Sterol Demethylation Inhibitor Research Articles

CbCyp51-Mediated Demethylation Inhibitor Resistance Is Modulated by Codon Bias.

Cercospora leaf spot, caused by the fungus Cercospora beticola, is the most destructive foliar disease of sugarbeet worldwide. Resistance to the sterol demethylation inhibitor (DMI) fungicide tetraconazole has been previously correlated with synonymous and nonsynonymous mutations in CbCyp51. Here, we extend these analyses to the DMI fungicides prothioconazole, difenoconazole, and mefentrifluconazole in addition to tetraconazole to confirm whether the synonymous and nonsynonymous mutations at amino acid positions 144 and 170 are associated with resistance to these fungicides. Nearly half of the 593 isolates of C. beticola collected in the Red River Valley of North Dakota and Minnesota in 2021 were resistant to all four DMIs. Another 20% were resistant to tetraconazole and prothioconazole but sensitive to difenoconazole and mefentrifluconazole. A total of 13% of isolates were sensitive to all DMIs tested. We found five CbCyp51 haplotypes and associated them with phenotypes to the four DMIs. The most predominant haplotype (E170_A/L144F_C) correlated with resistance to all four DMIs with up to 97.6% accuracy. The second most common haplotype (E170_A/L144) consisted of isolates associated with resistance phenotypes to tetraconazole and prothioconazole while also exhibiting sensitive phenotypes to difenoconazole and mefentrifluconazole with up to 98.4% accuracy. Quantitative PCR did not identify differences in CbCyp51 expression between haplotypes. This study offers an understanding of the importance of codon usage in fungicide resistance and provides crop management acuity for fungicide application decision-making.

Bioproducts of Pseudomonas chlororaphis Suppress DMI Fungicide-Induced CsCYP51A and CsCYP51B Gene Expression in Colletotrichum siamense and Generate Synergistic Effects with Metconazole and Propiconazole.

Mixtures of fungicides with different modes of action are commonly used as disease and resistance management tools, but little is known of mixtures of natural and synthetic products. In this study, mixtures of metabolites from the rhizobacterium Pseudomonas chlororaphis strain ASF009 formulated as Howler EVO with below-label rates (50 µg/ml) of conventional sterol demethylation inhibitor (DMI) fungicides were investigated for control of anthracnose of cherry (Prunus avium) caused by Colletotrichum siamense. Howler mixed with metconazole or propiconazole synergistically reduced disease severity through lesion growth. Real-time PCR showed that difenoconazole, flutriafol, metconazole, and propiconazole induced the expression of DMI target genes CsCYP51A and CsCYP51B in C. siamense. The addition of Howler completely suppressed the DMI fungicide-induced expression of both CYP51 genes. We hypothesize that the downregulation of DMI fungicide-induced expression of the DMI target genes may, at least in part, explain the synergism observed in detached fruit assays.

Characterization, Molecular Mechanism of Prochloraz-Resistance in Fusarium fujikuroi and Development of Loop-Mediated Isothermal Amplification Rapid Detection Technique Based on the S312T Genotype of Resistances.

Rice bakanae disease (RBD) is a typical seed-borne fungal disease caused by Fusarium fujikuroi. Prochloraz is a sterol demethylation inhibitor, which is among the most important classes of active ingredients for the management of RBD. In 2022, the total resistance frequency of F. fujikuroi to prochloraz in Zhejiang Province was 62.67%. The fitness of the prochloraz-resistant population was lower than that of the susceptible population, but its pathogenicity was slightly stronger. The S312T and F511S double mutations of Ffcyp51b were detected in the resistant isolates. Loop-mediated isothermal amplification (LAMP) technology based on S312T was established to rapidly determine prochloraz resistance in F. fujikuroi. LAMP primer mismatch design was performed based on the cyp51b gene, and 100-300 bp sequences containing a mutation at codon 312 were amplified. In a 25 µL reaction tube, 1 pg/µL DNA of F. fujikuroi could be detected. The detection limit for the frequency of prochloraz resistance was 0.498% using this method. We performed LAMP detection on rice seedlings inoculated with prochloraz-sensitive and -resistant isolates and treated them with prochloraz. Prochloraz demonstrated good control in rice seedlings. A chromogenic reaction was observed in seedlings treated with prochloraz-resistant isolates, and the results were verified using electrophoresis. It has been demonstrated that LAMP technology based on the S312T genotype can quickly and specifically detect prochloraz-resistant isolates in rice seedlings.

Ameliorative effects of gallic acid on tebuconazole–induced adverse effects in the cerebellum of adult albino rats: histopathological and immunohistochemical evidence

ABSTRACT Tebuconazole (TEB) is a common triazole sterol demethylation inhibitor fungicide utilized to manage a variety of diseases in crops like cereals, fruits, and vegetables. The aim of this work was to assess the effects of TEB on the structure of the cerebellum in adult albino rats and possible protective impact of co-administration of Gallic acid (GA). Four groups of forty adult male albino rats were randomly selected, and the rats in group I received corn oil through daily gavage for 4 weeks. Group II received GA dissolved in the normal saline at a dose of 100 mg/kg through daily gavage for 4 weeks, group III administered with TEB dissolved in corn oil at its acceptable daily intake dose (0.02 mg/kg body weight) through daily gavage for 4 weeks, group IV rats received both TEB and GA. For light microscopic, ultrastructural, and immunohistochemical investigations, cerebellar specimens were prepared. TEB exposure led to neuronal damage in the form of degenerated Purkinje cells with vacuolated cytoplasm, areas of lost Purkinje cells, the basket cells appeared vacuolated with degenerated neuropil, the granule cells clumped with congested areas between them, dilated cerebellar islands, weak positive bcl2 immunoreactions in the Purkinje cells, and numerous GFAP-positive astrocytes. GA mitigated TEB-mediated histological changes in the cerebellar cortex. We concluded that TEB caused Purkinje neurons in the rat cerebellar cortex to degenerate and undergo apoptosis. GA had a neuroprotective benefit against TEB toxicity in the rat cerebellar cortex.

Visual detection of fungicide resistance by combining RPA and CRISPR/Cas12a in peach Brown rot fungus Monilinia fructicola.

Peach brown rot caused by Monilinia fructicola severely affects the quality and yield of peach, resulting in large economic losses worldwide. Methyl benzimidazole carbamate (MBC) fungicides and sterol demethylation inhibitor (DMI) fungicides are among the most applied chemical classes used to control the disease but resistance in the target pathogen has made them risky choices. Timely monitoring of resistance to these fungicides in orchards could prevent control failure in practice. In the current study, we developed methods based on recombinase polymerase amplification (RPA) and CRISPR/Cas12a systems to detect MBC and DMI resistance based on the E198A mutation in the β-tubulin (MfTub2) gene and the presence of the Mona element in the upstream region of the MfCYP51, respectively. For MBC resistance, RPA primers were designed that artificially incorporated PAM sites to facilitate the CRISPR/Cas12a reaction. Subsequently, specific tcrRNAs were designed based on the E198A mutation site. For the detection of the Mona element, we designed RPA primers M-DMI-F2/M-DMI-R1 that in combination with crRNA1 detected 'Mona' and distinguished resistant from sensitive strains. Both methods exhibited high sensitivity and specificity, requiring only a simple isothermal device to obtain results within 1 h at 37 °C. The FQ-reporter enabled visualization with a handheld UV or white light flashlight. This method was successfully used with purified DNA from lab cultures and crude DNA from symptomatic fruit tissue, highlighting its potential for on-site detection of resistant strains in orchards. © 2024 Society of Chemical Industry.

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.

Reevaluation of Sensitivity of Monilinia fructicola Isolates to the DMI Fungicide Propiconazole in the Southeastern United States and Investigation of the Genetic Element Mona.

Sterol demethylation inhibitor (DMI) fungicides continue to be essential components for the control of brown rot of peach caused by Monilinia fructicola in the United States and worldwide. In the southeastern United States, resistance to DMIs had been associated with overexpression of the cytochrome P450 14α-demethylase gene MfCYP51 as well as the genetic element Mona, a 65 bp in length nucleotide sequence located upstream of MfCYP51 in resistant isolates. About 20 years after the first survey, we reevaluated sensitivity of M. fructicola from South Carolina and Georgia to propiconazole and also evaluated isolates from Alabama for the first time. A total of 238 M. fructicola isolates were collected from various commercial and two experimental orchards, and sensitivity to propiconazole was determined based on a discriminatory dose of 0.3 μg/ml. Results indicated 16.2, 89.2, and 72.4% of isolates from Alabama, Georgia, and South Carolina, respectively, were resistant to propiconazole. The detection of resistance in Alabama is the first report for the state. All resistant isolates contained Mona, but it was absent from most sensitive isolates. It was unclear if the resistance frequency had increased in South Carolina and Georgia. However, the resistance levels (as assessed by the isolate frequency in discriminatory dose-based relative growth categories) did not change notably, and no evidence of other resistance genotypes was found. Analysis of the upstream MfCYP51 gene region in the resistant isolate CF010 revealed an insertion sequence described for the first time in this report. Our study suggests that current fungicide spray programs have been effective against increasing resistance levels in populations of M. fructicola and suppressing development of new resistant genotypes of the pathogen.

Efficacy of Single- and Multi-Site Fungicides Against Neopestalotiopsis spp. of Strawberry.

Recently, the Florida strawberry industry faced unprecedented outbreaks of an emerging disease caused by the fungus Neopestalotipsis spp. Currently, there are no fungicides labeled to control this disease in the U.S. and the efficacy of single- and multi-site fungicides is unknown. Therefore, this study aimed to determine the in vitro sensitivity of Neopestalotiopsis spp. isolates to fungicides with different modes of action and to evaluate the efficacy of these products on detached fruit and in the field. In preliminary in vitro tests, 30 commercially available fungicides were screened using discriminatory doses. The effective concentration that inhibited mycelial growth by 50% (EC50) was determined for the most effective single-site fungicides. Four field experiments were conducted during the 2019-20, 2020-21, and 2021-22 seasons to determine product efficacy in managing the disease. The single-site fungicides fludioxonil, fluazinam, the sterol de-methylation inhibitors (DMIs), and the multi-sites captan, thiram, and chlorothalonil were the most effective in inhibiting pathogen growth and suppressing disease development. Conversely, products in FRAC groups 1 (MBC) and 7 (SDHI), except for benzovindiflupyr, were not effective against Neopestalotiopsis spp. Resistance to fungicides from FRAC group 11, e.g., azoxystrobin, was confirmed by the presence of the G143A mutation in the cytochrome b gene together with inoculation tests and field trials. Our results provide information to support or discourage the registration of fungicides to manage Neopestalotiopsis fruit rot and leaf spot in strawberry production. .

Baseline Sensitivity and Resistance Mechanism of Colletotrichum Isolates on Tea-Oil Trees of China to Tebuconazole.

Colletotrichum fungi could cause anthracnose, a destructive disease in tea-oil trees. The sterol demethylation inhibitor (DMI) tebuconazole has been widely used in controlling plant diseases for many years. However, the baseline sensitivity of Colletotrichum isolates on tea-oil trees to tebuconazole has not been determined. In this study, the sensitivity to tebuconazole of 117 Colletotrichum isolates from tea-oil trees of seven provinces in southern China was tested. The mean effective concentration resulted in 50% mycelial growth inhibition (EC50), 0.7625 μg/ml. The EC50 values of 100 isolates (83%) were lower than 1 μg/ml, and those of 20 isolates (17%) were higher than 1 μg/ml, which implied that resistance has already occurred in Colletotrichum isolates on tea-oil trees. The EC50 values of the most resistant and sensitive isolates (named Ca-R and Cc-S1, respectively) were 1.8848 and 0.1561 μg/ml, respectively. The resistance mechanism was also investigated in this study. A gene replacement experiment indicated that the CYP51A/B gene of resistant isolates Ca-R and Cf-R1 cannot confer Cc-S1 full resistance to DMI fungicides, although three single point mutants, Cc-S1CYP51A-T306A and Cc-S1CYP51A-R478K, exhibited decreased sensitivity to DMI fungicides. This result suggested that resistance of Colletotrichum isolates was partly caused by mutations in CYP51A. Moreover, the expression level of CYP51A/B was almost identical among Ca-R, Cf-R1, Cc-S1, and Cc-S1CYP51A point mutants, which indicated that the resistance was irrelevant to the expression level of CYP51A, and other nontarget-based resistance mechanisms may exist. Our results could help to guide the application of DMI fungicides and be useful for investigating the mechanism of resistance.

Bioactivity of the DMI fungicide mefentrifluconazole against Sclerotium rolfsii, the causal agent of peanut southern blight.

Sclerotium rolfsii, the causal agent of peanut southern blight, has become increasingly prevalent and harmful in China, causing serious economic losses to the peanut industry. To effectively manage peanut southern blight, this study evaluated the bioactivity of the new-generation sterol demethylation inhibitor (DMI) fungicide mefentrifluconazole against peanut S. rolfsii. In this study, the DMI fungicide mefentrifluconazole exhibited excellent inhibitory activity against the mycelial growth of S. rolfsii, with a mean EC50 value of 0.21 ± 0.11 mg L-1 and a range of 0.02 to 0.55 mg L-1 for 261 isolates collected from Hebei, Henan and Shandong provinces. Mefentrifluconazole significantly reduced the biomass of mycelia and affected the morphology of hyphae. Although sclerotia were more tolerant to mefentrifluconazole than mycelial growth, mefentrifluconazole greatly inhibited the formation and germination of sclerotia. In addition, sclerotia produced by mefentrifluconazole-treated mycelia were deficient in nutrients (e.g., protein, carbohydrate and lipid). These results indicated that mefentrifluconazole may reduce the population of S. rolfsii in the following year. In greenhouse experiments, mefentrifluconazole showed control efficacy and good persistence against peanut S. rolfsii. The preventative and curative activities of mefentrifluconazole at 200 mg L-1 against southern blight still reached 95.36% and 60.94%, respectively, after 9 days of application. No correlation was observed for the sensitivity of S. rolfsii to mefentrifluconazole and the tested DMI, quinone outside inhibitor and succinate dehydrogenase inhibitor fungicides. All data indicated that mefentrifluconazole could provide favorable control efficacy against S. rolfsii from peanuts and reduce the infection and population of S. rolfsii in the following year. © 2023 Society of Chemical Industry.

Involvement of CYP51A and CYP51B in Growth, Reproduction, Pathogenicity, and Sensitivity to Fungicides in Colletotrichum siamense

Strawberry crown rot is a serious fungal disease that poses a great threat to strawberry production in the growth cycle. The dominant pathogens of strawberry crown rot pathogens were different in different periods. The main pathogen of strawberry crown rot at the seedling stage is unclear. In this study, 74 Colletotrichum spp. were isolated from 100 strawberry plants at the seedling stage. Based on the morphological observations and phylogenetic analysis of multiple genes (ACT, CAL, CHS, GAPDH, and ITS), all 74 tested isolates were identified as C. gloeosporioides species complex, including 69 isolates of C. siamense and 5 isolates of C. fructicola. Colletotrichum siamense is the main pathogen of strawberry crown rot at the seedling stage in Zhejiang, China. The sterol demethylation inhibitors (DMIs) were used to control strawberry crown rot, and their target was the CYP51 gene. The role of the homologous CYP51 gene in growth, reproduction, pathogenicity, and sensitivity to DMI fungicides in C. siamense has not been determined. Our study found that the pathogenicity of CsCYP51A deletion mutants to strawberry leaves and stems was weakened. The hyphae growth rate of CsCYP51B deletion mutants was significantly slower than that of the wild type, but the sporulation and appressorium production rates increased. CsCYP51B deletion mutants had significantly increased pathogenicity to the stem. Deletion of CsCYP51A led to increased sensitivity to prothioconazole, ipconazole, hexaconazole, triadimefon, prochloraz, tebuconazole, metconazole, propiconazole, and difenoconazole. CsCYP51B deletion mutants were more insensitive. Our results indicate that the effect of the homologous CsCYP51 gene on hyphae growth, pathogenicity, and sensitivity to DMI fungicides differs.

Sterol demethylation inhibitor fungicide resistance in Colletotrichum siamense from chili is caused by mutations in CYP51A and CYP51B

Anthracnose, caused by fungi of the genus Colletotrichum, is a serious disease of chili worldwide. Sterol 14α-demethylation inhibitors (DMIs) are a class of chemical fungicides that can effectively control anthracnose diseases. In this study, 22 Colletotrichum isolates collected from commercial chili fields in Zhangzhou, Fujian Province, China, were identified as Colletotrichum siamense. The sensitivities of the 22 C. siamense isolates to tebuconazole were determined based on the EC50 (50% effective inhibition concentration) value. The results showed that the EC50 values of the two isolates to tebuconazole were 0.039 ± 0.0036 and 0.042 ± 0.0012 mg/L, while the other 20 isolates showed significantly decreased sensitivities to tebuconazole, with EC50 values ranging from 0.61 ± 0.056 to 1.94 ± 0.11 mg/L. Sequence analysis of CYP51A and CYP51B revealed five genotype mutations (i. e., CYP51AV46L, D115V, P163S, R306K, E397D, CYP51AD115V, S164Y, R306K, E397D, CYP51AD115V, R306K, P339T, E397D, CYP51AD115V, R306K, E397D, S400N, and CYP51AD115V, R306K, E397DCYP51BR266H) in the resistant isolates. The tebuconazole-resistant isolates of five genotypes suffered a fitness penalty and exhibited cross-resistance to difenoconazole, prochloraz, and propiconazole. Additionally, the five genotype mutations were validated as being responsible for tebuconazole-resistance in C. siamense by construction of replacement mutants. Overexpression of CYP51A and CYP51B was not detected in the replacement mutants of the five genotypes. Overall, the present study is the first to report DMI resistance in C. siamense and provides significant information for rational use of DMIs to control chili anthracnose.

Status of Fungicide Resistance and Physiological Characterization of Tebuconazole Resistance in Rhizocotonia solani in Sichuan Province, China.

The resistance prevalence of chemical fungicides has caused increasingly serious agro-ecological environmental problems. However, there are few previous reports about resistance to succinate dehydrogenase (SDHI) or sterol demethylation inhibitor (DMI) in Rhizoctonia solani, one of the main agro-diseases. In this study, the fungicide resistance of 122 R. solani isolates in Sichuan Province was monitored by the mycelial growth rate method. Results showed that all isolates were susceptible to hexaconazole and most isolates were susceptible to thifluzamide, except for the field isolate MSRS-2-7 due to a moderate resistance to thifluzamide (16.43-fold resistance ratio, RR), compared to the sensitivity baseline of thifluzamide (0.042 μg/mL EC50 values). On the contrary, many isolates showed moderate or high resistance to tebuconazole (10.59- to 60.78-fold RR), reaching EC50 values of 0.54~3.10 μg/mL, especially for a highly resistant isolate LZHJ-1-8 displaying moderate resistance to epoxiconazole (35.40-fold RR due to a 3.54 μg/mL EC50 value). The fitness determination found that the tebuconazole-resistant isolates showed higher fitness cost with these characteristics, including a lower growth rate, higher relative electric conductivity, an increased ability to tolerate tebuconazole, and high osmotic pressure. Four new mutations of cytochrome P450 sterol 14α-demethylase (CYP51), namely, S94A, N406S, H793R, and L750P, which is the target for DMI fungicides, was found in the tebuconazole-resistant isolates. Furthermore, the lowest binding energy with tebuconazole was also found in the LZHJ-1-8 isolate possessing all the mutations through analyses with Discovery Studio software. Therefore, these new mutation sites of CYP51 may be linked to the resistance against tebuconazole, and its application for controlling R. solani should be restricted in some areas.

Reduced Sensitivity to Tebuconazole in Botryosphaeria dothidea Isolates Collected from Major Apple Production Areas of China.

China has the largest acreage and is the greatest producer of apples in the world. Apple ring rot (ARR) caused by Botryosphaeria dothidea is one of the major diseases affecting apple fruit production. Tebuconazole, a sterol demethylation-inhibiting (DMI) fungicide, has been increasingly applied to manage ARR in China. Resistance to tebuconazole in field populations of B. dothidea may be selected and accumulate to higher degrees over time. Establishment of tebuconazole sensitivity monitoring programs is critically important for resistance management and effective ARR control. However, the resistance level of B. dothidea to tebuconazole in China remains largely unknown. In this study, invitro mycelial growth assays of B. dothidea in media amended with tebuconazole were conducted, and the sensitivity of B. dothidea to tebuconazole was determined with a set of 390 isolates collected from the major apple production provinces in China between 2006 and 2014. Results showed that the 50% effective concentration (EC50) value ranged from 0.011 to 0.918, 0.040 to 1.621, and 0.052 to 1.925 μg ml-1 with a median value of 0.194, 0.386, and 0.782 μg ml-1 in the isolates collected in 2006, 2010, and 2014, respectively. The frequency distribution of EC50 for tebuconazole was a nonnormal distribution (P < 0.05), suggesting that subpopulations with reduced sensitivity to tebuconazole had emerged in these B. dothidea isolates. The frequency distribution of the B. dothidea isolates collected in 2006 fit a unimodal curve and could be regarded as the baseline sensitivity to tebuconazole. The resistance levels increased over time with the average occurrence frequency of 43.3% and resistance index of 0.38. Positive cross-resistance was observed between tebuconazole and metconazole, which is another DMI fungicide, but multiple resistance was not detected between tebuconazole and non-DMI fungicides. Our results demonstrated that regular long-term resistance monitoring combining with prudent fungicide use should be implemented to prolong the lifespan of tebuconazole in management of ARR in apples.

Baseline Sensitivity and Control Efficacy of Two Quinone Outside Inhibitor Fungicides, Azoxystrobin and Pyraclostrobin, Against Ustilaginoidea virens.

Rice false smut caused by the filamentous fungus Ustilaginoidea virens is a devastating grain disease in rice. Fungicides have been an important measure for the control of this disease. In this study, baseline sensitivities of 179 isolates of U. virens to the quinone outside inhibitor (QoI) fungicides azoxystrobin and pyraclostrobin were established. The distribution of the 50% effective concentration (EC50) values of each fungicide was unimodal. The frequency distribution of logarithmically transformed EC50 values fit or fit closer to a normal distribution. The ranges of EC50 values for azoxystrobin and pyraclostrobin were 0.001 to 0.864 and 0.001 to 0.569 μg/ml, with means and standard errors of the mean values of 0.203 ± 0.012 and 0.079 ± 0.006 μg/ml, respectively. There was a statistically significant and moderately positive correlation (n = 100, r = 0.469, P = 0.001) in sensitivity between these two fungicides. No cross-resistance was found between azoxystrobin, pyraclostrobin, and carbendazim or sterol demethylation inhibitor fungicides. Each fungicide had a significantly higher mean preventive efficacy compared with its curative efficacy. Field assays showed that the control efficacy of pyraclostrobin against rice false smut was greater than that of azoxystrobin. Pyraclostrobin had the best control of rice false smut in three rice varieties, with the control efficacy ranging from 81.5 to 95.5%, whereas azoxystrobin decreased the disease index by 64.1 to 69.2% under the same conditions. These results provide us a reference point in the management of U. virens and future QoI fungicide resistance monitoring programs.

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.

Fungicide resistance in Pyricularia oryzae populations from southern and northern Brazil and evidence of fitness costs for QoI-resistant isolates

Rice blast management is heavily reliant on chemical control, and the rise of fungicide resistance is contingent on fitness costs. This study contrasts the responses of 322 blast isolates from southern (n = 224) and northern Brazil (n = 98) to fungicides in the Quinone outside inhibitor (QoI), Sterol demethylation inhibitor (DMI), and Melanin biosynthesis inhibitor (MBI) groups, and associated epidemiological traits. Resistance was more prevalent in northern Brazil, where conditions are most conducive to blast. Northern populations displayed higher EC50 for spore germination, faster mycelial growth rates, and higher rates of appressorium formation for fungicides in all chemical groups. Results were most striking for the QoI fungicide azoxystrobin. We examined possible fitness penalties and related in planta competitive abilities associated with the gain of azoxystrobin resistance. QoI-resistant (QoI-R) genotypes, carrying the G143A cytochrome b gene mutation, were found in 90% of the northern isolates. The EC50 values of these QoI-R isolates exceeded 10 μg L−1. However, QoI-R isolates had a reduced number of conidia, longer latent period, reduced infection efficiency and aggressiveness, compared to the sensitive isolates. These impairments were reflected in a decrease of competitive ability in planta in the absence of azoxystrobin. Nevertheless, under continuous fungicide applications, the proportion of resistant and sensitive isolates in mixed cultures in planta remained constant after four generations. The results are valuable for managing strategies as the increase of resistance may hamper the competitive abilities of QoI-R mutants in the absence of the respective active ingredient.

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.

Sensitivity of Pyricularia oryzae Populations to Fungicides Over a 26-Year Time Frame in Brazil.

The long-term dynamics of fungicide resistance of the rice blast fungus Pyricularia oryzae was monitored by examining the reaction of the fungal field isolates, collected over a period of 26 years, to the active ingredients of commercially relevant fungicides. The in vitro sensitivity of all isolates was measured against quinone outside inhibitors (QoI), melanin biosynthesis inhibitors, and sterol demethylation inhibitor (DMI) fungicides, namely azoxystrobin (as a QoI), tricyclazole (as a melanin biosynthesis inhibitor), tebuconazole (as a DMI), and trifloxystrobin + tebuconazole (QoI + DMI). Over the 26-year collection period, a gradual rise in the EC50 estimates for mycelial growth sensitivity was observed for all fungicides, but most strikingly for azoxystrobin. A rise in conidial germination and appressorium formation was also noted, most markedly for azoxystrobin. Consistently, the earlier isolates were much more sensitive to the active ingredients than the more contemporary isolates. The sequencing of the amplified cyt b fragment distinguished two haplotypes, H1 and H2. Haplotype H1 (six isolates) contained the G to C transversion at codon 143 (resulting in change G143A), linked to the resistant phenotype QoI-R. Haplotype H2 (40 isolates), gathered the isolates sensitive to QoI. This work documents the gradual rise in the frequency of fungicide-resistant isolates in P. oryzae rice populations on a long-term basis.

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.