance was assessed in Z. tritici-infected wheat leaf samples collected from commercial crops (n= 140) across 14 European countries prior to fungicide application in the spring of 2019. This revealed the presence of a West to East gradient in the frequencies of the most common key alterations conferring azole (S524T) and SDHI resistance (T79N and N86S), with the highest frequencies measured in Ireland and Great Britain. These observations were corroborated by sequencing (CYP51 and SDH subunits) and sensitivity phenotyping (prothioconazole-desthio and fluxapyroxad) of Z. tritici isolates collected from a selection of field samples. Additional sampling made at the end of the 2019 season confirmed the continued increase in frequency of the targeted alterations. Investigations on historical leaf DNA samples originating from different European countries revealed that the frequency of all key alterations (except C-T79I) has been gradually increasing over the past decade. Whilst these alterations are quickly becoming dominant in Ireland and Great Britain, scope still exists to delay their selection throughout the wider European population, emphasizing the need for the implementation of fungicide antiresistance measures. © 2021 Society of Chemical Industry.

Genome-Wide Association and Selective Sweep Studies Reveal the Complex Genetic Architecture of DMI Fungicide Resistance in Cercospora beticola.

The rapid and widespread evolution of fungicide resistance remains a challenge for crop disease management. The demethylation inhibitor (DMI) class of fungicides is a widely used chemistry for managing disease, but there has been a gradual decline in efficacy in many crop pathosystems. Reliance on DMI fungicides has increased resistance in populations of the plant pathogenic fungus Cercospora beticola worldwide. To better understand the genetic and evolutionary basis for DMI resistance in C. beticola, a genome-wide association study (GWAS) and selective sweep analysis were conducted for the first time in this species. We performed whole-genome resequencing of 190 C. beticola isolates infecting sugar beet (Beta vulgaris ssp. vulgaris). All isolates were phenotyped for sensitivity to the DMI tetraconazole. Intragenic markers on chromosomes 1, 4, and 9 were significantly associated with DMI fungicide resistance, including a polyketide synthase gene and the gene encoding the DMI target CbCYP51. Haplotype analysis of CbCYP51 identified a synonymous mutation (E170) and nonsynonymous mutations (L144F, I387M, and Y464S) associated with DMI resistance. Genome-wide scans of selection showed that several of the GWAS mutations for fungicide resistance resided in regions that have recently undergone a selective sweep. Using radial plate growth on selected media as a fitness proxy, we did not find a trade-off associated with DMI fungicide resistance. Taken together, we show that population genomic data from a crop pathogen can allow the identification of mutations conferring fungicide resistance and inform about their origins in the pathogen population.

The FgCYP51B Y123H Mutation Confers Reduced Sensitivity to Prochloraz and Is Important for Conidiation and Ascospore Development in Fusarium graminearum.

Fusarium graminearum is one of the most important causal agents of Fusarium head blight disease and is controlled mainly by chemicals such as demethylation inhibitor (DMI) fungicides. FgCYP51B is one of the DMI targets in F. graminearum, and Tyrosine123 (Y123) is an important amino acid in F. graminearum CYP51B, located in one of predicted substrate binding pockets based on the binding mode between DMIs and CYP51B. Previous studies suggest that resistance to DMI fungicides is attributed primarily to point mutations in the CYP51 gene and that the Y123H mutation in F. verticillioides CYP51 confers prochloraz resistance in the laboratory. To investigate the function of FgCYP51B Y123 residue in the growth and development, pathogenicity, and DMI resistance, we generated and analyzed the FgCYP51B Y123H mutant. Results revealed that the Y123H mutation led to reduced conidial sporulation and affected ascospore development; moreover, the mutation conferred reduced sensitivity to prochloraz. Quantitative PCR and molecular docking were performed to investigate the resistance mechanism. Results indicated that Y123H mutation changed the target gene expression and decreased the binding affinity of FgCYP51 to prochloraz. These results will attract more attention to the potential DMI-resistant mutation of F. graminearum and increase our understanding of the DMI resistance mechanism.

DMI-Fungicide Resistance in Venturia nashicola, the Causal Agent of Asian Pear Scab-How Reliable Are Mycelial Growth Tests in Culture?

Scab, caused by Venturia nashicola, is among the most serious diseases of Asian pears and control of this disease largely relies on sterol demethylation inhibitor (DMI) fungicides. However, pear growers have complained about field performance of DMIs since the mid-2000s. In this study, to evaluate pathogen sensitivity, mycelial growth tests and inoculation tests were conducted using DMI-amended culture medium and fungicide-sprayed potted pear trees, respectively. Results confirmed distribution of isolates resistant to fenarimol, hexaconazole, and difenoconazole in the field populations. Importantly, results from tests in culture did not fully correlate with those from tests in planta. Due to phenotypic instability of resistance and poor sporulation of this pathogen in culture, resistance is generally assessed by laborious and time-consuming inoculation with conidia collected from a field. To improve the result interpretation from in vitro tests, the isolates were genotyped: the CYP51 gene which encodes the target sterol 14α-demethylase was sequenced and various mutations have been detected in the coding sequence of DMI-resistant isolates. In addition to the detected single nucleotide polymorphisms, alternative mechanisms, not based on changes in the structure of the target protein, may also increase DMI resistance. Development of molecular methods for the diagnosis of DMI resistance seems to be challenging in V. nashicola.

A world-wide analysis of reduced sensitivity to DMI fungicides in the banana pathogen Pseudocercospora fijiensis.

BACKGROUND Pseudocercospora fijiensis is the causal agent of the black leaf streak disease (BLSD) of banana. Bananas are important global export commodities and a major staple food. Their susceptibility to BLSD pushes disease management towards excessive fungicide use, largely relying on multisite inhibitors and sterol demethylation inhibitors (DMIs). These fungicides are ubiquitous in plant disease control, targeting the CYP51 enzyme. We examined sensitivity to DMIs in P. fijiensis field isolates collected from various major banana production zones in Colombia, Costa Rica, Dominican Republic, Ecuador, the Philippines, Guadalupe, Martinique and Cameroon and determined the underlying genetic reasons for the observed phenotypes.RESULTSWe observed a continuous range of sensitivity towards the DMI fungicides difenoconazole, epoxiconazole and propiconazole with clear cross‐sensitivity. Sequence analyses of PfCYP51 in 266 isolates showed 28 independent amino acid substitutions, nine of which correlated with reduced sensitivity to DMIs. In addition to the mutations, we observed up to six insertions in the Pfcyp51 promoter. Such promoter insertions contain repeated elements with a palindromic core and correlate with the enhanced expression of Pfcyp51 and hence with reduced DMI sensitivity. Wild‐type isolates from unsprayed bananas fields did not contain any promoter insertions.CONCLUSIONThe presented data significantly contribute to understanding of the evolution and global distribution of DMI resistance mechanisms in P. fijiensis field populations and facilitate the prediction of different DMI efficacy. The overall reduced DMI sensitivity calls for the deployment of a wider range of solutions for sustainable control of this major banana disease. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Survey and Genetic Analysis of Demethylation Inhibitor Fungicide Resistance in Monilinia fructicola From Michigan Orchards.

Resistance to sterol demethylation inhibitor (DMI) fungicides in Monilinia fructicola, causal agent of brown rot of stone fruit, has been reported in the southeastern and eastern United States and in Brazil. DMI resistance of some M. fructicola isolates, in particular those recovered from the southeastern United States, is associated with a sequence element termed "Mona" that causes overexpression of the cytochrome demethylase target gene MfCYP51. In this study, we conducted statewide surveys of Michigan stone fruit orchards from 2009 to 2011 and in 2019, and we determined the sensitivity to propiconazole of a total of 813 isolates of M. fructicola. A total of 80.7% of Michigan isolates were characterized as resistant to propiconazole by relative growth assays, but the Mona insert was not uniformly detected and was present in some isolates that were not characterized as DMI resistant. Gene expression assays indicated that elevated expression of MfCYP51 was only weakly correlated with DMI resistance in M. fructicola isolates from Michigan, and there was no obvious correlation between the presence of the Mona element and elevated expression of MfCYP51. However, sequence analysis of MfCYP51 from 25 DMI-resistant isolates did not reveal any point mutations that could be correlated with resistance. Amplification and sequencing upstream of MfCYP51 resulted in detection of DNA insertions in a wide range of isolates typed by DMI phenotype and the presence of Mona or other unique sequences. The function of these unique sequences or their presence upstream of MfCYP51 cannot be correlated to a DMI-resistant genotype at this time. Our results indicate that DMI resistance was established in Michigan populations of M. fructicola by 2009 to 2011, and that relative resistance levels have continued to increase to the point that practical resistance is present in most orchards. In addition, the presence of the Mona insert is not a marker for identifying DMI-resistant isolates of M. fructicola in Michigan.

Resistance to demethylation inhibitors in Cercospora beticola, a pathogen of sugar beet in Japan, and development of unique cross-resistance patterns

Cercospora beticola Sacc. is the most destructive pathogen of sugar beet (Beta vulgaris L.) and causes Cercospora leaf spot (CLS). Since 1986, fungicides that function as demethylation inhibitors (DMIs) have been used to control CLS in Hokkaido, which is the only area in Japan where sugar beet is grown. Reduced sensitivity of C. beticola to DMI fungicides, based on the half maximal effective concentration (EC50), was first reported in Hokkaido in 1999, however the fungicides continued to be used effectively until 2014. In a field experiment in 2016, we found that the efficacy of difenoconazole against the field population of C. beticola was greatly reduced. We subsequently tested over 600 isolates collected throughout the sugar beet-growing region of Hokkaido and revealed that the mean resistance factor of four DMI fungicides (difenoconazole, fenbuconazole, tebuconazole, and tetraconazole) were high, which indicates that DMI-resistant isolates were distributed throughout the beet cultivation area. Moreover, we identified three types of isolates that have unique cross-resistance patterns between difenoconazole and fenbuconazole, with their EC50 rate (= difenoconazole EC50/ fenbuconazole EC50) converged to 31, 4.0, and 0.40, respectively, which appeared to be affected by the local history of fungicide usage. The F144L substitution in CbCYP51 was only found in the group whose EC50 rate was 0.40. This is the first report of DMI resistance in C. beticola in Japan, and the findings in this study could contribute to our understanding of the mechanism of DMI resistance.

Fungicide sensitivity and resistance in the blackleg fungus,

Fungicide use has become a fundamental part of many crop protection systems around the world, including to control blackleg disease on canola (Brassica napus L.). In Australia, most canola growers routinely apply at least one fungicide, and potentially multiple fungicides with different modes of action, in a single growing season. There is evidence for the emergence of fungicide resistance in Leptosphaeria maculans, the causal agent of blackleg disease, to the demethylation inhibitor (DMI) class of fungicides in Australia. However, it is not known whether resistance exists towards other chemical classes such as the succinate dehydrogenase inhibitors (SDHI). In this work, 397 samples were screened for resistance towards seven fungicide treatments in stubble-borne L. maculans populations collected from eight canola-growing agro-ecological regions of Australia from 2018 to 2020, a time frame that bridges the introduction of new chemicals for blackleg control. We confirmed that DMI resistance in L. maculans is pervasive across all of the sampled canola-growing regions, with 15% of fungal populations displaying high levels (resistance scores >0.5) of resistance towards the DMI fungicides. Although resistance to newly introduced SDHI fungicides was low, we found evidence of positive cross-resistance between established DMI-only fungicides and a newly introduced combined DMI and quinone outside inhibitor fungicide, suggesting that the efficacy of the latter may be limited by widespread DMI resistance. Proactive surveillance, as performed here, may provide a means to avoid the rapid loss of fungicide efficacy in the field.

Detection and Biological Characteristics of Alternaria alternata Resistant to Difenoconazole from Paris polyphylla var. chinensis, an Indigenous Medicinal Herb.

Black spot caused by Alternaria alternata (BSAA) is one of the most common diseases of Paris polyphylla var. chinensis, causing yield losses in China. Demethylation inhibitors (DMIs) have been used to control this disease in China for decades. Some farmers have complained about the decreased efficacy of DMIs against BSAA. The objective of this study was to detect and characterize the resistance of A. alternata against difenoconazole from P. polyphylla var. chinensis during 2018. Of the 22 isolates of A. alternata obtained from Sichuan Province in the southwest of China, 20 were resistant to difenoconazole. Mycelial growth rates and sporulation of the difenoconazole-resistant (DfnR) isolates were not different from those of the difenoconazole-sensitive (DfnS) isolates. No cross resistance between difenoconazole and tebuconazole or propiconazole was observed. Mutations were identified at gene AaCYP51 of DfnR isolates based on the sequence alignment of the DfnR and DfnS isolates. All of the mutations could be divided into three resistant genotypes, I (K715R + Y781C), II (K715R + D1140G + T1628A), and III (no mutation). The docking total score of the DfnS isolates was 5.6020, higher than the resistant genotype I (4.4599) or the resistant genotype II (3.8651), suggesting that the DMI resistance of A. alternata may be caused by the decreased affinity between AaCYP51 and difenoconazole.

Difenoconazole Resistance Shift in Botrytis cinerea From Tomato in China Associated With Inducible Expression of CYP51.

Gray mold caused by Botrytis cinerea is one of the most important diseases in tomato. It can be controlled effectively by demethylation inhibitor (DMI) fungicides, but their resistance status after long-term use in the field is unclear. The baseline sensitivity to difenoconazole of 142 B. cinerea isolates from China with no history of DMI usage was characterized, with a mean effective concentration for 50% mycelial growth inhibition (EC50) of 0.97 ± 0.50 μg/ml. EC50 values for difenoconazole sensitivity of another 248 isolates collected in 2011 and 2016 ranged from 0.04 to 11.99 μg/ml, and the frequency of difenoconazole sensitivity formed a nonnormal distribution curve. Detached fruit studies revealed that isolates with EC50 values of approximately 6.00 μg/ml were not controlled effectively. The mean EC50 of the resistant isolates changed from 6.74 to 8.65 μg/ml between 2011 and 2016. Positive cross-resistance was only observed between difenoconazole and two DMIs. One dual resistant isolate and one triple resistant isolate were found among the difenoconazole-resistant isolates collected in 2016, associated with point mutations in corresponding target proteins of the fungicides azoxystrobin and fludioxonil. This indicated that B. cinerea not only showed higher difenoconazole resistance levels but gradually changed from single to multiple fungicide resistance over time. No amino acid variation was found in the CYP51 protein. In the absence of difenoconazole, the relative expression of CYP51 was not significantly different in sensitive and resistant isolates. Induced expression of CYP51 is an important determinant of DMI resistance in B. cinerea from tomato. However, nucleotide variants found in the upstream region had no association with the fungicide resistance phenotype. These results will be helpful for the management of B. cinerea in the field.

Characterization of tebuconazole resistance in Botrytis cinerea from tomato plants in China

Fungal pathogen Botrytis cinerea, the casual agent of gray mold of vegetables and fruits, has a high risk of developing resistance to fungicide. Tebuconazole, one kind of demethylation inhibitor (DMI) fungicides, has been increasingly applied for the control of tomato gray mold in China. However, very limited information is available on the resistance profile of B. cinerea to tebuconazole. In this study, the baseline sensitivity of B. cinerea to tebuconazole was determined based on 138 isolates from field sites having no history of DMI usage, with a mean EC50 value of 0.29 μg/mL. Another 159 B. cinerea isolates collected in the greenhouse and field from 2011 to 2016 were demonstrated to have a shifted sensitivity to tebuconazole, with a mean EC50 value of 0.66 μg/mL. EC50 values of 10 B. cinerea isolates with reduced sensitivity to tebuconazole were greater than 1.56 μg/mL, and these reduced-sensitive isolates had a fitness penalty in sporulation and conidial germination, but showed similar mycelial growth rate and pathogenicity with those of the sensitive isolates. Positive cross-resistance was observed only between tebuconazole and the other two DMIs difenoconazole and prochloraz, but not between tebuconazole and the non-DMIs iprodione, procymidone, or fludioxonil. In reduced-sensitive isolates, no amino acid variation was found in the BcCYP51 protein. When exposed to tebuconazole, the expression level of BcCYP51 increased in these reduced-sensitive B. cinerea isolates as compared to sensitive ones, thus contributing to the reduced sensitivity of the pathogen to tebuconazole. Additionally, the nucleotide mutation observed in the 1200 bp upstream region of BcCYP51 had no correlation with the development of fungicide resistance in B. cinerea isolates. These findings will be helpful for the understanding of DMI resistance mechnism in B. cinerea.

Monitoring and analysis of rice pathogen Ustilaginoidea virens isolates with resistance to sterol demethylation inhibitors in China

Rice false smut (RFS), caused by Ustilaginoidea virens (Cooke) Takah, is an important fungal disease of rice. In China, sterol demethylation inhibitors (DMIs) are common fungicides used to control RFS. In a previous study, we detected two propiconazole-resistant U. virens isolates in 2015 in Huai’an city, Jiangsu Province, China. In the current study, we detected six propiconazole-resistant isolates out of 180 U. virens isolates collected from rice fields in Jiangsu Province in 2017, and found they were from three different places (Xuzhou, Huai’an and Jintan). All these six propiconazole-resistant isolates were cross-resistant to three other sterol demethylation inhibitor (DMI) fungicides, i.e. difenoconazole, tebuconazole, and epoxiconazole. Among them, two isolates (2017–61 and 2017–170) had high fitness. Through sequencing and RT-qPCR analysis, we found that the expression levels of CYP51 and its encoded protein were significantly increased in the propiconazole-resistant isolates with a “CC” insertion mutation upstream of the CYP51 coding region compared to the propiconazole-sensitive isolates. In addition, propiconazole stimulated CYP51 expression in all isolates. Propiconazole also stimulated the accumulation of CYP51 protein in propiconazole-sensitive isolates and propiconazole-resistant isolates without mutation, but not in propiconazole-resistant isolates with the “CC” mutation. According to JASPAR database analysis, the predicated functional binding sites for propiconazole-resistant isolates with a “CC” insertion mutation and propiconazole-sensitive isolates were different. Given the high fitness of the propiconazole-resistant isolates, the development of resistance to DMIs in U. virens should be monitored. Furthermore, we speculated that the over-expression of CYP51 may contribute to DMI resistance in U. virens with the “CC” insertion mutation.

Mutations at sterol 14α-demethylases (CYP51A&B) confer the DMI resistance in Colletotrichum gloeosporioides from grape.

Grape anthracnose caused by the ascomycete fungus Colletotrichum gloeosporioides has been widely controlled by demethylation inhibitors (DMIs) for decades in China. The resistance status and mechanism of C. gloeosporioides against DMIs is not well understood. All difenoconazole-resistant (DfnR ) isolates from vineyards exhibited decreased fitness. Positive cross-resistance was detected between DMI triazoles. Sequence alignment results from the DfnR and DfnS isolates revealed that multiple mutations are distributed at CgCYP51A, concomitant with mutations at CgCYP51B. The half maximal effective concentration (EC50 ) values of single deleted and complemented mutants of CgCYP51A and CgCYP51B showed that ΔCgCYP51A became more sensitive to difenoconazole, but not ΔCgCYP51B. Furthermore, all single complemented mutants had a stronger biological fitness than the progenitor strain. All the defectives of ΔCgCYP51A and ΔCgCYP51B could be restored by complementation of the whole corresponding gene from the resistant strains. Relative gene expression of CgCYP51A and CgCYP51B in most of the mutants was greatly upregulated relative to the progenitor isolate when treated with difenoconazole at the same concentration. Moreover, the extension of five amino acids (GNETI) caused by mutation at the stop codon of CgCYP51A, concurrent with other seven amino acid substitutions and the synonymous mutation P10P (CCG → CCT), significantly enhanced DMI resistance. The DMI resistance of C. gloeosporioides selected in vineyards is conferred by mutations at CgCYP51s, and validated by a genetics method. The roles of CgCYP51A and CgCYP51B overlap, and are counter-balanced, but cannot be replaced reciprocally. © 2020 Society of Chemical Industry.

Sterol Demethylation Inhibitor Fungicide Resistance in Leptosphaeria maculans is Caused by Modifications in the Regulatory Region of ERG11.

Blackleg is a worldwide disease of canola (Brassica napus), caused by a complex of fungal species in the genus Leptosphaeria, that impacts canola production and seed quality. Demethylation inhibitor (DMI) fungicides that target sterol 14α-demethylase are an integral part of disease control. Here, we report six DMI-resistant isolates of Leptosphaeria maculans and two different types of genetic modification related to the resistance. Analysis of the regulatory region of the DMI target gene ERG11 (also known as CYP51) revealed a 275-bp insertion in two of the isolates and three long terminal repeat retrotransposons (5,263, 5,267, and 5,248 bp) inserted in the promoter region of three resistant isolates. Genetic approaches confirmed that these elements are responsible for DMI resistance in L. maculans and crosses show segregation consistent with a single locus. Reverse-transcription quantitative PCR assays demonstrated that the 275-bp insertion increases ERG11 gene expression, conferring DMI fungicide resistance both in vitro and in planta. Moreover, transformation of a susceptible isolate of L. maculans with ERG11 driven by a promoter containing the 275-bp insertion increased resistance to tebuconazole. A minimal shift of the values of concentration whereby 50% of the mycelial growth is inhibited in vitro was observed in resistant isolates containing long terminal repeat retrotransposons; nevertheless, these isolates were able to develop significant lesions on cotyledons from fungicide-treated seedlings. This is the first report of genetic modifications in L. maculans relating to DMI fungicide resistance.

Induced expression of CYP51 associated with difenoconazole resistance in the pathogenic Alternaria sect. on potato in China.

Early blight caused by Alternaria spp. is amongst the most important diseases in potato. Demethylation inhibitor (DMI) fungicides are widely used to control the disease but long-term use may decrease its control efficacy due to fungicide resistance. This study investigated the occurrence of difenoconazole resistance in Alternaria spp. and molecular resistant mechanisms. EC50 values of 160 isolates to difenoconazole ranged from 0.026 μg mL-1 to 15.506 μg mL-1 and the frequency of difenoconazole sensitivity formed a non-normal distribution curve with a major and a minor peak. Isolates with EC50 values of 4.121 and 5.461 μg mL-1 were not controlled effectively at fungicide doses of 50 and 100 μg mL-1 . Cross-resistance was observed between DMI fungicides difenoconazole and propiconazole, but not between difenoconazole and other fungicide groups, including boscalid, iprodione, or carbendazim. The CYP51gene was 1673 bp encoding 525 amino acids in length and contained two introns. All sensitive and resistant isolates had the identical amino acid sequence of CYP51, with the exception of one resistant isolate carrying a mutation of R511W. A 6bp insertion in the upstream region was observed in half of the resistant isolates. In the absence of propiconazole, the relative expression of CYP51 was not significantly different in sensitive and resistant isolates. In the presence of difenoconazole, expression of CYP51 gene was induced significantly in the DMI-resistant isolates but not in the sensitive ones. Induced expression of CYP51 in resistant isolates exposed to difenoconazole is an important determinant for DMI resistance in potato pathogens Alternaria sect. © 2019 Society of Chemical Industry.

Characterization of difenoconazole resistance in Lasiodiplodia theobromae from papaya in Brazil.

Stem-end rot caused by Lasiodiplodia theobromae is one of the most important diseases of papaya in northeastern Brazil. It can be controlled effectively by demethylation inhibitor (DMI) fungicides, but the occurrence of DMI resistance may decrease fungicide efficacy. Detached fruit studies revealed that isolates with EC50 values of 6.07 and 6.28 μg mL-1 were not controlled effectively, but reduced virulence and ability to grow at temperatures ranging from 12 to 32 °C suggesting fitness penalties were observed. Cross-resistance was observed only between difenoconazole and propiconazole. The entire cytochrome P450 sterol 14α-demethylase (LtCYP51) gene and its flanking regions were cloned. The gene was 1746 bp in length and contained three introns. The predicted protein contained 525 amino acids. Phylogenetic tree analysis showed that the LtCYP51 belongs to the CYP51B clade. No amino acid variation was found between sensitive and resistant isolates; however, the gene was constitutively more highly expressed in resistant isolates. Resistance to DMI fungicides in L. theobromae is based on LtCYP51 gene overexpression and fitness penalties may be present in difenoconazole-resistant isolates. © 2019 Society of Chemical Industry.