Demethylation Inhibitor Fungicides 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.

Global spread, genetic differentiation and selection of barley spot form of net blotch isolates.

Spot form of net blotch, caused by Pyrenophora teres f. maculata, is a significant necrotrophic disease of barley that spread world-wide in the 20th century. Genetic relationships were analysed to determine the diversity, survival and dispersal of a diverse collection of 346 isolates from Australia, Southern Africa, North America, Asia Minor and Europe. The results, based on genome-wide DArTseq data, indicated isolates from Turkey were the most differentiated with regional sub-structuring, together with individuals closely related to geographically distant genotypes. Elsewhere, population subdivision related to country of origin was evident, although low levels of admixturing was found that may represent rare genotypes or migration from unsampled populations. Canadian isolates were the next most diverged and Australian and South African the most closely related. With the exception of Turkish isolates, multiple independent Cyp51A mutation events (which confer insensitivity to demethylation inhibitor fungicides) between countries and within regions was evident, with strong selection for a transposable element insertion at the 3' end of the promoter and counter-selection elsewhere. Individuals from Western Australia shared genomic regions and Cyp51A haplotypes with South African isolates, suggesting a recent common origin.

Sensitivity of the causal agent of northern leaf blight of corn, Exserohilum turcicum, to the demethylase inhibiting fungicide flutriafol

Fungicides including active ingredients in the demethylation inhibitor group (Fungicide Resistance Action Committee Group 3) are used to manage corn diseases such as northern corn leaf blight, caused by Exserohilum turcicum. This study assessed the level of sensitivity to the demethylation-inhibiting fungicide flutriafol on a population of 81 Exserohilum turcicum isolates collected from across the United States in 2020. Sensitivity to flutriafol for E. turcicum was assessed using fungicide-amended media to determine the effective concentration needed to limit hyphal growth by 50%. Mean absolute EC50 values of the overall population calculated using the 4-parameter log-logistic and Weibull2 4-parameter models were 2.1881 and 3.2643 µg/ml, respectively. Growth of most isolates (n=70, 86.4%) was completely inhibited by 1 µg/ml flutriafol. However, growth of seven isolates (8.6%) was only completely inhibited at 10 or 100 µg/ml and for four isolates (4.9%) complete inhibition was not observed at 100 µg/ml. These results suggest that there may be isolates of E. turcicum in the U.S. that are resistant to flutriafol. Additional fungicide sensitivity screening is critical to monitoring for the development of fungicide resistance in U.S. populations of E. turcicum.

Influence of propiconazole and metconazole formulations on Bacillus subtilis vegetative cell growth and disease control of fruit crops.

Biological control agent (BCA) Bacillus subtilis formulated as Theia® is registered for control of fungal and bacterial diseases of fruit crops. Combinations of Theia® and strategic concentrations of two demethylation inhibitor (DMI) fungicides were investigated to explore potential synergisms. Bacteria were cultured in nutrient broth and combined with technical grades and two formulations of propiconazole (EC and WP) and metconazole (EC and WDG) at 0, 10, 50, 100, and 150 µg/ml active ingredient. After co-cultivation, optical density (OD600) and colony forming units (CFU/ml) were evaluated. In contrast to EC formulations, the WP or WDG formulations at 10 or 50 µg/ml of both DMIs did not affect vegetative cell growth. The mixture of Theia® and each formulated DMI at 50 µg/ml active ingredient resulted in a significant reduction of Monilinia fructicola lesion development on apple, Colletotrichum siamense lesion development on cherry, and Botrytis cinerea lesion development on cherry. The combination of Theia® with EC formulations showed weaker disease reduction due to antagonism. Only Theia® + non-EC formulated propiconazole and metconazole significantly reduced brown rot disease incidence of apple compared to the respective solo treatments and anthracnose disease incidence of cherry compared to the untreated control. Our results indicated that at least some DMI fungicides possess bactericidal effects depending on the formulation and concentration. The combination of Theia® with a lower than label rate concentration (50 µg/ml) of the DMI fungicides propiconazole and metconazole showed potential for synergistic effects especially when non-EC formulations were used.

Barley powdery mildew control in Western Australia and beyond

AbstractAustralia is one of the largest barley exporters in the world, with Western Australia accounting for some 40% of national production. The crop is predominantly grown in the south and south‐west of the state in winter and spring, where temperate conditions and higher rainfall levels are more suited to barley than northern and eastern regions. Between 2007 and 2013, prolonged outbreaks of barley powdery mildew (BPM) occurred. This was brought about by a combination of the extensive use of susceptible cultivars and an over‐reliance on a small number of single mode‐of‐action demethylation inhibitor fungicides, which select for mutations in the C14α‐demethylase (Cyp51A) gene. This review highlights the steps taken to reduce losses to BPM, breeding efforts to introduce resistance into cultivars and the success of pre‐breeding research to find new and durable resistance genes. We also draw comparisons with powdery mildew in Australian wheat, where similar factors are leading to substantial outbreaks.

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.

Workflows for detecting fungicide resistance in net form and spot form net blotch pathogens.

Fungicide resistance in Pyrenophora teres f. maculata and P. teres f. teres has become an important disease management issue. Control of the associated barley foliar diseases, spot form and net form net blotch, respectively, relies on three major groups of fungicides, demethylation inhibitors (DMIs), succinate dehydrogenase inhibitors (SDHIs) and quinone outside inhibitors (QoIs). However, resistance has been reported for the DMI and SDHI fungicides in Australia. To enhance detection of different resistance levels, phenotyping and genotyping workflows were designed. The phenotyping workflow generated cultures directly from lesions and compared growth on discriminatory doses of tebuconazole (DMI) and fluxapyroxad (SDHI). Genotyping real-time polymerase chain reaction (PCR) assays were based on alleles associated with sensitivity or resistance to the DMI and SDHI fungicides. These workflows were applied to spot form and net form net blotch collections from 2019 consisting predominantly of P. teres f. teres from South Australia and P. teres f. maculata from Western Australia. For South Australia the Cyp51A L489-3 and SdhC-R134 alleles, associated with resistance to tebuconazole and fluxapyroxad, respectively, were the most prevalent. These alleles were frequently found in single isolates with dual resistance. This study also reports the first detection of a 134 base pair insertion located at position-66 (PtTi-6) in the Cyp51A promoter of P. teres f. maculata from South Australia. For Western Australia, the PtTi-1 insertion was the most common allele associated with resistance to tebuconazole. The workflow and PCR assays designed in this study have been demonstrated to efficiently screen P. teres collections for both phenotypic and genetic resistance to DMI and SDHI fungicides. The distribution of reduced sensitivity and resistance to DMI and SDHI fungicides varied between regions in south-western Australia, suggesting the emergence of resistance was impacted by both local pathogen populations and disease management programmes. The knowledge of fungicide resistance in regional P. teres collections will be important for informing appropriate management strategies. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Evaluation of foliar fungicides for frogeye leaf spot control and yield protection in soybean in Arkansas

The efficacy of fifteen foliar fungicides to control frogeye leaf spot (caused by Cercospora sojina K. Hara) and protect soybean grain yield was investigated over three years (2014–2016) in two field experiments. Experiments were divided into two fungicide groups: premix fungicides, which included combinations of quinone outside inhibitors (QoI), demethylation inhibitors (DMI), succinate dehydrogenase inhibitors (SDHI), or methyl benzimidazole carbamates (MBC), and solo fungicides, all DMI fungicides. Fungicides were applied once at the full pod (R4) in 2014 and 2015 and at the beginning seed (R5) growth stage in 2016. A very susceptible frogeye leaf spot cultivar, Armor 48R40, was used. Frogeye leaf spot severity at fungicide application timing average 1.0, 0.5, and 8.0% in 2014, 2015, and 2016, respectively. Final disease severity averaged 21.7, 14.5, and 15.1% in 2014, 2015, and 2016, respectively on the non-treated control. Azoxystrobin provided no disease control, suggesting the presence of QoI-resistant C. sojina. Three fungicides: azoxystrobin + difenoconazole, flutriafol, and thiophanate-methyl + tebuconazole applied at R4 provided effective disease control compared to the non-treated control. Fungicides applied at R5 provided no disease control. No fungicide provided significant grain yield protection in either experiment. Overall, the average yield protection trend by fungicides applied at R4 and R5 was 6.8 and 3.0%, respectively compared to the non-treated control. These data suggest that although foliar fungicides can provide effective frogeye leaf spot control, yield protection may be limited when applied at the R4 growth stage, particularly when disease severity exceeds 0.5% on a very susceptible soybean cultivar.

Iminoctadine tris (albesilate) resistance in Geotrichum citri-aurantii and its management

The bis-guanidine fungicide iminoctadine tris (albesilate) (ITA) is widely used to control citrus sour rot pathogen Geotrichum citri-aurantii but its resistance status in the orchards is still unclear. In the present study, ITA resistance in G. citri-aurantii was assessed and resistance mechanisms and its alternative fungicides were explored. ITA resistance frequency in field isolates of G. citri-aurantii collected between 2018 and 2020 from orchards in China was 6.2%. Consecutively culturing on potato dextrose agar (PDA) for 10 generations showed that ITA resistance for field resistant isolates was relatively stable, whereas resistance for the laboratory induced resistant isolates declined by 53.9%. The competitive ability on citrus fruit for resistant isolates was substantially lower than that of the sensitive isolates. Resistance mechanism studies showed that an ATP-binding cassette (ABC) gene (EVM0003677) and two major facilitator superfamily (MFS) genes (EVM0000087 and EVM0003101) might be involved in ITA resistance only in the induced resistant isolates but not in the field resistant isolates. Bioassay of 26 fungicides found that the demethylation inhibitor fungicides metconazole and propiconazole, and the dinitro-aniline fungicide fluazinam were highly effective to ITA-sensitive isolates of G. citri-aurantii and their EC50 values were 0.33, 0.56, and 0.54 mg/L, respectively. The protective control efficacies of ITA, propiconazole, metconazole, and fluazinam all at 400 mg/L were 100%, 60.4%, 33.9%, and 18.5%, respectively. No positive cross resistance was detected between ITA and metconazole, propiconazole or fluazinam. The mixture of ITA and propiconazole at the mass ratio of 9:8 had a synergistic co-toxicity factor of 26. These results have significant implications for our awareness of ITA resistance in orchards and its management.

Characterization of the triadimefon resistant Puccinia striiformis f. sp. tritici isolates in China

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a devastating disease that seriously threatens the production of crops worldwide. Triadimefon is the widely-used fungicide for controlling the disease in China; however, as the fungicide targets a single site (position 401 in the 134th codon of the Cyp51 gene), the extensive application imposes a strong selection pressure on the pathogens, which may potentially lose the effect over time. In this study, 176 Pst field isolates sampled from different regions of Xinjiang were determined for their sensitivity to triadimefon because it is the few frequent Pst outbreak and representative area in China. The results showed that the Pst isolates collected from Yili, Xinjiang, exhibited a strong resistance to triadimefon with an average EC50 of 0.263 µg/mL, despite the rest of the isolates maintaining high sensitivity to triadimefon. The triadimefon-resistant and triadimefon-sensitive isolates did not display significant differences in sporulation, but the triadimefon-resistant isolates exhibited weaker adaptive traits in their latent period and urediniospore germination rate than the triadimefon-sensitive isolates. No cross-resistance was found for the other two fungicides, flubeneteram or pyraclostrobin; however, cross-resistance for the demethylation inhibitor (DMI) fungicides, tebuconazole and hexaconazole, was found. Genome sequencing revealed that the Tyrosine (Y) at 134 residue was mutated to Phenylalanine (F) in the Xinjiang isolates. Our study revealed that a natural mutation in Pst led to the efficacy loss of triadimefon to control the disease.

Leaf shredding as an alternative strategy for managing apple scab resistance to demethylation inhibitor fungicides

Within integrated apple scab control there is a strong focus on reduction of Venturia inaequalis primary inoculum. The hypothesis that leaf shredding as an orchard sanitation practice would reduce the effective population size of the fungus (resulting in lower genetic variation due to reduction in sexual offspring) was tested. Assuming the allele causing fungicide resistance is already present in the population, it will be widely distributed at the end of the season, since selection occurs when the demethylation inhibitor (DMI) fungicide was applied. For short-term disease management a reduction of inoculum size, (i.e. potential ascospore dose) is most important. In the long-term resistant isolates/genotypes would be less likely to survive the winter and/or to infect in the spring, if that inoculum (i.e. in fallen leaves) has been removed. To sustain the use of highly effective synthetic fungicides, such as the DMIs, fungicide resistance management practices have to be evaluated. Fungicide resistance, which negatively affects pathogen fitness, is hypothetically reversible, if the selection pressure by the fungicide is removed. This study quantified the effect of leaf shredding on changes in the pathogen’s flusilazole sensitivity and population genetic structure using SSR markers. Venturia inaequalis populations in orchard trials, where sanitation practices had been applied, were tested for flusilazole sensitivity in planta and in vitro. Significant shifts towards flusilazole resistance were identified in orchards with a history of DMI application without sanitation treatment, with a mean sensitivity of EC50= 0.208 ug/ml (n=49) compared to an unexposed V. inaequalis population (EC50= 0.104 ug/ml, n=55). However, the isolates from the same sanitation trial orchards, from leaf shredding treatment in combination with a fungicide spray programme, had a mean EC50 of 0.110 ug/ml (n=41), similar to an unexposed V. inaequalis population. Furthermore, V. inaequalis offspring after sanitation treatment, showed shifts in microsatellite allele frequency distribution patterns used as an indicator of sexual reproduction. This study concludes that sanitation treatments, i.e. leaf shredding, impact on fungicide sensitivity and therefore effectively contributes to fungicide resistance management.

Target and non-target site mechanisms of fungicide resistance and their implications for the management of crop pathogens.

Fungicides are indispensable for high-quality crops, but the rapid emergence and evolution of fungicide resistance have become the most important issues in modern agriculture. Hence, the sustainability and profitability of agricultural production have been challenged due to the limited number of fungicide chemical classes. Resistance to site-specific fungicides has principally been linked to target and non-target site mechanisms. These mechanisms change the structure or expression level, affecting fungicide efficacy and resulting in different and varying resistance levels. This review provides background information about fungicide resistance mechanisms and their implications for developing anti-resistance strategies in plant pathogens. Here, our purpose was to review changes at the target and non-target sites of quinone outside inhibitor fungicides (QoIs), methyl-benzimidazole fungicides (MBCs), demethylation inhibitor fungicides (DMIs), and succinate dehydrogenase inhibitor fungicides (SDHIs) and to evaluate if they may also be associated with a fitness cost on crop pathogen populations. The current knowledge suggests that understanding fungicide resistance mechanisms can facilitate resistance monitoring and assist in developing anti-resistance strategies and new fungicide molecules to help solve this issue. This article is protected by copyright. All rights reserved.

Resistance to QoI and DMI Fungicides do not Reduce Virulence of C. beticola Isolates in North Central USA.

Cercospora leaf spot (CLS) is a destructive disease limiting sugar beet production and managed using resistant cultivars, crop rotation, and timely applications of effective fungicides. Since 2016, its causal agent, Cercospora beticola, has been reported to be resistant to Quinone outside inhibitors (QoIs) and to have reduced sensitive to Demethylation inhibitors (DMIs) in sugar beet growing areas in North Dakota and Minnesota. Isolates of C. beticola resistant to QoIs, DMIs, and both QoIs and DMIs were collected from Foxhome fields, Minnesota in 2017. Fitness of these resistant isolates was compared to that of QoI- and DMI-sensitive isolates in laboratory and greenhouse studies. In the lab, mycelial growth, spore production, and spore germination were measured. The results showed that resistant isolates had significantly less mycelial growth and spore production than sensitive isolates, while no significant difference in spore germination was detected. In the greenhouse, six leaf-stage sugar beets were inoculated with a spore suspension made from each resistant group and then incubated in separate humidity chambers. CLS disease severity was evaluated visually at 7, 14, and 21 days after inoculation (DAI) and the areas under disease progress curve (AUDPC) were calculated. Resistant isolates had significantly smaller AUDPC but still caused high disease severity as the sensitive ones at 21 DAI. Although QoI and/or DMI resistant isolates had a relatively slower disease development, they still caused high disease severity and need to be factored in disease management practices.

Deep amplicon sequencing reveals extensive allelic diversity in the erg11/CYP51 promoter and allows multi-population DMI fungicide resistance monitoring in the canola pathogen Leptosphaeria maculans

Continued use of fungicides provides a strong selection pressure towards strains with mutations to render these chemicals less effective. Previous research has shown that resistance to the demethylation inhibitor (DMI) fungicides, which target ergosterol synthesis, in the canola pathogen Leptosphaeria maculans has emerged in Australia and Europe. The change in fungicide sensitivity of individual isolates was found to be due to DNA insertions into the promoter of the erg11/CYP51 DMI target gene. Whether or not these were the only types of mutations and how prevalent they were in Australian populations was explored in the current study. New isolates with reduced DMI sensitivity were obtained from screens on DMI-treated plants, revealing eight independent insertions in the erg11 promoter. A novel deep amplicon sequencing approach applied to populations of ascospores fired from stubble identified an additional undetected insertion allele and quantified the frequencies of all known insertions, suggesting that, at least in the samples processed, the combined frequency of resistant alleles is between 0.0376% and 32.6%. Combined insertion allele frequencies positively correlated with population-level measures of in planta resistance to four different DMI treatments. Additionally, there was no evidence for erg11 coding mutations playing a role in conferring resistance in Australian populations. This research provides a key method for assessing fungicide resistance frequency in stubble-borne populations of plant pathogens and a baseline from which additional surveillance can be conducted in L. maculans. Whether or not the observed resistance allele frequencies are associated with loss of effective disease control in the field remains to be established.

Widespread Resistance to Tebuconazole and Cross-Resistance to Other DMI Fungicides in Stagonosporopsis citrulli Isolated from Watermelon in South Carolina.

Tebuconazole, a demethylation-inhibitor (DMI) fungicide, is widely used on watermelon and muskmelon because it is inexpensive and has been effective against Stagonosporopsis citrulli, the primary causal agent of gummy stem blight in the southeastern United States. Most isolates (94% of 251) collected from watermelon in South Carolina in 2019 and 2021 were moderately resistant to tebuconazole at 3.0 mg/liter in vitro. Ninety isolates were identified as S. citrulli, and no isolates of S. caricae were found in this study. On watermelon and muskmelon seedlings treated with the field rate of tebuconazole, sensitive, moderately resistant, and highly resistant isolates were controlled 99, 74, and 45%, respectively. In vitro, tebuconazole-sensitive isolates were moderately resistant to tetraconazole and flutriafol but sensitive to difenoconazole and prothioconazole, while highly resistant isolates were highly resistant to tetraconazole and flutriafol and moderately resistant to difenoconazole and prothioconazole. On watermelon seedlings treated with field rates of five DMI fungicides in the greenhouse, severity of gummy stem blight did not differ significantly from the nontreated control when seedlings were inoculated with a highly resistant isolate, while severity was lower with all DMIs on seedlings inoculated with a sensitive isolate, although severity was greater with tetraconazole than with the other four DMIs. In the field, tetraconazole rotated with mancozeb did not reduce severity of gummy stem blight caused by a tebuconazole-sensitive isolate when compared to the nontreated control, while the other four DMIs did. With a highly resistant isolate, all DMIs rotated with mancozeb reduced severity of gummy stem blight compared to the nontreated control, but severity with tetraconazole and tebuconazole was greater than with mancozeb alone, and severity with flutriafol, difenoconazole, prothioconazole, and difenoconazole plus cyprodinil did not differ from mancozeb applied alone. Results from in vitro, greenhouse, and field experiments with the five DMI fungicides were highly correlated with each other. Thus, determining relative colony diameters with a discriminatory dose of 3 mg/liter of tebuconazole is an effective way to identify isolates of S. citrulli highly resistant to tebuconazole.

Sensitivity of Puccinia triticina f. sp. tritici from China to Triadimefon and Resistance Risk Assessment.

Wheat leaf rust, caused by Puccinia triticina f. sp. tritici (Pt), is distributed widely in wheat-producing areas and results in serious yield losses worldwide. In China, leaf rust has been largely controlled with a demethylation inhibitor (DMI) fungicide, triadimefon. Although high levels of fungicide resistance in pathogens have been reported, no field failure of wheat leaf rust to DMI fungicides has been reported in China. A resistance risk assessment of triadimefon to Pt was investigated in the present study. The sensitivity of 197 Pt isolates across the country to triadimefon was determined, and the density distribution of EC50 values (concentration at which mycelial growth is inhibited by 50%) showed a continuous multimodal curve because of the extensive use of this fungicide in wheat production, with a mean value of 0.46 μg/ml. The majority of the tested Pt isolates were sensitive to triadimefon, whereas 10.2% developed varying degrees of resistance. Characterization of parasitic fitness revealed that the triadimefon-resistant isolates exhibited strong adaptive traits in urediniospore germination rate, latent period, sporulation intensity, and lesion expansion rate. No correlation was observed between triadimefon and tebuconazole and hexaconazole, which have the similar mode of action, or pyraclostrobin and flubeneteram, which have different modes of action. Overexpression of the target gene Cyp51 led to the triadimefon resistance of Pt. The risk of resistance to triadimefon in Pt may be low to moderate. This study provided important data for fungicide resistance risk management against wheat leaf rust.

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.

Analysis of the difenoconazole-resistance risk and its molecular basis in Colletotrichum truncatum from soybean.

Anthracnose disease, caused by Colletotrichum truncatum, is a destructive fungal disease in soybean worldwide and some demethylation inhibitor fungicides are used to manage it. In this study, the sensitivity of C. truncatum to difenoconazole was determined, and the risk for resistance development of C. truncatum to difenoconazole was also assessed. The results showed that the mean EC50 value was 0.9313 μg/mL and the frequency of sensitivity formed a unimodal distribution. Six stable mutants with a mutation frequency of 8.33 × 10-5 were generated, and resistance factors ranged from 3.00 to 5.81 after 10 successive culture transfers. All mutants exhibited fitness penalties in reduced mycelial growth rate, sporulation and pathogenicity, except for the Ct2-3-5 mutant. Positive cross-resistance was observed between difenoconazole and propiconazole, but not between difenoconazole and prochloraz, pyraclostrobin or fluazinam. One point mutation I463V in CYP51A was found in five resistant mutants. Surprisingly, the homologous I463V mutation has not been observed in other plant pathogens. CYP51A and CYP51B expression increased slightly in the resistant mutants as compared to wild-types when exposed to difenoconazole, but not in the CtR61-2-3f and CtR61-2-4a mutants. In general, a new point mutation, I463V in CYP51A, could be associated with low resistance to difenoconazole in C. truncatum. In the greenhouse assay, control efficacy of difenoconazole on both parental isolates and the mutants increased in a dose dependent manner. Collectively, the resistance risk of C. truncatum to difenoconazole is regarded to be low to moderate, suggesting that difenoconazole can still be reasonably used to control soybean anthracnose.

Efficacy of quinone outside inhibitors and demethylation inhibitors fungicides against false smut of rice (Oryza sativa)

The present study was carried out at research farm of Bihar Agricultural University, Bhagalpur, Bihar during rainy (kharif) season of 2018–19 and 2019–20 to study the efficacy of four quinone outside inhibitors, demethylation inhibitors and benzimidazole group of fungicides, viz. trifloxystrobin 25% + tebuconazole 50% wg, flusilazole 25% + carbendazim 12.5% se, azoxystrobin 18.2% + difenoconazole 11.4% sc and azoxystrobin 11% + tebuconazole 18.3% W/W at different growth stage of rice (Oryza sativa L.), i.e. booting stage (BS), 50% flowering stage (FS) and 100% flowering stage. Among them, trifloxystrobin 25% + tebuconazole 50% wg @1 g/litre at 50% flowering stage was found superior in minimizing the per cent of disease infection. The number of infected tillers/m2 (18.33 and 13.33), number of smut balls/panicle (1.96 and 1.80), smut balls per panicle (0.48% and 0.51%), disease incidence (5.22%and 3.71%) and disease severity (2.86% and 1.78%) respectively were observed in trifloxystrobin 25% + tebuconazole 50% wg @1 g/litre at 50% flowering stage as compared to control. Out of the tested fungicides, trifloxystrobin 25% + tebuconazole 50% wg was found significantly superior in terms of reducing the infection with highest grain yield (90.58 q/ha) and thereby the grain yield upto 43.05% when it was sprayed at 50% flowering stage of crop @1 ml/litre under the field condition in both years. Hence, it is concluded that, trifloxystrobin 25% + tebuconazole 50% wg belonging to two different chemical family group with broad mode of action fungicide significantly (P<0.05%) reduced the infection at 50% flowering stage of crop against false smut disease.

Frequency of Alternaria Genotypes Resistant to SDHI Fungicides in California Pistachio Orchards Determined by Real-Time PCR.

Real-time PCR methods were developed to quantify the frequency of SDHC-H134R and SDHB-H277Y mutants associated with succinate dehydrogenase inhibitors (SDHI) resistance in Alternaria populations from pistachio. The linearity of the standard curves demonstrated the applicability in the quantification of the assays. The accuracy and reliability of the qPCR protocols to determine the frequency of mutants in real samples were corroborated. Orchards visibly affected by Alternaria late blight were sampled. The frequency of mutants was determined using the qPCR assays, while the frequency of resistant phenotypes was determined using a single discriminatory dose. The statistical analysis showed that the frequencies of the mutation SDHC-H134R determined with the qPCR assay were highly correlated with those estimated with the conventional method. The survey also evidenced that resistance to boscalid is still widespread in California. Results also indicated the possible contribution of other mutations to SDHI resistance. Our results confirmed the prevalence of SDHC-H134R mutants and the occurrence of mutation SDHB-H277Y at low frequencies. The real-time PCR methods developed in this study were able to detect differences in the frequencies of resistant mutants caused by the use of chemical fungicides. Finally, the effects of two fungicide programs on the frequency of mutants resistant to SDHI and quinone outside inhibitors fungicides were studied using qPCR assays. The experiments demonstrated that the use of anilinopyrimidine and demethylation inhibitors fungicides in the same program reduced the frequency of these mutations in Alternaria populations. The qPCR methods developed and used in this study can be used to track resistance levels in the pistachio orchards on a large scale.